CN100385013C - Solid support assay systems and methods utilizing non-standard bases - Google Patents
Solid support assay systems and methods utilizing non-standard bases Download PDFInfo
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- CN100385013C CN100385013C CNB018205151A CN01820515A CN100385013C CN 100385013 C CN100385013 C CN 100385013C CN B018205151 A CNB018205151 A CN B018205151A CN 01820515 A CN01820515 A CN 01820515A CN 100385013 C CN100385013 C CN 100385013C
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Abstract
Solid support assays using non-standard bases are described. A capture oligonucleotide comprising a molecular recognition sequence is attached to a solid support and hybridized with a target oligonucleotide the solid support. In some instances, the molecular recognition sequence includes one or more non-standard bases and hybridizes to a complementary tagging sequence of the target oligonucleotide. In other instances, incorpoaration of a non-standard base (e.g., via PCR or ligation) is used in the assay.
Description
Background of invention
For oligonucleotide, comprise DNA and RNA fragment, developed multiple different analytical procedure of all kinds, these methods can directly detect whether comprise the oligonucleotide with particular target oligonucleotide sequence in the sample usually.In some cases, many allelotrope sequences, the difference of oligonucleotide sequence only are the differences of several Nucleotide.Single nucleotide polymorphism (SNPs) refers to the difference of the single Nucleotide of allelotrope.At least under certain conditions, even the difference of single Nucleotide also causes the change of corresponding heredity response or proterties.Accordingly, in order there to be which kind of allelotrope in the test sample, this detection technique must have enough sensitivity to distinguish closely-related sequence.
Many analytical technologies comprise a plurality of compositions, and every kind of composition will become phase-splitting hybridization with other in analytical test.Non-specific hybridization in the analytical test between the composition (that is: the hybridization of two incomplementarity sequences) produces high hybrid context.Such as, closely similar but not on all four two sequences can not form complete complementary two strands, wherein the base of two strands not the base pairing of complementary position be interrupted.When the composition of two similar sequences is hybridized mutually, described as top situation, a lot of allelotrope sequence particularly contains the allelotrope sequence of SNP, and non-specific hybridization is increased.Therefore, reducing non-specific hybridization from method, perhaps reduce the influence of non-specific hybridization in analysis, is that benifit is arranged very much to having which kind of allelic hybridization analysis in the working sample for example.
Summary of the invention
Generally speaking, the present invention relates to analyze the method for oligonucleotide, test kit and composition.In addition, the invention still further relates to method, test kit and the composition that uses non-standard bases analysis oligonucleotide.In one embodiment, provide a kind of method that is used to analyze the assay specific sequence.Catch oligonucleotide for one and comprise a molecular recognition sequence, this molecular recognition sequence contains at least one non-standard bases and is coupled at (for example single solid support on the support, as chip, wafer or particle support), it contact with sample and under suitable hybridization conditions with target oligonucleotide sequence hybridization, if this sample contains the target oligonucleotide.The target oligonucleotide comprises a flag sequence, and it is complementary to the complementary sequence of catching oligonucleotide molecular recognition sequence and analyte distinguished sequence or analyte distinguished sequence.The hybridization that detects the target oligonucleotide and catch.
Another embodiment of the present invention provides another to detect the method for assay distinguished sequence.Catch oligonucleotide and support coupling mutually, this is caught oligonucleotide and comprises a molecular recognition sequence, this molecular recognition sequence is identical or complementary to small part with the assay distinguished sequence, and capture oligo contacts and target oligonucleotide hybridization with sample under hybridization conditions.The target oligonucleotide comprises flag sequence and the distinguished sequence of assay or the complementary sequence of analyte distinguished sequence that contains at least one non-standard bases.Catching oligonucleotide is that template is carried out enzymatic and extended with the target oligonucleotide, with the non-standard bases relative position of flag sequence on mix the complementary non-standard bases.Reporter group also can be incorporated into the extension of capture oligo.Detect target oligonucleotide and the hybridization of catching oligonucleotide.
Another embodiment of the present invention provides another to detect the method for assay distinguished sequence.The assay that contains the assay distinguished sequence contacts with second primer with first primer.First primer comprises the complementary sequence of flag sequence and assay first sequence.Second primer comprises complementary sequence and non-standard bases of assay second sequence.The first and second primer enzymatics extend and form the target oligonucleotide and second oligonucleotide respectively.One of target oligonucleotide and second oligonucleotide contain the assay distinguished sequence, and another contains the complementary sequence of assay distinguished sequence.The extension of first primer stops at non-standard bases with second primer basically when meeting.Be complementary to the second primer non-standard bases and be incorporated into first primer of extension and relative with the non-standard bases of second primer.It is identical or complementary to small part with the assay distinguished sequence to catch the oligonucleotide molecular recognition sequence, analyte distinguished sequence and support coupling mutually, under hybridization conditions, catch the contact of oligonucleotide molecular recognition sequence and target oligonucleotide and with hybridize mutually with the target oligonucleotide that comprises flag sequence and assay distinguished sequence or assay distinguished sequence complementary sequence.Detect target oligonucleotide and the hybridization of catching oligonucleotide.
Another embodiment of the present invention is one to be applied to the test kit of aforesaid method.This test kit comprises support and catches oligonucleotide.This test kit also comprises the target oligonucleotide or make the reagent of target oligonucleotide from assay.These reagent comprise, for example, polysaccharase, are complementary to first, second primer of assay sequence, and perhaps first or second primer comprises flag sequence.In certain methods, this test kit also comprises off-gauge base or mixes the triphosphopyridine nucleotide of non-standard bases.
More than relevant general introduction of the present invention be not to be description to all disclosed embodiments of the present invention or application.Accompanying drawing and following detailed Description Of The Invention will be to these embodiment more detailed descriptions.
The accompanying drawing summary
With reference to the detailed description and the diagram of the various embodiments of the present invention, meeting of the present invention is understood well, wherein:
Put on display the chemical structure of various non-standard bases among Fig. 1, wherein A is the point that links to each other with main polymer chain, and X is N or C-Z, and Y is N or C-H, and Z is alkyl H or replacement or non-replacement;
Fig. 2 A and 2B are two examples that oligonucleotide of the present invention is hybridized on solid support;
Fig. 3 has illustrated that the present invention analyzes the step of assay distinguished sequence for the first time;
Fig. 4 has illustrated that the present invention analyzes the step of assay distinguished sequence for the second time;
Fig. 5 has illustrated that the present invention analyzes the step of assay distinguished sequence for the third time;
Fig. 6 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 4th time;
Fig. 7 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 5th time;
Fig. 8 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 6th time;
Fig. 9 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 7th time;
Figure 10 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 8th time;
Figure 11 has illustrated that the present invention analyzes the step of assay distinguished sequence for the 9th time;
Figure 12 is the detailed annotation of 3D exterior view, 98 molecular recognition sequences (y axle) has been described, with the hybridization of the complementary indicia sequence (x axle) of each in 100 molecular recognition sequences;
Figure 13 is the detailed annotation of 3D exterior view, the recognition sequence (y axle) of 50 molecules has been described, with the hybridization of the complementary indicia sequence (x axle) of each in 50 molecular recognition sequences;
Figure 14 shows the diagram detailed annotation that an allelotrope analysis of the present invention obtains;
Figure 15 shows the diagram detailed annotation that the genetic analysis of another time of the present invention equipotential obtains;
Figure 16 has illustrated that the present invention analyzes the step of assay distinguished sequence for the tenth time;
Figure 17 shows the diagram that an allelotrope analysis of the present invention obtains.
Though the various modifications that the present invention has provided in the accompanying drawings by way of example, selection form and special case are listed one by one and are described in detail.But it must be understood that the present invention is not limited to the specific embodiments that the present invention describes.On the contrary, the present invention has covered all modifications without prejudice to spirit and scope of the invention, the of equal value replacement and the form change, and they all belong to category of the present invention.
The detailed description of optimum implementation
The application's related application is, application number is 60/240,397 U.S. Provisional Application, and the date of application is on October 14th, 2000; Application number is 60/282,831 U.S. Provisional Application, and the date of application is April 10 calendar year 2001; Application number is 09/861,292 U.S. Provisional Application, and the date of application is May 18 calendar year 2001; Application number is 60/293,259 U.S. Provisional Application, and the date of application is May 22 calendar year 2001, and these patents are hereby incorporated by reference.
What the present invention relates to is a kind of oligonucleotide analysis and analytical procedure thereof.What the present invention relates in particular, is a kind of test and analytical procedure of using one or more non-standard bases to test oligonucleotide.Though the present invention is not limited thereto, the benefit of relevant all respects of the present invention will obtain by following discussion.Other analytical procedures of using non-standard bases are described in application number is 60/240,397 United States Patent (USP) provisional application book, and the date of application of this application is on October 14th, 2000.
Employed as us, " nucleic acid " comprises polymerizable molecular, resemble thymus nucleic acid (DNA), Yeast Nucleic Acid (RNA), peptide nucleic acid(PNA) (PNA), or other any sequences, other any sequences are often referred to by chemical backbone and connect base, and wherein the base of this sequence can form base pair or hybridize with the complementary chemical structure.Suitable non-nucleic acid chemistry main chain comprises, such as: polymeric amide and poly morpholine main chain.Term " nucleic acid " comprises oligonucleotide, Nucleotide or polymerized nucleoside acid sequence and fragment and partial sequence thereof.Nucleotide can obtain with any suitable form, for example separates from natural origin, and reorganization preparation, synthetic can be strand or two strands, also can be sense strand or antisense strand.
Term " oligonucleotide " (for example is commonly referred to as short nucleotide sequence, length is less than 100 Nucleotide, normal length is 6 to 50 Nucleotide), this sequence can use the following technology in this field to obtain usually, as the solid support synthetic technology of nucleic acid, dna replication dna, reverse transcription, digestion with restriction enzyme, second takes turns and transcribes etc.The definite size of oligonucleotide is determined that by various factors the size of oligonucleotide is determined by the basic function or the purposes of oligonucleotide again conversely.
" sequence " refers to the order of Nucleotide.
Term " sample " comprises sample or substratum (for example, microbiological culture media), or biological specimen, derives from the biological specimen of biological fluid and the sample in abiotic source.
Term " assay " refers to the nucleic acid to be detected in the sample.Assay is the target (for example, analysis is to detect whether assay exists concentration or amount in the sample) of analytical test.Assay can tested directly or indirectly analysis.At least in some embodiments the indirectly testing assay if assay occurs in sample, is the synthetic target oligonucleotide of template with this assay, for example uses round pcr.The target oligonucleotide is used to analyze then, illustrates whether analyte in sample exists, concentration or amount.
Term " target oligonucleotide " refers to oligonucleotide actual tested in analysis test method.The target oligonucleotide can be, assay or contain the oligonucleotide of assay specific sequence for example, and this specific sequence is identical with the assay sequence or complementary.Such as, the target oligonucleotide can be the part of the pcr amplification product or the assay of assay.
Term " catch oligonucleotide " and refer to have the identification molecular sequences and with solid support surface link coupled oligonucleotide sequence mutually, this sequence can with contain flag sequence or be complementary to the assay distinguished sequence hybridization of molecular recognition sequence, thereby at solid support surface acquisition target oligonucleotide.
" molecular recognition sequence " that the application mentioned refers to the oligonucleotide sequence that is complementary to target oligonucleotide assay distinguished sequence or flag sequence.
Here, term " complementation " or " complementarity " be when being used for nucleic acid, and (that is, as the nucleotide sequence of oligonucleotide nucleic acid or target nucleic acid) refers to the basepairing rule of sequence.For the standard base, basepairing rule is that fertile gloomy Watson and Ke Like Crick propose.For non-standard bases, will describe to some extent below, thereby the non-standard bases pair principle refers to form hydrogen bond or to form special base pairing by hydrophobicity, entropy or Van der Waals force to the Wo Sen mode similar with the Ke Like basepairing rule.For example, the complementary sequence of sequence " T-G-A " is " A-C-T ".Complementation can be " part " complementation, wherein has only the base of part nucleotide sequence to meet base and joins principle and mate.Another kind of mode is complete or all complementary between the nucleic acid.Article two, the complementary degree of nucleic acid interchain affects the efficient and the intensity of two nucleic acid chains intermolecular hybrids.
Term " hybridization " refers to the pairing of two complementary nucleic acid chains.Hybridization and intensity for hybridization (that is, associating intensity between nucleic acid) are influenced by multiple factor, such as, complementary degree between nucleic acid molecule, the rigorous degree of hybridization conditions comprises the melting temperature (Tm) (T that forms hybridization
m), the G in the nucleic acid molecule: the ratio of C.
Carry out analytical test and come whether to contain in the test sample assay that has specific nucleic acid sequence (or its complementary sequence).This nucleotide sequence refers to " assay distinguished sequence ".At least in some instances, original sample can not be tested by direct analysis.But, if this assay exists, with assay clone or amplification (for example, using round pcr) so that analytic sample to be provided, but in sample, contain the target oligonucleotide that has the assay distinguished sequence of detection limit.Other amplification technique comprises, for example, nucleotide sequence base amplification (NASBA, for example Guatelil etc., " American Academy of Sciences's progress " (Pro.Natl.Acad.Sci.) 87,1874 (1990), incorporated by reference here), chain is replaced amplification (SDA, for example Wakler etc. " American Academy of Sciences's progress " (Pro.Natl.Acad.Sci.) 89,392-96 (1992), incorporated by reference here), ligase chain reaction (LCR) (LCR, Kalin etc. for example, " mutation research " (mutat.Res.), 283,119-23 (1992), here incorporated by reference), the amplification of transcriptive intermediate (TMA, LaRocco etc. for example, clinical (Eur.J.Clin.Microbiol.Infect.Dis) 13 of European infected by microbes disease, 726-31 (1994), here incorporated by reference), rolling circle amplification (RCA, for example Lizardi etc., " natural genetics " (Nat.Genet), 19,225-32 (1998), incorporated by reference here).At least, the part of target oligonucleotide generally corresponds to following wherein a kind of, a) assay, and the b) part of assay c) is complementary to the material of assay, d) is complementary to the part of assay.The detected target oligonucleotide of this analytical test has illustrated the existence of assay in the primary sample.
Usually, the analyzing and testing system that is used to detect one or more assay distinguished sequences comprises solid support (for example, the perhaps many solid particulates of chip, wafer).Catch oligonucleotide and be fixed on the solid support (for example, utilize the position on chip or wafer or utilize particle characteristics to link to each other) with the special oligonucleotide of catching in a kind of mode that allow to identify oligonucleotide.Catch oligonucleotide and comprise molecular recognition sequence.The difference that has different molecular recognition sequences is caught oligonucleotide and is used to detect different assay distinguished sequences.Therefore, in the single test macro, design is used and different caught oligonucleotide and can analyze the single sample that contains a plurality of assay distinguished sequences.
Contain the target oligonucleotide of assay distinguished sequence and catch the oligonucleotide serial response, target oligonucleotide sequence also comprises flag sequence except the assay distinguished sequence.Special flag sequence is connected with each assay distinguished sequence.Flag sequence is complementary to one of them molecular recognition sequence usually.Therefore, under hybridization conditions, target oligonucleotide sequence is hybridized mutually with suitable acquisition sequence.In addition, in ad hoc approach more of the present invention, the assay distinguished sequence can be complementary to one of molecular recognition sequence.
Target oligonucleotide or its complementary strand typically contain a reporter gene or are used to connect the coupling agent of reporter gene.Whether exist on the solid support and the specific mutually associating reporter gene of oligonucleotide of catching by observing, determine whether the special analytical sequence of assay special in the sample exists.Suitable reporter gene includes, but not limited to vitamin H, fluorescein, chemiluminescent substance, digoxin, spin label, radio-labeled thing, DNA cutting part, chromophoric group, or fluorescence chromophoric group.Some suitable coupling agents include but not limited to amine, thio-alcohol, Sodium Hydrosulphite (hydrosines) alcohol or alkyl.
The example of appropriate analysis of test system is as shown in synoptic diagram 2A and the 2B.One of in this analytical test, catch oligonucleotide 100a, on 100b coupling one solid support 120, such as, single solid substrate 120a (as, chip or wafer), or the member of solid particulate 120b.Usually, have at least one catch oligonucleotide (as, catch oligonucleotide 100a) contain a part recognition sequence 102, it is complementary to the flag sequence 112 of target oligonucleotide 110, therefore, under hybridization conditions, target oligonucleotide 110 with catch oligonucleotide 100a and hybridize mutually.
Although this analysis method of testing can react all oligonucleotides of catching with same spherical molecular recognition sequence, typical, that uses two groups or how different group catches oligonucleotide 100a, 100b.Each group is caught oligonucleotide and is all had a different molecular recognition sequence.On a solid-phase matrix, catch oligonucleotide for every group and typically be arranged in one or several specific zone of matrix, these zones and specific molecular recognition sequence association.When using the particulate support, catch oligonucleotide 100a, each group of 100b is arranged on the particulate 120b of special properties, on one of at least one group of 120c, like this, the oligonucleotide on the specific particle can be from particulate character that it connected and is determined.Can use following test, for example, a) by making different oligonucleotides (with zones different in the matrix, different particulates) associate mutually with each allelotrope, have which kind of allelotrope to exist in the test sample, b) analyze multiple relevant or incoherent oligonucleotide sequence or c) implement these two.Shown in Fig. 2 A and 2B, the target oligonucleotide preferentially and is accordingly caught oligonucleotide hybridization, whether exist by observing target oligonucleotide (Fig. 2 B) on particular space on the single support (Fig. 2 A) or the specific particle that connects, the existence of determining the assay distinguished sequence whether.
As described below, a supplementary component of this analysis test macro is a reporter gene 130, it and target oligonucleotide molecule 1 10 (or its complementary sequence 120) coupling mutually.Reporter gene 130 is moietys of analytical test, this moiety can detected technology (as, colorimetry, fluorometry, electrophoresis, electrochemical process, spectroscopic analysis, red, orange, green, blue, yellow (ROGBY), optical densitometric method or radiation technique) detect, with existing or concentration of display target oligonucleotide.Reporter gene determines that by detection technique (for example, fluorescence technique is used the fluorescence reporter gene, and radiation technique is used radio-labeled usually.)
In some test analysis, catch a kind of in them of oligonucleotide and target oligonucleotide or all comprise at least one non-standard bases.The use of non-standard bases can improve the analysis specificity of the analytical test process that comprises hybridization, because non-standard bases is preferentially hybridized mutually with other complementary non-standard bases.Use longer oligonucleotide also can increase the specific ratio of hybridization.About three to four complete complementary samples of base are got in the hybridization of nucleic acid usually.This has just formed the nucleation site.If the nucleation site forms, hybridization will extend to whole chain.Can not be complementary after if the base on the chain is extended, two chains will discharge.Because nucleation process is wanted spended time, the use of non-standard bases can reduce the possibility that the nucleation site forms, and mates and the sampling step number of needs thereby reduced in order to form fully.
In addition, the use meeting of non-standard bases is directly mixed sequence (for example using round pcr) to another non-standard bases.The non-standard bases that is impregnated in comprise reporter gene and and reporter gene link coupled reagent, what this reagent can highly selective mixes reporter gene to be used to detect the target oligonucleotide.
Oligonucleotide and base
DNA and RNA are oligonucleotides, and they are respectively the polymers that deoxynucleoside or nucleosides are linked to each other by phosphodiester bond.Each deoxynucleoside or nucleosides comprise a base coupled with glycosyl.Naturally the base of mixing among DNA of Cun Zaiing and the RNA has VITAMIN B4 (A), guanine (G), thymus pyrimidine (T), cytosine(Cyt) (C) and uridylic (U).These five kinds of bases are called " natural base ".According to Wo Sen and the described basepairing rule of Ke Like, natural base can hybridize that the formation purine---pyrimidine bases are right, G and C pairing, A and T or U pairing.This pair principle is exactly oligonucleotide and its complementary oligonucleotide specific hybrid.
The form of these base pairings of natural base produces by forming two or three hydrogen bonds between two bases.Each base all has two or three hydrogen bond donors and hydrogen bond receptor.Each hydrogen bond between base pair is that at least one hydrogen bond donor of a base and the hydrogen bond receptor of another base interact and generation.Hydrogen bond donor comprises for example heteroatoms (for example, Sauerstoffatom, nitrogen-atoms), and it has a continuous hydrogen atom at least.Hydrogen bond receptor comprises for example heteroatoms (for example, Sauerstoffatom or nitrogen-atoms), and it has a pair of lone-pair electron at least.
Natural base, A, G, C, T and U are substituted in the site of non-hydrogen bond, are derivatized to by the natural base of modified.For example, natural base can by derivatize link to each other with support be by reactive functionality (as, mercaptan, hydrazine, alcohol or amine) with base on the coupling of non-hydrogen bond atom form.Other possible substituting group comprises vitamin H, digoxin, and the plain group of English light, alkyl (such as, methyl or ethyl).
Non-standard bases also can form base pair by hydrogen bond, can be with reference to the description of following information, for example, United States Patent (USP) 5,432,272,5,965,3
64, 6,001,983 and 6,037,120 and Application No. 08/775,401 all these patent documentations be hereby incorporated by reference." non-standard bases " refers to a kind of base, and it is not A, G, and C, T, or U but can be incorporated in the oligonucleotide chain, can pass through hydrogen bond, or hydrophobic interaction, entropy, Van der Waals force interacts and is complementary to complementary base with it, forms base pairing.Fig. 1 be several suitable bases and with the example of the several base pairings of they complementary.The object lesson of these bases is included in the base in the following base pair complex body (iso-C/iso-G, K/X, H/J, and M/N).
Wherein A is the point that the other parts with glycosyl or main polymer chain are linked to each other, and R is alkyl H or replacement and non-replacement.The non-standard bases of recognizing other can prepare by hydrogen bonded, perhaps mixes the non-standard bases more than the modified with functional group in the non-hydrogen bond atom of base bonded position and prepares.In Fig. 3 to Fig. 9, non-standard bases is indicated by following symbol: X represents iso-C, and Y represents iso-G.
Non-standard bases paired hydrogen bond is similar to the hydrogen bond of natural base pairing, has wherein formed two or three hydrogen bonds between right hydrogen bond donor of non-standard bases and the hydrogen bond receptor.A difference between natural base and non-standard bases is hydrogen bond donor and acceptor quantity and position.For example, cytosine(Cyt) is considered to one donor/acceptor/acceptor base, and guanine is complementary acceptor/donor/donor base.Described as reference United States Patent (USP) 6,037,120, Iso-C is one acceptor/acceptor/donor base, and iso-G is complementary donor/donor/acceptor base, and this patent is here in the lump by incorporated by reference.
Other non-standard bases that uses in the oligonucleotide includes, for example, and naphthalene, phenanthrene, pyrene derivatives.Can consult for information about, for example, Ren etc. are published in " American Chemical Society " (J.Am.Chem.Soc.), 118,1671 (1996) article and McMinn etc., at " American Chemical Society " (J.Am.Chem.Soc.) 121,11585 (1999) article, these two pieces of documents are hereby incorporated by reference.These bases are not to form stable base pair by hydrogen bond, but rely on hydrophobic interaction, and entropy, Van der Waals force interact and form base pair.
Solid support
The enforcement of this analysis test has been used solid support in part at least.Usually, catch oligonucleotide and be coupled to or be attached to the support surface.Can use multiple different support.In some embodiments, solid support is single solid phase support body, for example chip, wafer, test tube, the internal surface of Erlenmeyer flask or other article or outside surface.Solid support can be made by any suitable material, as long as this material can provide such as stability, and size, shape, the combination of best attributes such as surface flatness.Preferable material will not influence nucleic acid hybridization, also not can with a large amount of non-specific binding of nucleic acid.The material that is fit to is zoic, abiotic, organic or inorganic materials.For example, microarray can be by any suitable plastic, polymkeric substance, and silicon, glass, pottery or metallic substance are made, and also can be made into solid simultaneously, or polymkeric substance, colloid, ebonite sheet, various forms such as Elastic Film.The suitable polymers material comprises that for example, polystyrene gathers (alkyl) methacrylic ester, poly-(ethene benzophenone), polycarbonate, polyethylene, polypropylene, polymeric amide, poly(vinylidene fluoride) and other analogue.Preferable material comprises polystyrene, glass and silicon.
As shown in Figure 3, in some embodiments, single solid support 300 has been divided into one by one independently zonule 310, catches oligonucleotide and is arranged on the support in each district.On each individual region or each particulate support, catch oligonucleotide significant (for example, at least 75%) and contain identical molecular recognition sequence.Preferably, in each district or all basically on each particle support (such as, at least 90%, better, at least 99%) catch the oligonucleotide molecule and all contain identical molecular recognition sequence.Under the usual condition, the oligonucleotide of catching of different zones has different sequences, even in certain embodiments, identical catch oligonucleotide and can be used in two or more zones, for example, in contrast or be used to confirm the result.
There is the solid support of different zones can make rule or irregular array, is used for specimen and measures whether exist multiple different assay distinguished sequence.For example, microarray can detect 10,100,1000 or more different assay distinguished sequence.
The size of solid support is by the quantity in desired point sample zone and factors such as the number decision of the assay distinguished sequence that will analyze.For example, solid support can be long from about 0.5cm to about the 7.5cm, wide from the two dimensional surface to about the 7.5cm about 0.5cm.Solid support can also one or more layers be placed on other support, such as microslide (for example, long for about 7.5cm, wide is about 2.5cm).The size of solid support can be adjusted in some concrete enforcements as required easily.
Also can use the solid support of some other type.In some embodiments, solid support is some particulate supports.In these embodiments, catching oligonucleotide is coupled on the particulate.Usually, particulate divides in groups, and the particulate in same group has particular characteristics, such as, color, fluorescence, and frequency, density, size, or shape can be distinguished or separate not on the same group particulate with these character.Preferably, these particulates can with technical point from, for example, flow cytophotometer.
Desired according to the present invention, particulate can be to be made by any insoluble or solid material.Such as, particulate can be by silica gel, glass, nylon, resin, sephadex
TM, agarose
TM, Mierocrystalline cellulose, magneticsubstance, metal (as, steel, gold, silver, aluminium, copper or alloy), metallization, plastics (as, polyethylene, polypropylene, polymeric amide, polyester, poly(vinylidene fluoride) (PVDF)) etc., and their composition is made.Appropriate example is a microballon, and it is at United States Patent (USP) 5,736, describes in 330,6,046,807 and 6,057,107, draws in the lump at this to be reference.The example of suitable particulate is available, for example from Luminex company, Austin, Dezhou.
In one embodiment, be placed in the container with catching the particulate support that oligonucleotide links to each other, container can be an ampoule for example, test tube, orifice plate.The target oligonucleotide joins in the container, carries out analytical test under hybridization conditions.Based on every group of all characteristic of support the particulate support is separated again.And then the target oligonucleotide that is connected on every group of support is tested in research respectively.Choose wantonly, can also the wash-out support lower the influence of phase mutual cross.As following description, the wash-out of one or many can carry out under identical or different rigorous condition, and is as mentioned below.Optionally, before catching oligonucleotide and support contacts, contain the solution of target oligonucleotide in addition, size exclusion chromatography, relative settlement method can for example be used, column spinner, methods such as Filter column remove the primer that is not amplified or with target oligonucleotide other material in different size.
In some embodiments, use a plurality of containers (as, bottle, test tube etc.), analyze a plurality of analytic samples in each container or different binding substances (containing associating support) of catching oligonucleotide.Under other situations, each container only comprise one type catch oligonucleotide (with associating support).
In another embodiment, support can be some independently support surfaces, and they can be randomly together coupled.Can comprise individual optical fiber or some other support members such as, this support, they respectively coupling different catch oligonucleotide, interconnect again, form an independent support, such as matrix.
Usually, support (no matter being one support or particulate support) can make the capture oligo combination or remain on its surface in a kind of sufficiently stable mode, to realize purpose described herein.The top combination of mentioning can comprise, for example, at support with catch and form covalent linkage between the oligonucleotide, ionic linkage, coordinate bond, hydrogen bond or Van der Waals key or attracted by positively charged or negative charge support.Catch oligonucleotide and directly or by connecting joint be connected the solid support surface.In some embodiments, catch oligonucleotide and directly be connected the support surface, the surface that provides support, or oligonucleotide, or they both, perhaps use one or more reactive groups with the surface, the oligonucleotide derivatize.For example, Luminex
TMMicroparticle surfaces can be used, for example, carboxylicesters, maleimide, functional group such as hydrazides or avidin are modified.Glass surface can be used silane, and acetaldehyde is handled (forming schiff base base DNA aldehyde-amine conjugate).In some embodiments, the material on support or the support (for example, the bag tegillum on support surface) has response function group, and it can be coupled to the response function group that catches on the oligonucleotide.For example, support can functionalised (that is, the polymer surface of the metal or the function that responds) or contain functionalized group (that is, containing the polymkeric substance of debatable functional group), so that scheme site and oligonucleotide coupling mutually to be provided.
In another embodiment, catch oligonucleotide the surface with catch oligonucleotide and be cross-linked with each other.Preferably, the crosslinked oligonucleotide of catching has comprised that a crosslink part and one catch part, wherein catches part and comprises molecular recognition sequence, and it can be hybridized mutually with the flag sequence of target oligonucleotide.
In the another embodiment, support can part or all of combined reagent place wrap quilt, such as, streptavidin, antibody, antigen, enzyme, the cofactor of enzyme or supressor, hormone, or hormone receptor.Binding reagents is biomolecules or synthetic molecules normally, and they have high avidity by covalent linkage or non covalent bond to other molecule or macromole.Catch oligonucleotide be coupled to the binding reagents complement (as, vitamin H, antigen, antibody, the cofactor of enzyme or supressor, enzyme, hormone receptor, or hormone).When catching oligonucleotide when contacting with binding reagents, catching oligonucleotide will be retained on the support.The known coupling technology of other some also can be adjusted employing easily and be implemented in native system and method.
Catch oligonucleotide and target oligonucleotide
Catch oligonucleotide and comprise molecular recognition sequence, this sequence can be caught the target oligonucleotide with complementary indicia sequence by hybridization.The molecular recognition sequence of catching oligonucleotide is hybridized mutually with the flag sequence of target oligonucleotide, causes the target oligonucleotide to be connected on the solid support.Molecular recognition sequence and flag sequence associate mutually with specific assay distinguished sequence (also being the part of target oligonucleotide), if there is hybridization to take place, it has the existence or the concentration of the assay of assay distinguished sequence (or its complementary sequence) with in the display analysis sample.
Typically, coding and flag sequence comprise at least six Nucleotide, in certain embodiments, comprise at least 8,10,15,20, or more Nucleotide.In some analytical tests, as following description, molecular recognition sequence and flag sequence comprise one or more non-standard bases.In other analytical tests, molecular recognition sequence and flag sequence do not comprise any non-standard bases.
Catch oligonucleotide and generally also comprise official's group, this group makes catches oligonucleotide and solid support surface, and the functional group of perhaps arranging on the solid support or extend is connected.This functional group can directly connect on the main polymer chain or a base of nucleotide sequence on.Optionally, as last description, catch the oligonucleotide molecule and can comprise that is used for a crosslinked cross-linking zone, it can be combined in the surface by electrostatic force.Catching oligonucleotide can produce with various technology, as, comprise solid phase synthesis, dna replication dna, reverse transcription, restriction enzyme digestion, second takes turns and transcribes etc.
Except flag sequence, the target oligonucleotide comprises an assay distinguished sequence, and it is consistent or complementary with interested sequence in the assay.The assay distinguished sequence can be and the diverse sequence of flag sequence that perhaps some or all flags sequence can be the parts of assay distinguished sequence.
Catching the length of oligonucleotide can optimize by the intensity and the kinetics of expectation hybridization.Usually, the length of molecular recognition sequence is in scope is the individual Nucleotide scope of 6 to 20 (being preferably 8 to 12).In optimum implementation, the differing molecular recognition sequence of catching oligonucleotide is not complementary each other, preferred situation be and any known natural gene sequence or testing sample in the most possible a large amount of gene fragment that occurs not complementary.The result is that the capture molecules recognition sequence of catching oligonucleotide is mainly hybridized mutually with corresponding target oligonucleotide complementary indicia sequence.
The target oligonucleotide oligonucleotide of (or be complementary at least a portion of target oligonucleotide) includes a reporter gene or is used to connect the coupling reagent of reporter gene.Reporter gene or coupling reagent can combine with main polymer chain, or combine with any base of target oligonucleotide or its complementary oligonucleotide, and the technology that reporter gene and nucleotide base (comprise natural with non-standard bases) are connected is known.The example of some reporter gene groups comprises, vitamin H, and digoxin, the spin labeling group, radio-labeled, DNA divides fragment, and chromophoric group and fluorescence chromophoric group be fluorescein for example.The example of coupling reagent comprises, vitamin H or contain the substituent of reactive functionality.The reporter gene group connects streptavidin then or comprises and the interactional reactive functionality of coupling agent, thereby reporter group is attached on target oligonucleotide or its complementary oligonucleotide molecule.
Polymerase chain reaction (PCR) technology
As following description, multiple polymerase chain reaction (PCR) technology is known and can be used for this analysis test.Usually round pcr at least a portion of oligonucleotide that is used to increase.Exist the testing sample of assay distinguished sequence to contact: oligonucleotide first and second primers with following reagent; Nucleic acid polymerase; The corresponding nucleoside triphosphate of the Nucleotide that adds in the PCR process.The nucleoside triphosphate of natural base comprises: dATP, dCTP, dGTP, dTTP, and dUTP.If necessary, the nucleoside triphosphate of non-standard bases also can add.Known suitable PCR polysaccharase, and comprise for example heat-stabilised poly synthase, for example, no matter be protoenzyme or transformed Thermus Thermus kind polysaccharase, these genus are planted and are included but are not limited to Thermus aquaticus thermus aquaticus (Taq), the Thermus flavus Huang hot bacterium (Tfl) that dwells, Thermus thermophilus thermus thermophilus (Tth), and the Klenow segment of dna polymerase i and HIV-1 polysaccharase.
First and second primers are complementary to the different positions of the different templates chain of oligonucleotide two strands to be amplified.The oligonucleotide sequence that amplifies comprise and two primer sequences of analyte hybridization and two primers between sequence.Primer can be synthetic by several different methods, comprises solid phase synthesis technique, dna replication dna, and reverse transcription, digestion with restriction enzyme, second takes turns and transcribes etc.
First oligonucleotide primer and the annealing of second oligonucleotide primer of the extension products that forms in that the circulation step that round pcr comprises has a double chain oligonucleotide that (i) will increase or the previous cycles; (ii) extend annealing back first and second oligonucleotide primers, synthetic primer extension products by nucleic acid polymerase; (iii) make the product sex change, obtain the nucleic acid of strand.By revise reactions steps and various condition (as, time and temperature), developed multiple round pcr.Though in some special detections, some round pcrs are perhaps more handy than other generally speaking, in following the description, multiple PCR method all is applicable to this analysis test.
" fast-shot PCR " described below is that some analyses that are suitable for improve one of PCR method, and the primer extension time of this method shortens or eliminates.Employed as us, term " fast-shot polymerase chain reaction " or " fast-shot PCR " refer to when PCR extends termination, have also stopped the annealing and the step of unwinding simultaneously, and the time of use is very short or say there is not PCR consuming time.Usually, use this method, 3 ' end of two primers does not separate with template nucleic acid can surpass 10 bases.
Use the specificity that fast-shot PCR circulation can intensified response, this technology is extended when stopping at PCR, has also stopped the annealing and the step of unwinding simultaneously, the time of use very short or say do not have consuming time.In some embodiments, PCR/ mixed solution Rapid Cycle at most only stops the time in an about second in each temperature, thereby makes polysaccharase have no time to extend the mispairing primer between about 90 to 100 ℃ and about 55 to 65 ℃.In some embodiments, between 95 ℃ to 58 ℃, each temperature approximately will stop a second to the temperature of reaction cycle greatly.
This Rapid Cycle is easy to generate short PCR product, usually, stays the room of about 0 to 10 base between 3 ' end of the template nucleic acid and first and second primers.Preferably, primer is designed to the Tm value at about 55 to 60 ℃.In some embodiments, 37 circulations just generally are fit to detect the target oligonucleotide molecule that size is 30 Nucleotide approximately altogether.
Allele-specific PCR primer can be used to differentiate SNP (single nucleotide polymorphism) and other allelotrope.Detect for SNP, primer should be designed to be complementary to each allelotrope, interested polymorphism base is positioned at or approaches the 3 ' end (typical, in 3 to 5 bases) of first or second primer.High-caliber allelotrope is differentiated, part because during Taq polysaccharase primer extension, the mispairing of 3 ' terminal nucleotide and target DNA and being limited to, that is, primer bonded allelotrope is not special.Other polysaccharase also can be used.
In addition, the misfit energy by other position in the displacement allele-specific primers realizes the allelotrope resolution.Selective amplification can be carried out with following two kinds of main modes in other positions of primer Nucleotide mispairing, 1) simply by reducing the Tm value (melting temperature(Tm)) of primer, primer can not be hybridized on dna profiling when thermal cycling, finally cause polysaccharase can not extend primer.2) set up a disadvantageous primer/formwork structure, polysaccharase can not be extended.
The embodiment of assay determination
Use non-standard bases analytical test coding region sequence and flag sequence
In the analysis as shown in Figure 4, two or more groups are caught oligonucleotide molecule 202 and are produced.Catch oligonucleotide molecule 202 for every group and comprise specific molecular recognition sequence 204.The sub-recognition sequence of every component contains (being typically two or more) non-standard bases (dotting in the drawings) at least.The use of non-standard bases significantly reduced catch oligonucleotide with only by the possibility of the sequence hybridization of natural based composition.Compare with the similarity analysis test of using the oligonucleotide that natural base is only arranged, generally cause non-specific hybridization to reduce far away.Catch oligonucleotide and generally also comprise a reactive functionality, in order to connect solid support 206, certainly, according to described above, the method that also has other is in order to connect.
Test employed support, can be, for example, a kind of single solid support, as, glass, metal, plastics or inorganic chip.Catching oligonucleotide is arranged on the support, and generally (for example be fixed on the support with one of above described method, catch oligonucleotide and support by the reactive group coupling, use binding reagents to handle support, the perhaps crosslinked oligonucleotide of catching).Every kind of group is arranged in the zone of the one or more uniqueness of solid support, to such an extent as to should can associate mutually with specific oligonucleotide in the zone.
(not diagram) in another embodiment, the support of analytical test is particulate support (for example a, pearl).Any analytical test as described herein can on the particulate support, or carry out on other any support on single solid support, and this can be understood.The particulate support can be divided into several groups, each the group all have be different from other characteristic of respectively organizing particulate (as, color, shape, size, density or other chemistry and physical properties).Every kind of oligonucleotide can with a group or more groups of particulate couplings.One group of specific particulate and one group of special oligonucleotide produce association, can utilize particulate support peculiar property to identify and catch oligonucleotide accordingly.
Get back to Fig. 4, target oligonucleotide 208, if it exists in testing sample, it comprises an assay distinguished sequence 210 and a flag sequence 212, this flag sequence is complementary to a kind of molecular recognition preface example 204 of catching oligonucleotide 202.Flag sequence 212 has a kind of non-standard bases at least, otherwise this flag sequence can not be complementary to the molecular recognition sequence of catching oligonucleotide.The binding reagents (not diagram) that the oligonucleotide sequence 214 that is complementary to target oligonucleotide 208 parts comprises a reporter gene 216 or is connected with reporter gene.
Get back in the analytical test, target oligonucleotide 208 is general to have the support 206 of catching oligonucleotide 202 (or fixedly the container of particulate support) to contact with association.If there be (shown in Fig. 4 D) in the suitable oligonucleotide molecule of catching on support, the control by condition promotes the flag sequence of target oligonucleotide and catches oligonucleotide mutually by the molecular recognition sequence selective cross.Also add reporter gene (remove non-complementary oligonucleotide 214 reporter gene has been arranged), be used for coupling complementary oligonucleotide 214.Alternatively, before hybridization, use following technology to remove the primer that does not mix, such as, size exclusion chromatography, relative settlement method, column spinner, Filter column or hybridization back wash-out.
For some plane supports, whether analytical test can have the existence of reporter gene to obtain detecting by reading each isolated area of support.The existence of reporter gene illustrates and contains the assay that has the assay distinguished sequence in this primary sample, and specific marker sequence and molecular recognition sequence associate mutually on this assay distinguished sequence and the support zone.Not the existing of reporter gene shows does not contain the assay that has specific assay distinguished sequence in the sample.
For the analyzing and testing of particle support, particulate can according to they characteristic and separated, determine then which kind of particle contains to have by catching oligonucleotide and target oligonucleotide to be coupled to reporter gene on the particulate.Finish isolating technology and comprise, for example, the fluidic cell photometer.The existence of reporter gene group has shown that sample contains the target oligonucleotide, and this target oligonucleotide contains the assay distinguished sequence, and this sequence is associated with the specific molecular recognition sequence of catching oligonucleotide mutually with the specific markers sequence.
The method that Fig. 4 kind is adopted can be adjusted and be used for the allelic existence of test sample.Comprise corresponding to two or more allelic allele-specific primerses (comprise first primer 2 18 or second primer 2 22, and they both) such as, this analysis.Each allele-specific primers comprise one only with the sequence of allele-specific hybridization.The flag sequence or the reporter gene (or coupling agent) that are connected in allele-specific primers also are special concerning each allelotrope.If there is allelotrope in the sample, can be extended with the associating allele-specific primers of allelotrope, and by by with complementary, allele-specific is caught oligonucleotide hybridization on the support, perhaps observes the existence of allele-specific reporter gene group and detects.Same understanding being clipped to, and whether the nonallelic assay distinguished sequence that this test also can be used for testing assay exists.
This analysis method can be used for detecting the SNP (single nucleotide polymorphism) of allelotrope.Be that first or second primer all is that SNP is specific.Usually, the SNP Auele Specific Primer that two (or a plurality of) are different is used to analytical test.Preferably, the SNP site in the SNP Auele Specific Primer is positioned at or near the elongated end of (for example three to five bases) primer." Fast-shot PCR " technology can be used for this snp analysis and detect, because the short extension time has reduced the possibility of incomplementarity primer extension basically.
The hybridization of catching between oligonucleotide and the target oligonucleotide is the feature of analytical test described herein.As many traditional hybridizing methods, this hybridization occurs in the hybridization mixture, this mixture comprise salt (as; sodium salt or magnesium salts); damping fluid (as, TRIS, TAPS; BICINE or MOPS); non-specific closed reagent (as, SDS, the genomic dna of BSA or shearing); with the protection reagent (as, EDTA or trinitride).Typically, hybridization occur in sodium ion (or other positively charged ion) concentration at least 0.01 to 1.0M and pH at 7.0 to 8.3 o'clock.Generalized case, hybridization and elution step are carried out under temperature that can satisfy the required rigorous degree of maintenance hybridization and salt concn.Rigorous condition depends on sequence.If be ready, on the basis of rigorous condition, can increase the wash-out number of times.
" the low rigorous condition " selected be meant, than specificity sequence at the pH of hybridization solution and the pyrolysis chain temperature (T under the ionic strength
m) low about 10 to 15 ℃.T
mValue is meant 50% flag sequence and the hybridization of complementary molecular recognition sequence, and the temperature (fixed ionic strength, pH and nucleic acid concentration) when being in balance.
" in rigorous condition " selected be, than specificity sequence at the pH of hybridization solution and the pyrolysis chain temperature (T under the ionic strength
m) low about 5 to 10 ℃.
" the high rigorous condition " selected is at the pH of hybridization solution and the pyrolysis chain temperature (T under the ionic strength than specificity sequence
m) low less than about 5 ℃.
Another kind of check and analysis scheme as shown in Figure 5, in Fig. 5 A, the oligonucleotide 252 of catching of two or more groups is produced, they link to each other with support 256.Each group of catching oligonucleotide 252 all comprises a specific molecular recognition sequence 254.Every group molecular recognition sequence comprises at least one (generally two or more) non-standard bases.Target oligonucleotide 258 and complementary oligonucleotide 264 can, by for example, pcr amplification contains the assay of assay distinguished sequence or its complementary sequence and forms.In pcr amplification, use two kinds of different primers (shown in Fig. 5 B, 5C).First primer 2 68 comprises the sequence that is complementary to assay 270 first chains first sequence, and second primer 2 72 comprises the sequence that is complementary to assay 270 second chains second sequence, and second chain, second sequence is positioned at the upstream or the downstream of first sequence.Typically, the assay distinguished sequence comprises the extension sequence of assay and the sequence (or its complementary sequence) that primer is hybridized.First primer 2 68 comprises that flag sequence 262, the second primer 2s 72 comprise reporter gene 266 (or coupling agent of reporter gene).
Use enzyme 280, can with complementary oligonucleotide 264 with catch oligonucleotide 252 covalency and link to each other.The enzyme that is suitable for has ligase enzyme.Selectable, shown in Fig. 5 E, from 264 sex change of complementary oligonucleotide, other composition in target oligonucleotide and the analytical test can be removed by wash-out with target oligonucleotide 258, thereby stays the complementary oligonucleotide molecule 264 that is fixed on the support 256.Detect reporter gene 266 then.
In another analytical test as shown in Figure 6, target oligonucleotide 314 forms hairpin structure or neck ring structure 321,323 (or the structure beyond the typical double-spiral structure).In this method, each of first and second primers 318,322 comprises the complementary sequence of part flag sequence 312b or part flag sequence 312a respectively.In addition, one of them primer 322 has a reporter gene 316 coupling agent of reporter gene (or be used for) to be incorporated into part flag sequence 312b.Utilize, for example round pcr with first and second primers, 318,322 amplification assays 320, prepares target oligonucleotide 314 and its complementary sequence 308.The flag sequence 312b of target oligonucleotide 314,313a is positioned at the two ends of target oligonucleotide.
Another kind of check and analysis scheme as shown in Figure 7, in Fig. 7 A, assay 420 and initial primers 440,442 contacts, each primer all has the sequence that is complementary to assay 420.One of them initial primers 440 also comprises a coupling group 444 (for example, vitamin H, or comprise the substituting group of reactive functionality), is used for substrate 450 and is connected.Shown in Fig. 7 B, initial primers 440,442 for example utilizes round pcr to extend.The initial primers 446,448 of extending comprises assay distinguished sequence or its complementary sequence respectively.
Then, the initial primers 446,448 of extension contacts with substrate 450, the coupling group of the initial primers 446 of this substrate and extension 444 interacts, and then the initial primers 446 that will extend is connected on the substrate 450, shown in Fig. 7 C.For example, substrate can be by streptavidin bag quilt, the initial primers bag bag vitamin H of extension.
Then, first and second primers 418,422 contact with the initial primers 446,448 of extending, shown in Fig. 7 C.First primer 418 has a flag sequence 412, the second primers 422 that a reporter gene 416 coupling agent of reporter gene (or be used for) is arranged.Article two, primer also all comprises and initial primers 446,448 a part of complementary sequences of extending.Detection shown in Figure 7 shows that simultaneously other primer 422a also can be added.This is not an essential characteristic that detects, but is used to describe in detail an embodiment that detects allelic analytical test.The application of allele specific primer can be used to other any detection described herein.
In the analyzing and testing that describes in detail, primer 422,422a are to have allele specific reporter gene 416, the allele specific primer of 416a.In the example that describes in detail, allelotrope is the difference of a Nucleotide, but this can be understood as these technology of application and also can detect other the allele specific that an above nucleotide difference is arranged.It is because it is a complementary with one section sequence of the initial primers 446 of extending that primer 422 is extended.Primer 422a can not extend with initial primers 446 complementations of extending because of it.Recognize that another top detection includes several different allele specific primers, they have allele specific flag sequence (relative with the allele-specific reporter gene).Recognize that also another kind of analytical test comprises the non-allelic genes primer, the existence that they are used for detecting analyzed sample non-allelic genes assay specific sequence whether.
Primer 418,422 is extended, and forms target oligonucleotide 408 that has flag sequence 412 and the complementary oligonucleotide 414 that has reporter gene 416 (or coupling agent of a reporter gene).Initial primers 446,448 sex change of target oligonucleotide 408 and complementary oligonucleotide 414 from extending, and and on solid support 406 (as chip, wafer or particle), catch oligonucleotide 402 contacts.Target oligonucleotide 414 and catch oligonucleotide 402 hybridization is caught oligonucleotide 402 and is contained and flag sequence 412 complementary molecular recognition sequences 404.Whether catch the reporter gene that oligonucleotide links to each other and exist by observing with specific, the existence of determining the analyte in sample distinguished sequence whether.
In another embodiment of this analysis test, first primer 468 contacts and extends to form target oligonucleotide 458 and its complementary oligonucleotide 464 with second primer 472 and assay 470.In the example that describes in detail, first and second primers 468,472 all are allele specific, but different allelotrope is had specificity.Remove first and second primers, outside 468,472, other first and second primers, 469,473, if in sample, there is other allelotrope that can increase.
First primer 468 comprises the 462a of first part of flag sequence, and second primer 472 comprises the second section 462b of flag sequence.One of 462a and 462b part comprise a reporter gene 466 (or coupling agent of reporter gene).Usually, the 462a of flag sequence and 462b part will have certain form and primer 468,472 is not extended by flag sequence.For example, 462a and 462b part can comprise a non-standard bases, and it connects primer 468,472 extendable portions and flag sequence part as a base.In this embodiment, the complementary base nucleoside triphosphate of non-standard bases is not included among the pcr amplification reaction.As selection, chemical joint can be used to flag sequence partly is coupled to the extendable portion of primer.The example of suitable joint include but not limited to the n-propyl, triethylene glycol, six ethylidene glycols, 1 ', 2 ' bi-deoxyribose, 2 '-O-methyl ribonucleotides, the different cytidine of deoxidation, or any chain that can stop polysaccharase.
A link coupled oligonucleotide 452 is provided on support 456.Link coupled oligonucleotide 452 comprises 453a, the 453b part, and the 462a of they and flag sequence and 462b part are complementary.These 453a, 453b partly by chemistry or joint 454 couplings of Nucleotide, 454 it can with 5 ' (or 3 ') end coupling of two nucleotide sequences.
Target oligonucleotide 458 and its complementary oligonucleotide 464 are with support 456 and catch oligonucleotide 452 reactions, the special part 462a of flag sequence, 462b with catch the corresponding section 453a of oligonucleotide, 453b hybridization.The remainder of target oligonucleotide 458 and complementary oligonucleotide 464 typically form a structure as shown in Figure 8.
The assay determination that adds non-standard bases by round pcr
Although the natural nucleotide base of mark has many purposes, relevant with the natural nucleotide of mark also have a shortcoming.For example, the fixed point that is difficult to accomplish the natural nucleotide base of a mark is mixed.Usually, for site of mark on the oligonucleotide that contains VITAMIN B4 at, the adenosine triphosphate [(dATP of mark
*)] be used as in the substrate adding reaction mixture, comprise oligonucleotide templates [dGTP] in the reaction mixture, [dCTP] and [dTTP], and polysaccharase.If dATP all in the reaction mixture are labeled, all the VITAMIN B4 residues on oligonucleotide sequence will be labeled.If dATP has only part to be labeled in the reaction mixture, so, the VITAMIN B4 residue of random site is labeled in sequence.Thereby an independent nucleotide residue is very difficult in the labeled oligonucleotide.
The bi-deoxyribose nucleic acid of serviceable indicia overcomes the relevant problem of nucleotide residue of mixing a plurality of marks.Because bi-deoxyribose nucleic acid lacks 3 ' hydroxyl, like this oligonucleotide can only partly introduce mark endways bi-deoxyribose nucleic acid.Available race ladder is surveyed oligonucleotide sequence to determine the position of labeled nucleotide.Because oligonucleotide stops in the position of introducing bi-deoxyribose nucleic acid, bi-deoxyribose nucleic acid can not be used in combination with the oligonucleotide chain amplification usually.
Fig. 9 has shown that a class of the present invention detects, and comprises by PCR non-standard bases is mixed.First and second primers 518,522 and assay 520 hybridization and extension.One of primer 522 comprises a non-standard bases 550, when extending, becomes target oligonucleotide 508.Selectable, can also the outer base of additionalamount behind the non-standard bases 550.Target oligonucleotide 508 has non-standard bases 550, its then with solid support 506a, 506b contact, solid support 506a, 506b include capture oligo 502a, 502b.The solid support that shows in Fig. 9 is a particulate support discussed above, yet, recognize also can use an independent solid support (as, chip or wafer).
After target oligonucleotide 508 hybridization, capture oligo 502a is including dATP, dUTP, dGTP, extend in the PCR reaction solution of dCTP, second non-standard bases ribonucleoside triphosphote (as, diso-GTP) 552 non-standard bases 550 that are complementary on the target oligonucleotide 508.Second non-standard bases 552 is by reporter gene 516 (or coupling agent of reporter gene) mark.Along with capture oligo is extended, be incorporated in the capture oligo of extension by second non-standard bases 552 of reporter gene 516 marks, and relative with the position of non-standard bases 550.Like this, whether reporter gene exists on specific support particle, shown with the existence of capture oligo link coupled particular target oligonucleotide whether.
Figure 10 shows that another analysis.In this analyzed, first primer 618 comprises a flag sequence 612, the second primers 622 had a non-standard bases (or a sequence that comprises non-standard bases) 621 at its 5 ' end.Primer 618,622 is at dATP, dCTP, and dGTP, dTTP existence condition be amplification assay 620 down, and the non-standard bases ribonucleoside triphosphote is complementary to non-standard bases 621.This non-standard bases ribonucleoside triphosphote is by a reporter gene 616 (or coupling group of reporter gene) mark, and is incorporated into the position corresponding to non-standard bases 621, forms target oligonucleotide 608.
Target oligonucleotide 608 contacts with solid support 606, and solid support 606 has capture oligo 602, and capture oligo 602 contains molecular recognition sequence.If flag sequence 612 complementations of molecular recognition sequence one of them and target oligonucleotide 608, target oligonucleotide 608 will with capture oligo 602 hybridization.Detection will be carried out with the method for discussing among the embodiment of front.
Shown in Figure 11ly be still another analysis.In this analyzed, first primer 718 comprises a flag sequence 712, the second primers 722 had a non-standard bases 721 to follow a natural base (or a urao basic sequence) 723 at its 5 ' end.Primer 718,722 is at dATP, dCTP, and amplification assay 720 under the situation that dGTP and dTTP exist only forms target oligonucleotide 707 that part extends and its complement 714.The extension of the target oligonucleotide that this part is extended is limit by non-standard bases 721.After the beginning is opened in amplification, clean to remove dATP, dCTP, dGTP and dTTP amplified production 707,714.
Second is extended step and then carries out at the non-standard bases triphosphoric acid that is complementary to non-standard bases 721 with under the situation that has at least the natural base triphosphoric acid that is complementary to natural base 723 to exist.This natural base triphosphoric acid is by reporter gene 716 (or coupling group of reporter gene) institute mark, and is incorporated into the position relative with natural base 723, forms target oligonucleotide 708.
Target oligonucleotide 708 contacts with solid support 706, and solid support 706 has the capture oligo 702 that has molecular recognition sequence.If one in the molecular recognition sequence is and flag sequence 712 complementary of target oligonucleotide 708 that then target oligonucleotide 708 will be hybridized with capture oligo 702.Subsequently detection is according to the carrying out of discussing among the embodiment above.
In a concrete example, allele specific second primer uses jointly with the first identical primer.Allele specific second primer is different from the part at annealed second primer from assay.Each allelotrope is selected different natural bases 723.In second step of extending, in the base that adds with respect to non-standard bases 721 and natural base 723 places, two or more urao yl nucleosides triphosphoric acids are added into and extend in the mixture.Different ribonucleoside triphosphotes is by different reporter gene marks.Therefore, if natural base 723 can be A or C, this depends on allelotrope, and [dTTP] that use in extending step used different reporter gene marks with [dGTP].The evaluation of reporter gene can be judged the allelic existence of a certain relevant.And then for example four kinds of different allelotrope can detect in this way simultaneously, under the selection of suitable reporter gene, can utilize four kinds of different colours and show.
Other analyses
In the analysis shown in Figure 16, prepare two or more sets capture oligos 902.Every group of capture oligo 902 comprises the molecular recognition sequence 904 of a uniqueness.This molecular recognition sequence of every group randomly includes at least one or a plurality of non-standard bases.Typically, capture oligo also comprises a reactive functionality that can be attached to solid support 906, and as above-mentioned, other mode of connection also can be utilized.
In an object lesson, the support of detection is particulate support (as a pearl).Be appreciated that any detection described herein can on a particle support, perhaps carry out on other any support on an independent solid support.The particle support is divided into some groups of particles, and every group of particle has a characteristic (as color, shape, size, density or other physics-chem characteristic), and this characteristic makes a distinction them separately.Every group of capture oligo and one or more groups particle coupling.This has caused the coupling of one group of specific particle and one group of specific capture oligo, allows to differentiate capture oligo by observing unique particle support characteristic.
(do not provide) in another embodiment, the support of analysis can be, for example, an independent solid support, as glass, metal, plastics, or inorganic chip.Capture oligo is arranged on the support, and fixes with a kind of (as reactive group coupling on capture oligo and support and support, with the wedding agent on the support, or capture oligo is crosslinked) in the aforesaid method.Each group is arranged in the specific region of one or more solid supports, so that can associate mutually with specific capture oligo in the zone.
Get back to Figure 16, target oligonucleotide 908 if exist in the sample that detects, contacts with second primer 911 with first primer 909.First and second primers 909,911 can be allele specific, or preferably, be not with target oligonucleotide allele-specific part complementary (as, interested allele-specific partly be positioned in the target oligonucleotide and two primer hybridizations between the zone).Second primer 911 has also comprised incomplementarity joining region 905, and this incomplementarity reporter gene joining region 905 is selectable to comprise one or more non-standard bases.Use first and second primers 909,911 and round pcr that target oligonucleotide 908 is amplified, obtain an amplified production 907, it contains reporter gene joining region 905.
Amplified production 907 then with allele-specific primers 920a, extend 920b contact back, if there is specific allelotrope, then uses the reaction conditions close with PCR and be reacted into and assign to produce allele specific extension products 922.Each allele- specific primers 920a, 920b have the allele-specific flag sequence that is complementary to different molecular recognition sequence 904 and capture oligo 902.As allele specific primer 920a, when 920b extends, be complementary to joining region 905 one or more bases mark Nucleotide 925 (or oligonucleotide) just can be produced.Nucleotide 925 that is labeled or oligonucleotide can comprise a reporter gene or coupling agent, as vitamin H, are used for adhering to of reporter gene.
After extension products 922 forms, with capture oligo 902 and reporter gene 930 phase reactions (unless reporter gene connects).Capture oligo 902 and support 906 differentiate and have which allelotrope in the sample that reporter gene provides the detectability of extension products 922.For the detection of particle support, particle can be separated according to unique property, and determining then has reporter gene to be connected on the particulate by capture oligo 902 and extension products 922 on which particulate 906.Realize that isolating technology comprises, for example, flow cytometry.The existence of reporter gene group shows and contains the allelotrope that has specific allele-specific flag sequence in the sample.
The selection of molecular recognition sequence
When a plurality of molecular recognition sequences are used, have just formed and to have detected the analytical system of a plurality of assay specific sequences by simple sample is increased.The set of differing molecular recognition sequence is necessary.Preferably, molecular recognition sequence has enough differences, to permit under the rigorous degree condition of the hope assay distinguished sequence being carried out reliable detection.Various method can be used to select the set of molecular recognition sequence.Describe some method that can use and standards below in detail.These methods and standard can be used separately also can unite use.
Following Example is the standard that can be used for creating the set of molecular recognition sequence: the base number of sequence, non-standard bases number in the sequence, the number of the natural base that links up in the sequence, the number of identical continuous base in any two sequences (not being that forward is exactly reverse), the sequence of special requirement (for example, the GC at 3 ' end or 5 ' end or two ends presss from both sides), estimate or actual melting temperature(Tm).The example of a method of measuring Tm is by descriptions such as Peyet, Biochemistry, and 38,3468-77 (1999), incorporated by reference in the lump here.Non-standard bases can be through estimating or calculate and use, for example, and the value of other bases (can be by estimating) with G/C as, iso-G/iso-C, or use as experimental data cited below.
Below be the series of steps that can be used to set up the molecular recognition sequence set:
1) with the non-standard bases of natural base and expectation (as or iso-C, or iso-G or both) set up the series of an oligonucleotide, the series of this oligonucleotide comprises having length n
1The all possible oligonucleotide of (as 8,9 or 10 Nucleotide).
2) randomly require to make oligonucleotide have a special subsequence (as, be positioned at the GC folder on 3 ' or 5 ' or two ends).
3) remove and do not bring to few n
2Individual non-standard bases (as, do not bring to few two iso-C bases) or have more than n
3Individual non-standard bases (as, have more than two iso-C bases) or comprehensively both (as, only accepting proper crossed belt has two iso-C bases) oligonucleotide.
4) randomly in one group, remove and have n
4(as, 4 or 5) oligonucleotide of individual natural base.
5) select a residue oligonucleotide, remove that other are any, identical sequence n is arranged on the oligonucleotide sequence
5The residue oligonucleotide of individual base (as 5 or 6 bases).Each oligonucleotide of not removing is repeated this operation.
6) randomly select one the residue oligonucleotide and survey it reverse complementary sequence (as, the reverse complemental of " ACT " is " AGT "), remove other any n of having then
6The oligonucleotide of the individual continuous base identical (as, 4 or 5 bases) with the reverse complementary sequence part.All carry out repetition for each oligonucleotide of not removing.
7) randomly only select to estimate or actual melting temperature(Tm) (T
m) in the preferred temperature scope, more than the preferred temperature lower limit, or the residue oligonucleotide below the preferred temperature upper limit.For example, the oligonucleotide that the melting temperature(Tm) that has is lower than room temperature (about 22 ℃) can be removed.
Embodiment
The cross hybridization of coding and flag sequence detects
The instrument that uses in this analysis comprises Luminex
100 and Luminex
Microballon, dna synthesizer (Northern Engineering company), spectrophotometer, thin-layer chromatography post (TLC) (the SI250F TLC silica-gel plate in the synthetic field of point measurement, JTBaker), be used for the qualitative control of oligonucleotide, whizzer, ultrasonoscope (Ney Dental), vortex instrument (Vortex) and various pipettor (2,20,200 and 1000 μ l)
One group of at least 100 oligonucleotide chain (molecular recognition sequence) and complementary sequence (flag sequence) thereof are designed and are synthetic.These two groups of oligonucleotides comprise non-standard Nucleotide (isoC and isoG) (EraGenBiosciences, Inc., Madison, WI) and natural nucleotide (A, G, C and T) (Perkin-Elmer/ABI), long 9 to 10bp.5 ' end of first group of oligonucleotide chain that is designed to molecular recognition sequence is with an amido modified thing (C6-TFA, Glen Reasch) mark.Another group complementary oligonucleotide is designed to flag sequence, and holds then by Cy3 (Glen Reasch) 5 ') mark.
With the reagent of molecular recognition sequence below the specific mutually coupled use of Luminex microballon: 0.1mM 2-[N-morpholine] ethyl sulfonic acid (MES) (pH4.5) (Sigma)
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloric acid (EDC) (Pierce), 0.02% (v/v) Tween (Sigma), 0.1% (W/v) SDS (Sigma)
Hybridization step comprises hybridization buffer: Sourav 0.5, comprises 10mM Tris (Sigma), 1mM EDTA (Sigma), 200mM NaCl (Aldrich), 10mM MgCl
2(Aldrich), reach 1% (W/v) PEG8000 (Sigma).
Article 98, molecular recognition sequence is diluted to 1nmol/ μ l with MES.Prepare 98 specific specificity
Microballon is used for coupling.Microballon will be through ultrasonication 20 seconds vortex vibration 10 seconds again before experiment.From 1.25 * 10
7Take out 5 * 10 in the microballon solution of pearl/ml
7Microballon, put into the Eppendorf tube of a 1.5mL.Microballon is with 10, centrifugal 1 minute of 000rcf.With the microballon decant, carefully do not stir microballon, then microballon is moved among the 50 μ L MES, carry out the vibration of ultrasonication and vortex.For molecular recognition sequence being coupled on the different microballons, add the various molecular recognition sequences of 1nmol in every kind of unique microballon, add the EDC (20mgEDC/1mL ddH2O) of the new preparation of 1.75 μ l again to mixture, carry out the vibration of ultrasonication and vortex.Then mixed solution was placed 30 minutes in room temperature, dark, vortex vibration in per 10 minutes once.After 30 minutes, add the EDC of the new preparation of 1.75 μ l again.Incubation 30 minutes, vortex vibration in per 10 minutes once.
After the coupling, add 400 μ l Tween-20 and wash microballon, the vortex vibration, 10,000rct/ minute is centrifugal, abandons supernatant.Add 400 μ l SDS again, centrifugal, abandon supernatant, add 100 μ l MES at last, then counting.
Complementary oligonucleotide (flag sequence) comes quantitatively with TLC and polyacrylamide gel and is qualitative, and is that final concentration is the working solution of 50fmol/ μ l with the MOPS dilution.
After the counting,
Microballon/molecular recognition sequence is bonded on one 98 microballon plate (98beadset) (1000 microballons/microballon district/hole) and analyzes.On the basis of 98 microballon plates, there are a kind of 50 microballon plates (2500 microballons/microballon district/hole) to come out.Table 1 has been enumerated the molecular recognition sequence of 50 microballon plates, and table 2 has been enumerated the molecular recognition sequence of 98 microballon plates.
Carrying out cross hybridization when experiment, the flag sequence (1-＞98) of getting 50fmol moves in the hole of two 96 orifice plates (the 1st and the 2nd hole in contrast).Existing
100 limitation is, the data set of layout is that 98 flags sequence and 2 (do not have flag sequence) in contrast to eliminate background.
Add to every hole and to become owner of mixed solution (98 kinds of mixing), 10 μ l/ holes add 31 μ L, 2 * Sourav, 0.5 hybridization buffer to every hole again, and complement to 62 μ l/ hole cumulative volumes with distilled water.Mix reagent and be incubated about 10 minutes in room temperature.Sample immediately
Analyze on 100 the flow cytometer.
50 microballon master mixed solutions also apply its corresponding complementary molecule recognition sequence and flag sequence, and just the consumption of flag sequence is the 500fmol/ hole.
Below data report the fluorescent media intensity of every microballon on two blocks of microballon plates (MedianFluorescence Intensity, MFI).Figure 12 has shown the 3D exterior view of drawing with 98 microballon master combined experiments gained data.Y-axis is represented molecular recognition sequence, X-axis expressive notation sequence.Figure 13 has shown the 3D exterior view that 50 microballon master combined experiments gained data are drawn.
Table 1
The molecular recognition sequence of 50 pearls (Y=iso, X=iso-G)
Table 2
The molecular recognition sequence of 98 pearls (Y=iso-C, X=iso-G)
The effect of preliminary definite non-standard bases in prediction nucleic acid duplex stability parameter
The beckman DU-7500 spectrometer that has temperature control system and specimen holder is used in this experiment.Accurate temperature controlling system can be measured the temperature of six samples simultaneously.Quartz curette is selected U.S. Hellma product for use, and in order to cover 100 times scope of sample concentration, its path length is respectively 0.1cm, 0.2cm, 0.5cm, and 1.0cm.DNA is synthetic on 392 type dna synthesizers of Perkin-Elmer/ABI company.TLC chromatography liquid holding groove (Fisher), and the TLC flat board (Si250F, JTBaker).Use Sanvant SpeedVac to prepare DNA, also used Sep-pakC-18 purification column (Waters), ultraviolet lamp, vortex vibrator, 100cc syringe, and micro-liquid getting device some (2,20,200,1000 μ l).
Oligonucleotide synthetic used natural nucleotide (A, G, C and T) (Perkin-Elmer/ABI) and isoC and isoG (EraGen Biosciences, Inc., Madison, WI).Table 3 and table 4 have been enumerated self complementary sequence and non-self complementary sequence of synthetic.
Table 3 self complementary sequence (isoG=X, isoC=Y)
| 3A | GGA CGT CC | Contrast |
| 3B | GGA YXT CC | Placed in-line isoC-isoG effect |
| 3C | GXA YXT YC | IsoC-isoG is in penultimate |
| 3D | GGA GCT CC | Contrast |
| 3E | GGA XYT CC | Exchange placed in-line isoC-isoG effect |
Non-self the complementary sequence of table 4 (isoG=X, isoC=Y)
| 4A | SEQ ID NO：99 | 5′GCC AGT TTA A3′ 3′CGGTCAAATT5′ | Contrast |
| 4B | SEQ ID NO：100 | 5′GCC AXT TTA A3′ 3′CGG TYA AAT T5′ | Single isoC-isoG is positioned at AT, between TA |
| 4C | SEQ ID NO：101 | 5′GCX AGT TTA A3′ 3′CGY TCA AAT T5′ | Single isoC-isoG is mixed in GC, between AT |
| 4D | SEQ ID NO：102 | 5′GYC AGT TTA A3′ 3′CXG TCA AATT5′ | Single isoC-isoG is mixed in GC, between CG |
| 4E | SEQ ID NO：103 | 5′GYY AGT TTA A3′ 3′CXX TCA AAT T5′ | The last series connection of isoC-isoG substitutes |
Use following reagent in the oligonucleotide purifying and the experiment of unwinding: with n-propyl alcohol/ammonia/water (volume ratio 55: 35: 10) (following document Chou of reference, S.-H., Flynn, P., and Reid, B. (1989) " biological chemistry " (Biochemistry) 28,2422-2435,) to TLC wash-out 5-6 hour, with purifying TLC.Hybrid experiment carries out (1.0M NaCl (Fisher), 10mM sodium dimethylarsonate (Fisher), 0.5mM Na in 1 * SL degassing damping fluid
2EDTA, pH7) (reference SantaLucia, J., Allawi, H, and Seneviratne, P.A., (1996) " biological chemistry " (Biochemistry) 35,3555-3562, incorporated by reference here).
Use Meltwin
TMV3.0, the data of thermokinetic parameters by melting curve determine, and as Petersheim, M., and Turner, D.H. (1983) " biological chemistry " (Biochemistry) 22, and 253-263 is described, and is incorporated by reference here.
After synthetic, oligonucleotide chain places ammoniacal liquor, and 50 ℃ are spent the night, again through freeze-drying, each sample is dissolved with 175 μ l distilled waters, and through the TLC purifying, wash-out 5-6 hour.Wash-out Shaoxing opera is strong, and moving belt can be few more, is not easy to do to spend flat board more.Wash-out will carry out three times with the 3ml distilled water.Use Sep-pak
TMPost carries out further desalination and purifying to oligonucleotide, uses 30% acetonitrile, 10mM bicarbonate of ammonia, pH7 (SantaLucia, J., Allawi, H., and Seneviratne, P.A., (1996) " biological chemistry " (Biochemistry) 35 3555-3562) carried out wash-out, uses SpeedVac at last
TMDry.
Self complementary oligonucleotide is collected and OD by quantitatively
260Be 2.0, use SpeedVac then
TMDry again.Oligonucleotide carries out gradient dilution to form 100 times dilution intervals with 1 * SL damping fluid.By the distribution plan of Beckman DU-7500 spectrophotometer measurement photoabsorption and temperature, also need multiple routine little cuvette, specimen holder and temperature control unit in the process.Table 5 and table 6 have been enumerated one group of diluted sample gradient, and its dilution gradient is according to each specimen preparation of table 3 and table 4.
Table 5: gradient A
| Sample | Volume (μ L) | Add-on (μ L) | Put into the amount (μ L) of cuvette |
| A1 | 0.0 | 94.5 | 34.5 |
| A2 | 57.5 | 40.2 | 34.5 |
| A3 | 63.2 | 44.3 | 34.5 |
| A4 | 73.0 | 51.2 | 69.0 |
| A5 | 55.2 | 38.5 | 69.0 |
After measuring the A1-A5 sample, second group of diluting soln is set up.In the B group, remaining 24.71 μ l are added be in the same place (about altogether 172.5 μ l) with dilution in A-4 and the A-5 cuvette and merge by at A-3 in last once sample, add 345 μ l lx SL damping fluids again.
Table 6: gradient B
| Sample | Volume (μ L) | Add-on (μ L) | Put into the amount (μ L) of cuvette |
| B1 | 542.2 | 0.0 | 172.5 |
| B2 | 369.8 | 230.0 | 172.5 |
| B3 | 427.2 | 270.0 | 345.0 |
| B4 | 352.5 | 224.0 | 345.0 |
| B5 | 231.5 | 132.2 | 345.0 |
Pack into behind the liquid, about 4% the heat radiation of space to guarantee to separate sample in the chain process will be reserved in each cuvette (cuvette) volume top.
In each operation, sample all will further outgas, and improves temperature to 85 ℃ annealing 5 minutes then, then cools to 10 ℃ of insulations more than 5 minutes.Under cold condition, it is concentrated to limit as blank to get dry argon.A, B measure OD for two groups simultaneously
260And OD
280When temperature when 10 ℃ rise to 90 ℃, sample heats up with the constant speed of 1.0 ℃/min.
Collection unwind the experiment data and use Meltwin
TMSoftware is In (G) and Tm
-1The optimum curve analysis, wherein G is the concentration of total chain, Tm
-1Be melting temperature(Tm) inverse (Borer, P.N., Dengler, B., Tinoco, I .Jr., and Uhlenbeck, O.C. (1974) " molecular biology journal " (J.Mol.Biol) 86,843-853, be incorporated herein as with reference to).
Non-self complementary oligonucleotide merges with identical molar weight, OD
260Be the optical density(OD) under the 2.0260nm, and with table 5 and table 6 in the identical mode of self complementary oligonucleotide dilution gradient dilute.Same, non-self mutually by the data of unwinding of oligonucleotide by Meltwin
TMCollect and analyze.
Table 7 and table 8 have been summed up by Meltwin
TMThe thermokinetic parameters of self complementary oligonucleotide that obtains and non-self complementary oligonucleotide.
The thermokinetic parameters of table 7 self complementary oligonucleotide sequence (isoC=Y, isoG=X)
| -ΔG 37 (kcal/mol) | -ΔH (kcal/mol) | -ΔS(cal/K·mol) | T M(℃) 1.0e-4M | ||
| 1A | GGA CGT CC | 8.27 | 53.5 | 145.9 | 52.8 |
| 1B | GGA YXT CC | 9.41 | 57.62 | 155.4 | 58.5 |
| 1C | GXA CGT YC | 10.89 | 662.7 | 178.6 | 63.5 |
| 1D | GGA GCT CC | 8.10 | 51.04 | 138.5 | 52.4 |
| 1E | GGA XYT CC | 9.70 | 57.77 | 155.0 | 60.2 |
The thermokinetic parameters of non-self the complementary oligonucleotide sequence of table 8 (isoC=Y, isoG=X)
| -ΔG 37 (kcal/mol) | -ΔH (kcal/mol) | -ΔS (cal/K·mol) | T M(℃) 1.0e-4M | |||
| 4A | SEQ ID NO：99 | 5′GCC AGT TTA A3′ 3′CGG TCA AAT T5′ | 8.43 | 69.22 | 196.0 | 45.8 |
| 4B | SEQID NO：100 | 5′GCC AXT TTA A3′ 3′CGG TYA AAT T5′ | 9.56 | 56.66 | 151.9 | 54.5 |
| 4C | SEQ ID NO：101 | 5′GCY AGT TTA A3′ 3′CGX TCA AAT T5′ | 9.36 | 62.98 | 172.9 | 51.6 |
| 4D | SEQID NO：102 | 5′GYC AGT TTA A3′ 3′CXG TCA AAT T5′ | 9.62 | 54.30 | 144.1 | 55.7 |
| 4E | SEQ ID NO：103 | 5′GYY AGT TTA A3′ 3′CXX TCA AAT T5′ | 10.59 | 70.19 | 192.2 | 56.0 |
All samples all have concentration dependent T
MAnd the single-phase conversion of unwinding takes place.IsoC and isoG make that the duplex form is more stable, and each isoC/isoG is to making Watson-Crick oligonucleotide high 5 ℃ sample 3Bs and 4C to 10 ℃ (sample 3C and the 4E) of melting temperature(Tm) than natural (A, T, G, C).
Table 7 and 8 has shown the degree of some approach effects that taken place when the AEGIS base is sneaked into n DNA.
The site is controlled mixes
First primer, 5 ' AGAACCCTTTCCTCTTCC (SEQ ID NO:104) target sequence 5 ' AAGAACCCTTTCCTCTTCCGATGCAGGATACTTAACAATAAATATTT (SEQ ID NO:105)
The second primer CTACGTCCTATGAATTGTTATTTATAAAYAGGACAGACG5 ' (SEQ ID NO:106)
Y＝isoCTP
First primer and target sequence thereof and second primer are respectively SEQ ID NO:104, shown in SEQ ID NO:105 and the SEQ ID NO:106.
Carry out the PCR experiment with following mixed solution: add 0.2 μ M, first primer, 0.2 μ/M second primer, 50fM target sequence in the 20 μ l reaction volumes, dGTP, dATP, dTTP and dCTP, each 50 μ M, 10mM TrispH8,0.1%BSA, 0.1%Triton X-100, the salmon sperm dna of 0.1 μ g/ μ l degraded, 40mMKAc, 2mM MgCl
2, 1U Amplitaq Stoffel (Perkin Elmer Biosciences, Foster City, CA).Mixed solution is 95 ℃ of insulations earlier 2 minutes, circulates in 95 ℃ of 1 second and 58 ℃ 10 seconds, 30 PCR is set circulates.Finally 58 ℃ of insulations 2 minutes.
Prepare two kinds of PCR reaction mixtures.Every kind of PCR mixed solution all will be used Autoseq
TM(Amersham Pharmacia Biotech Inc., Piscataway NJ) carry out desalination to remove unconjugated dNTPs to microspin column, use post damping fluid and ddH before with the sample desalination
2The O exchange.Sample after the desalination is adjusted to final concentration with following reactive component: add 10mM Tris pH8 in 25 μ l reaction volumes, 0.1%BSA, 0.1%TritonX100, the salmon sperm dna of 0.1 μ g/ μ l degraded, 40mM KAc, 2mM MgCl
2, 1U/ reaction AmplitaqStoffel (Perkin Elmer Biosciences, Foster City CA) and 10 μ M Cy3-dTTP (NEN lifescience Products, Inc., Boston, MA).In addition, disoGTP adds by following concentration: 0 μ M (reference examples) or 40 μ M (embodiment 3).Reaction mixture is 68 ℃ of insulations 15 minutes, gets 5 μ l products with the detection of 10% denaturing polyacrylamide gel electrophoresis.(Molecu lar Dtbanucs, Sunnyvale CA) detect the extension products that contains Cy3 to gel with 595 fluorescence imaging instrument.
The result shows (not providing data), and when not having disoGTP, first primer can not cause extra extension (that is, with respect to the iso-C of primer 2, do not have or base mispairing seldom arranged) in final PCR reaction.
Synthesizing of 5 ' of mark-fast morpholine nucleoside acid of triphosphoric acid deoxidation iso bird
In the following chemical reaction, three fourth ammonium tetra-sodiums are bought from Sigma company, and vitamin H N-maloyl imines ester is bought from Pierce chemical company; All other pharmaceutical chemicalss are all available from Aldrich chemical company or Fisher chemical company, and the further purifying of process does not just use.Solvent is logical
Molecular sieve carries out drying.Be reflected in the dried glassware of baking of baking oven, in the exsiccant argon, carry out.Carry out column chromatography (230-425 order) with silica gel.
Abbreviation:
Ac
2The O acetic anhydride
DMFN, dinethylformamide
DMAP4, the 4-Dimethylamino pyridine
DMT4, the 4-dimethoxytrityl
Et
3The N triethylamine
The MeCN acetonitrile
MeOH methyl alcohol
Tol is right-toluyl
1-(p, p '-dimethoxytrityl)-hexanediamine (2)
Hexanediamine (10 equivalents, 375mmol, 43.5g) from pyridine by condistillation twice, and be dissolved in 100 milliliters the pyridine.Add DMAP (0.1 equivalent, 3.75mmol, 457mg), with the reaction flask ice bath.Be dissolved in 4 in 100 milliliters of pyridines, (1 equivalent, 37.5mmol 12.69g) are dropwise added 4-dimethoxytrityl-chlorine, and be consuming time more than 2 hours.Room temperature was stirred 4 hours, added MeOH (5ml), and reaction mixture concentrates, remaining resistates sodium bicarbonate aqueous solution/ethyl acetate extraction.Organic layer is washed 2 times with sodium bicarbonate aqueous solution, drying, and solvent is evaporated.The product that obtains is not further purified, and uses in next step.
Output: 14.895g (35.634mmol, 95%) thickness oily matter.
2-chloro-6-(6-p, the amino hexyl of p '-dimethoxytrityl)-aminopurine-2 '-deoxidation-3 ', 5 '-dimethylbenzoyl nucleosides (3)
(1.3 equivalents, 31.916mmol is 13.34g) with DMF coevaporation and be dissolved in 100ml DMF for compound 2.Add the diisopropylethylamine be dissolved in 100ml DMF (3.9 equivalents, 95.748mmol, 16.65ml) and compound 1 (1 equivalent, 24.551mmol, 13.282g), stirring at room 3 hours.Concentrate residuum sodium bicarbonate aqueous solution/ethyl acetate extraction, organic layer drying, evaporating solvent afterwards.Resistates grinds twice with ether, and the solids that obtains further uses after the vacuum-drying without being further purified.
2-benzyloxy-6-(6-p, p '-dimethoxy triphenyl amine hexyl)-aminopurine-2 '-dezyribonucleoside (4)
(1 equivalent, 19.23mmol 17.74g) are dissolved in DMF (25ml) and add to a kind of benzyl alcohol solution (128ml) of NaH (10 equivalents, 192.3mmol, 7.69g, 60% dispersion thing in mineral oil) to compound 3.120 ℃ of reaction mixtures heating 6 hours, then before filtering the C ore deposit in stirring at room 15 hours, evaporation leaches thing, residuum is with ethyl acetate/water extraction, organic layer NaHCO
3Solution cleans, drying, and evaporating solvent, residuum grinds 5 times with ether/normal hexane (1: 10).
TLC：CHCl
3/10％MeOHR
F＝0.26
Output: 10.280g (13.562mmol, 70.5% was used for for 2 steps) foam.
2-benzyloxy-6-(6-p, the amino hexyl of p '-dimethoxytrityl)-aminopurine-2 '-deoxidation-5 '-O-p, p '-dimethoxytrityl nucleosides (5)
Compound 4 (14.7388mmol 11.172g) uses the pyridine coevaporation, is dissolved in the 150ml pyridine, add DMAP (0.25 equivalent, 3.6847mmol, 450mg).Flask is placed on the ice bath, in 2 hours, slowly add DMTCl (1.5 equivalents, 22.108mmol, 7.484g).Stirring at room 22 hours adds MeOH (1ml), concentrated reaction mixture, residuum chloroform/NaHCO
3Aqueous solution extraction.Organic layer carries out drying, evaporating solvent, and residuum grinds to remove unnecessary DMT with ether/normal hexane at 1: 1, and insoluble decorating film carries out drying, and purifying can further not use in addition.
Output: 14.890g (14.047mmol, 95%) light brown foam.
2-benzyloxy-6-(6-p, the amino hexyl of p '-dimethoxytrityl)-aminopurine-3 '-O-ethanoyl-2 '-deoxidation-5 '-O-p, p '-dimethoxytrityl nucleosides (6)
Compound 5 (14.047mmol 14.89g) uses the pyridine coevaporation, is dissolved in the 200ml pyridine, add DMAP (0.25 equivalent, 3.5117mmol, 428mg), Et
3N (5 equivalents, 70.235mmol, 9.7ml) and Ac
2O (2.5 equivalents, 35.1175mmol, 3.582g).Stirring at room 4.5 hours adds MeOH (2ml), concentrated reaction mixture, residuum ethyl acetate/NaHCO
3Aqueous solution extraction.Organic layer carries out drying, and evaporating solvent, residuum chromatography column purifying adopts a step gradient, first ethyl acetate/normal hexane/Et
3N 30: 60: 1 is 65: 35: 3 then.Output: 5.93g (5.385mmol, 38%), yellow foam.
2-benzyloxy-6-(the amino hexyl of 6-)-aminopurine-3 '-O-acetyl-2 '-deoxynucleoside-(7)
(2.471mmol 2.723g) is dissolved in 50ml acetonitrile/2ml water to compound 6, adds Ce (NH
4)
2(NO
3)
3(0.3 equivalent, 0.74mmol, 406mg).Reflux 45 minutes, and then add 0.15 equivalent Ce (NH
4)
2(NO
3)
3(0.37mmol 205mg), continued reflux 1 hour.Evaporation then, residuum are ground with ether and are removed DMT, and insolubles carries out drying, and purifying is not standby in addition.
2-benzyloxy-6-(6-trifluoroacetamido hexyl)-aminopurine-3 '-O-ethanoyl-2 '-deoxynucleoside-(8)
Top gained compound 7 (5.385mmol at most) is dissolved in 30ml MeOH/50ml ethyltrifluoroacetate/5ml Et
3Among the N, reaction mixture was in stirring at room 21.5 hours.TLC (chloroform/17.5%MeOH): R
F=0.72) shows conversion fully.Evaporate then, residuum salt solution/ethyl acetate extraction, the organic layer drying, evaporating solvent, residuum adopt a step gradient by the silica gel column chromatography column purification, and chloroform/1.5%MeOH was 17.5%MeOH then before this.Output: 2.80g (4.714mmol, 87%) foam.
2-benzyloxy-6-(6-trifluoroacetamido hexyl)-aminopurine-3 '-O-ethanoyl-5 '-three phosphoryl-2 '-deoxynucleoside-(9).
Imidazoles (61 equivalents, 306mg, 4.5mmol, recrystallization) is dissolved in acetonitrile (3.6ml) and is cooled to 0 ℃.Add POCl
3(19 equivalents, 0.128ml) and triethylamine (61 equivalents 0.633ml), stirred 0.5 hour in 0 ℃, add then a part (0.309ml) give compound 8 (1 equivalent, 0.074mmol, 44mg).Stirred 0.5 hour under this mixture room temperature, add then and contain tributyl ammonium pyrophosphate salt (2 equivalents, 0.16mmol, DMF 73mg) (1.5ml).Use 2ml 10%NH after 24 hours
4COO termination reaction and lyophilized.Product uses 20%MeCN and (a NH by anion-exchange chromatography (Dionex ProPac SAX-10)
4)
2CO
3/ 20%MeCN gradient and purifying.The product of collecting repeatedly lyophilized to remove unnecessary salinity.Output: 0.007mmol (10%), white solid.
6-(the amino hexyl of 6-)-aminopurine-5 '-three phosphoryls-2 '-deoxynucleoside (10).
Compound 9 (0.007mmol) be dissolved in methyl alcohol (2.5ml) add then Pd/C (10%, 5mg) and NH
4COO (0.05mmol, 31mg).Suspension returning heating 1 hour filters catalyzer and evaporating solvent then.Residuum is handled with 28% ammonium hydroxide (1.5ml, 3 hours, room temperature), dry reagent contained within then, and product uses 20%MeCN and (a NH by anion-exchange chromatography (Dionex ProPac SAX-10)
4)
2CO
3/ 20%MeCN gradient purifying.The product of collecting repeatedly lyophilized to remove unnecessary salinity.Output: 0.0063mmol (90%), white solid.
6-(6-biotinyl amido hexyl)-aminopurine-5 '-three phosphoryls-2 '-deoxynucleoside (11).
(10.5 μ l, 1M pH8.5) add the compound 10 (0.88 μ mol, triethylammonium salts) that is dissolved in 40 μ l water to Sodium Tetraborate, add the DMF (216 μ l) that contains vitamin H N-maloyl imines ester (2.6 μ mol, 3 equivalents) then.React 55 ℃ and carried out 3 hours, with the 20%MeCN dilution, product makes water and a NH then
4HCO
3Solution gradient is by anion-exchange chromatography (Dionex ProPac SAX-10) purifying.Productive rate about 70%.
Mix the mark base and on solid phase is supported microballoon, be captured in application in the genomic dna diversification genotype detection
Genotype on 9 polymorphism seats is determined by following steps: amplification, and inquire about and catch from the target nucleic acid sequence in the genome DNA sample.The first step is a multi-PRC reaction, comprises that one group of diversified PCR primer is right.Every pair of PCR primer comprises one first primer A and one second primer B, they be designed to mouse gene group DNA in comprise the area hybridization of a known pleomorphism site, and this section zone of increasing.Second step was that a multiple allele specific primer extends (ASPE) reaction, comprised and organized the allele specific primer of mark more.The allele specific primer of each mark comprises a 5 ' flag sequence that contains non-standard Nucleotide (iso-G), the back is a C3 transcribed spacer (a n-propylene glycol), the back is one section 3 ' sequence again, and 3 ' sequence is designed to and hybridizes from the wherein chain in the DNA chain of front multiplex PCR step amplification.3 ' Nucleotide by every marker allele special primer is determined allele-specific.Many group echos allele specific primer is designed, to be used for inquiring about the known pleomorphism site that is included in the multiplex PCR extension increasing sequence.The triphosphoric acid of mark (dATP-vitamin H) is added in the ASPE reactant, thereby the extension of the allele-specific of marker allele Auele Specific Primer has caused mixing of dATP-vitamin H.Uncorporated dATP-vitamin H subsequently catch step before be removed.
In the 3rd step, catching of promptly multiple ASPE reaction product used to include and specific Luminex
TMThe acquisition sequence of the covalently bound non-standard Nucleotide of microballon (iso-C).Flag sequence complementation on the marker allele Auele Specific Primer that acquisition sequence and front ASPE reaction are used.Add phycoerythrobilin (Phycoerythrin) and be on the extension of marker allele special primer chain in conjunction with biotin labeling, thereby a kind of fluorescent signal is provided.After acquisition sequence and flag sequence hybridization, microballon is injected into Luminex 100
TMDevice is to detect and every group of relevant signal of specific microballon identity.
Genomic 9 polymorphic regions of mouse are used as target in the present embodiment:
| The target thing | SEQ ID NO： | Sequence | A/J | C57BL6/ |
|
| 2 | 104 | AGAAACAACCATCTAATCCCACACTAAAAT TCAAGGCTCCACAGACGAAACAGTGAAGAA TAATTGTTCAGCATACTAACCAACTGATTA CATATTTACCATACTCAGGTTTGTGCTTCA TACAAACCCAC/TAGTCCGGCGCTCCCTGTTA | CC | TT | |
| 3 | 105 | CTTCTCCCATTGCCCAGGGCACTCTCCTCT GTAGAA/GTAGACTGATC/TTTTGTGGAGACATC | GG | AA | |
| 4 | 106 | AGTGCCTGCTACCTGTCAGGTGAAAATTTC TTAGTGATCCC/TAAGCTCAATGGGTGCYGGC | CC | TT | |
| 5 | 107 | GGTTGGAATGTTTGCACATGCAGTGTTAGT TATTTGGGC/TGATAACTACTTAGCTTATCTA | TT | CC | |
| 6 | 108 | CTGATCTGACCTCAGACTGTTGTGCTAACA GATATAACACCAGTAAGTTGAC/GTCAAATAC | GG | CC | |
| 7 | 109 | GACTGCTGGAGAGCTGAGGGAGGCTGTGGA GAATAAGGAGAGAGCA/GTAGTCTCGTGCCCT | GG | AA | |
| 8 | 110 | GGACTGTCCAAAKGGATCTCAAGGAGAATA GT CCTTGCTATTAA/GGAGTATAAAGGCATAA AAGAGGTCATAGGGGACAACCATGACCAAG | AA | GG | |
| 9 | 111 | CCTTCCTGCAYTCCACAGTATAAACACAGA ATGCACACTGCA/GGTCGTTGTATTTGTGTTC GATGTGAATTAAAGATGCTTTGGCTAAGCC | AA | GG | |
| 10 | 112 | CACATACACCATGTCAGCCATCAGCGCAAA GCCTTCGAGTTTCAGCTGTGAGATGAAGGC TTGGAGAAGCACGTTGATCTGCAAAGAAGC AAAGGAGCTAGCGGAGGCC/TGGTCACTGACC GACTGCTCA | CC | TT |
In this example, following nucleic acid is used for the multiplex PCR step:
| Nucleic acid component | Sequence | SEQ IDNO |
| |
5′-CATCTAACAGGGAGCGCC-3′ | 113 |
| |
5′-6FAM-AGAAACAACCATCTAATCCCACA-3′ | 114 |
| PCR primer 2 A | 5′-6FAM-CTTCTCCCATTGCCCAGG-3′ | 115 |
| PCR primer 2 |
5′-TGATGTCTCCACAAAGATCAGTC-3′ | 116 |
| |
5′-AGTGCCTGCTACCTGTCAG-3′ | 117 |
| |
5′-6FAM-CCTGCAAGCCAGCACC-3′ | 118 |
| |
5′-6FAM-GGTTGGAATGTTTGCACATGC-3′ | 119 |
| |
5′-GCTGGACCAGGCTAGATAAGC-3′ | 120 |
| |
5′-6FAM-CTGATCTGACCTCAGACTGTTG-3′ | 121 |
| |
5′-GCAAGGCTCTACTTCCTGC-3′ | 122 |
| |
5′-6FAM-GACTGCTGGAGAGCTGAGG-3′ | 123 |
| |
5′-GTGTCTTGGCTGCTCAGTATG-3′ | 124 |
| |
5′-6FAM-GGACTGTCCAAAGGGATCTC-3′ | 125 |
| |
5′-CAACTTCTTGGTCATGGTTGTC-3′ | 126 |
| |
5′-Cy3-CCTTCCTGCAYTCCACAG-5′ | 127 |
| |
5′-6FAM-CAGTATTATCATCTCCTGGCTTAGC-3′ | 128 |
| |
5′-6FAM-CACATACACCATGTCAGCC-3′ | 129 |
| |
5′-TGAGCAGTCGGTCAGTG-3′ | 130 |
| |
Mouse gene group DNA; Strain: A/ | |
| Template | ||
| 2 | Mouse gene group DNA; Strain: C57BL6/J |
The PCR primer is synthesized and is diluted in 1mM MOPS pH7.5, in the 0.1mMEDTA solution.Some PCR primer adds 6F AM or Cy3 fluorescent mark, makes the detection of the multi-PRC reaction on polyacrylamide gel have possibility.
The mouse gene group DNA sample available from the Jackson laboratory (Bar Harbor, ME).All genome DNA samples are all used 1mM MOPS pH7.5, and 0.1mM EDTA solution dilution is 5ng/ μ l.The PCR reacted constituent is as follows:
| |
1 * concentration | The supplier and the place of production |
| 10XPCR Buffer II | 1.2X | Applied Biosystems，Foster City，CA |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| dATP | 200μM | Amersham |
| dGTP | 200μM | Amersham |
| dCTP | 200μM | Amersham |
| dTTP | 200μM | Amersham |
| Amplitaq TMGold DNA polysaccharase | 0.1U/μl | Applied Biosystems，Foster City，CA |
| PCR primer (each) | 0.1μM |
All female mixed solutions of listing component are prepared as 1.09 * concentration of 25 μ l end reaction volumes.Female mixed solution of 23 μ l and 2 μ l genomic dna templates (5ng/ μ l) are mixed in the one PCR pipe.Negative contrast water replaces the genomic dna template.The PCR reaction cycle is as follows:
After PCR is circulated throughout, respectively gets 2 μ l PCR reactants and react to multiple ASPE as template transfer.Following synthetic nucleic acid is used as marker allele special (TAS) primer in the multiple ASPE reaction:
| Nucleic acid component | Sequence | SEQ ID NO |
| TAS primer 1 | 5’-GTGYACAYGC-c3-GCTTCATACAAACCCAC-3’ | 131 |
| The TAS primer 2 | 5’-CGAYTCTGYC-c3-GCTTCATACAAACCCAT-3’ | 132 |
| TAS primer 3 | 5’-CTAYCAAYCC-c3-CACTCTCCTCTGTAGAA-3’ | 133 |
| TAS primer 4 | 5’-GAGAYCYAAG-c3-CACTCTCCTCTGTAGAG-3’ | 134 |
| TAS primer 5 | 5’-GTTCYTGAYG-c3-GAAAATTTCTTAGTGATCCT-3’ | 135 |
| TAS primer 6 | 5’-GCYTAYCTAC-c3-AAAATTTCTTAGTGATCCC-3’ | 136 |
| TAS primer 7 | 5’-GTTAYCYTCC-c3-AGTGTTAGTTATTTGGGT-3’ | 137 |
| TAS primer 8 | 5’-CACYATACYG-c3-GTGTTAGTTATTTGGGC-3’ | 138 |
| TAS primer 9 | 5’-CYTACCYATG-c3-TAACACCAGTAAGTTGAC-3’ | 139 |
| TAS primer 10 | 5’-GYCGAYAATC-c3-TAACACCAGTAAGTTGAG-3’ | 140 |
| TAS primer 11 | 5’-GYCGTAYTTG-c3-AGAATAAGGAGAGAGCA--3’ | 141 |
| TAS primer 12 | 5’-GTYTATYCCG-c3-GAATAAGGAGAGAGCG-3’ | 142 |
| TAS primer 13 | 5’-GACAYACYTC-C3-AGAATAGTCCTTGCTATTAA-3’ | 143 |
| TAS primer 14 | 5’-GGAAYAACYG-C3-AGAATAGTCCTTGCTATTAG-3’ | 144 |
| TAS primer 15 | 5’-GATYTYCAGC-c3-AGAATGCACACTGCA-3’ | 145 |
| TAS primer 16 | 5’-GTYATYTGCG-c3-GAATGCACACTGCG-3’ | 146 |
| TAS primer 17 | 5’-GATYGTCYYG-c3-GCTAGCGGAGGCC-3’ | 147 |
| TAS primer 18 | 5’-GGYCTYATGG-c3-GCTAGCGGAGGCT-3’ | 148 |
The composition of ASPE reaction is:
| |
1 * concentration | The supplier and the place of production |
| Bis-Tris-propane pH 8.9 | 10mM | Sigma，St.Louis，MO |
| Potassium acetate | 40mM | Sigma，St.Louis，MO |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| Vitamin H-11-dATP | 4μM | NEN，Boston，MA |
| dGTP | 200μM | Amersham Pharmacia，Piscataway，NJ |
| dCTP | 200μM | Amersham Pharmacia，Piscataway，NJ |
| dTTP | 200μM | Amersham Pharmacia，Piscataway，NJ |
| Amplitaq TMThe Gold archaeal dna polymerase | 0.067U/μl | Applied Biosystems，Foster City，CA |
| PCR primer (each) | 0.067μM | EraGen Biosciences，Inc.，Madison，WI |
A kind of comprise above except that the TAS primer female mixed solution of all components be made into 1.36X.Each ASPE reaction comprises the female mixed solution of 11 μ l, the multiple TAS primer mixture of 2 μ l (each 0.5 μ M), 2 μ l PCR reactants (from preceding step).The ASPE reaction cycle is as follows:
After the ASPE circulation, 10 μ l reactant volumes and 5 μ l comprise 40mM Tris, and the solution of 40mM EDTA mixes to stop the activity of polysaccharase.Reactant is removed unconjugated dATP-vitamin H with the G-50 column purification.The multiple ASPE reaction solution of purifying is by coupling Luminex then
TMThe acquisition sequence of microballon (Luminex company, Houston, Texas) untwists.The link coupled microballon is:
| The microballon identity | SEQ ID NO： | |
| 1 | 2 | |
| 2 | 9 | |
| 7 | 14 | CGGXATAXAC |
| 15 | 13 | CAAXTACGXC |
| 17 | 22 | GCTGXAXATC |
| 18 | 23 | CGCAXATXAC |
| 19 | 1 | GAXGTXTGTC |
| 20 | 3 | GGXTTGXTAG |
| 21 | 4 | CTTXGXTCTC |
| 22 | 5 | CXTCAXGAAC |
| 34 | 7 | GGAXGXTAAC |
| 35 | 8 | CXGTATXGTG |
| 37 | 6 | GTAGXTAXGC |
| 38 | 10 | GATTXTCGXC |
| 45 | 28 | CXXGACXATC |
| 47 | 29 | CCATXAGXCC |
| 61 | 36 | GCXTGTXCAC |
| 62 | 37 | GXCAGAXTCG |
The link coupled microballon mixes with mixture, this mixture the storage damping fluid (10mM MOPSpH7.5,200mM NaCl, 1mM EDTA, 1%PEG8000 contains each specific microballon of equivalent in 0.05%SDS).The composition of catching hybridization reaction solution is:
| |
1 * concentration | The supplier and the place of production |
| MOPS pH 7.5 | 10mM | Fisher Chemical，Fair Lawn，NJ |
| NaCl | 200mM | Fisher Chemical，Fair Lawn，NJ |
| MgCl 2 | 50mM | Sigma，St.Louis，MO |
| EDTA | 1mM | Fisher Chemical，Fair Lawn， |
| PEG8000 | ||
| 1％ | Sigma，St.Louis，MO | |
| SDS | 0.05％ | Fisher Chemical，Fair Lawn，NJ |
| The Pacific herring sperm DNA | 0.1mg/mL | Promega，Madison，WI |
| Mixture of microspheres | 1000 various microballoons | EraGen Biosciences，Inc.， Madison，WI |
Component listed above is to be in the reaction system of 60 μ L in the reaction soln final volume, and the concentration of female mixed solution is 1.2 times.The mother solution of 50 μ L mixes with the multiple ASPE reaction soln of 10 μ l purifying, hybridizes at ambient temperature ten minutes.10 μ L contain streptavidin phycoerythrobilin (Phycoerethrin) (Molecular Probes, Eugene, hybridization buffer OR) (10mM MOPS pH7.5,200mM NACI, the 50mM MgCl of 0.01mg/ml
2, 1mM EDTA, 1%PEG8000 0.05%SDS) is added into each and catches in the hybridization reaction solution, is then injected in the Luminex100TM instrument.
Catch hybridization for each, 55 μ L reactants are joined Luminex100 by the speed of dividing with 60 μ L/
TMIn, continue reading then, when can differentiate per 50 microballoons.Fluorescent media intensity is used as the measurement that is connected in fluorescent signal on the microballoon.The result is presented on Figure 14.
The locus specificity of mark non-standard bases mixes and is captured in the application in the genomic dna diversification genotype detection on solid phase support microballoon
Amplification by the target nucleic acid sequence on the genome DNA sample, check, catch, determined the genotype of these nine pleomorphism sites on the genome.The first step, polynary PCR reaction comprises many to paired PCR primer.Every pair of primer comprises the first primer A and the second primer B, and is comprising a non-standard Nucleotide (iso-C) at nearly 5 ' end of the second primer B.Each primer is to being designed to the area hybridization that comprises one section known pleomorphism site with mouse gene group DNA, and this section zone of increasing.Second step was that a multiple allele specific primer extends (ASPE) reaction, comprised many group echos allele specific primer.Each marker allele special primer comprises a 5 ' flag sequence that contains the accurate Nucleotide of tendering (iso-G), the back is-C3 transcribed spacer (n-propylene glycol), the back is one section 3 ' sequence again, and 3 ' sequence is designed to and hybridizes from the wherein chain in the DNA chain of front multiplex PCR step amplification.3 ' Nucleotide by every marker allele special primer is determined allelic specificity.Organize that the marker allele special primer is designed, to be used for inquiring about the known pleomorphism site that is included in the multiplex PCR extension increasing sequence more.The non-standard triguaiacyl phosphate of mark (iso-GTP-vitamin H) is added in the ASPE reactant, thereby the extension of the allele-specific of marker allele Auele Specific Primer has caused at the non-standard triphosphoric acid (iso-4GTP-vitamin H) that mixes mark with respect to non-standard Nucleotide (iso-C) position on the template strand, and this template strand is produced by the front multi-PRC reaction.Uncorporated iso-GTP-vitamin H subsequently catch step before remove.
The 3rd step, catch the product that multiple ASPE reacts, this step has been utilized and has been covalently bind in specific Luminex separately
TMOn the microballoon, contain the acquisition sequence of non-standard Nucleotide (iso-C).Flag sequence complementation on the marker allele Auele Specific Primer that acquisition sequence and front ASPE reaction are used.Add phycoerythrobilin (Phycoerythrin) and be on the extension of marker allele special primer chain in conjunction with biotin labeling, thereby a kind of fluorescent signal is provided.After acquisition sequence and flag sequence hybridization, microballon is injected into Luminex100
TMDevice is to detect the signal that combines with every group of specific microballon.
Genomic 9 polymorphic regions of mouse are used as target in this embodiment:
| The target thing | SEQIDNO： | Sequence | A/J | C57BL6/ | AB6F1 | |
| 2 | 104 | AGAAACAACCATCTAATCCCACACTAAAAT TCAAGGCTCCACAGACGAAACAGTGAAGAA TAATTGTTCAGCATACTAACCAACTGATTA CATATTTACCATACTCAGGTTTGTGCTTCA TACAAACCCAC/TAGTCCGGCGCTCCCTGTTA GATG | | TT | CT | |
| 3 | 105 | CTTCTCCCATTGCCCAGGGCACTCTCCTCT GTAGAA/GTAGACTGATYTTTGTGGAGACATC A | | AA | AG | |
| 4 | 106 | AGTGCCTGCTACCTGTCAGGTGAAAATTTC TTAGTGATCCC/TAAGCTCAATGGGTGCYGGC TTGCAGG | | TT | CT | |
| 5 | 107 | GGTTGGAATGTTTGCACATGCAGTGTTAGT TATTTGGGC/TGATAACTACTTAGCTTATCTA GCCTGGTCCAGC | | CC | CT | |
| 6 | 108 | CTGATCTGACCTCAGACTGTTGTGCTAACA GATATAACACCAGTAAGTTGAC/GTCAAATAC TGCAGGAAGTAGAGCCTTGC | | CC | CG | |
| 7 | 109 | GACTGCTGGAGAGCTGAGGGAGGCTGTGGA GAATAAGGAGAGAGCA/GTAGTCTCGTGCCC T GCCCTGCCCATACTGAGCAGCCAAGACAC | | AA | AG | |
| 8 | 110 | GGACTGTCCAAAKGGATCTCAAGGAGAATA GTCCTTGCTATTAA/GGAGTATAAAGGCATAA AAGAGGTCATAGGGGACAACCATGACCAAG AAGTTG | | GG | AG | |
| 9 | 111 | CCTTCCTGCAYTCCACAGTATAAACACAGA ATGCACACTGCA/GGTCGTTGTATTTGTGTTC GATGTGAATTAAAGATGCTTTGGCTAAGCC AGGAGATGATAATACTG | | GG | AG | |
| 10 | 112 | CACATACACCATGTCAGCCATCAGCGCAAA GCCTTCGAGTTTCAGCTGTGAGATGAAGGC TTGGAGAAGCACGTTGATCTGCAAAGAAGC AAAGGAGCTAGCGGAGGCC/TGGTCACTGACC GACTGCTCA | CC | TT | CT |
In this example, following nucleotide sequence is used to polynary PCR reactions steps.
| Nucleic acid component | Sequence | SEQ ID NO |
| PCR primer 1A | 5′-6FAM-AGAAACAACCATCTAATCCCACA-3′ | 113 |
| PCR primer 1B | 5′-TXCATCTAACAGGGAGCGCC-3′ | 114 |
| PCR primer 2 A | 5′-6FAM-CTTCTCCCATTGCCCAGG-3′ | 115 |
| PCR primer 2 B | 5′-TXTGATGTCTCCACAAAGATCAGTC-3′ | 116 |
| PCR primer 3A | 5′-6FAM-CCTGCAAGCCAGCACC-3′ | 117 |
| PCR primer 3B | 5′-TXCCTGCAAGCCAGCACC-3′ | 118 |
| PCR primer 4A | 5′-6FAM-GGTTGGAATGTTTGCACATGC-3′ | 119 |
| PCR primer 4B | 5′-TXGCTGGACCAGGCTAGATAAGC-3′ | 120 |
| PCR primer 5A | 5′-6FAM-CTGATCTGACCTCAGACTGTTG-3′ | 121 |
| PCR primer 5B | 5′-TXGCAAGGCTCTACTTCCTGC-3′ | 122 |
| PCR primer 6A | 5′-6FAM-GACTGCTGGAGAGCTGAGG-3′ | 123 |
| PCR primer 6B | 5′-TXGTGTCTTGGCTGCTCAGTATG-3′ | 124 |
| PCR primer 7A | 5′-6FAM-GGACTGTCCAAAGGGATCTC-3′ | 125 |
| PCR primer 7B | 5′-TXCAACTTCTTGGTCATGGTTGTC-3′ | 126 |
| PCR primer 8A | 5′-6FAM-CAGTATTATCATCTCCTGGCTTAGC-3′ | 127 |
| PCR primer 8B | 5′-TXCCTTCCTGCACTCCACAG-3′ | 128 |
| PCR primer 9A | 5′-6FAM-CACATACACCATGTCAGCC-3′ | 129 |
| PCR primer 9B | 5′-TXTGAGCAGTCGGTCAGTG-3′ | 130 |
| Template 1 | Mouse gene group DNA; Strain: A/J | |
| Template 2 | Mouse gene group DNA; Strain: C57BL6/J | |
| Template 3 | Mouse gene group DNA; Strain: AB6F1 |
The primer of synthetic PCR reaction and in MOPS, the 0.1mM EDTA of the 1mM of pH7.5, diluting.The mouse gene group DNA sample is that (Bar Harbor ME) buys from Jackson Laboratory in.All genome DNA samples all are diluted to 5ng/ μ l in MOPS, the 0.1mM EDTA of the 1mM of pH7.5.The component of PCR reaction is:
| |
1 * concentration | The supplier and the place of production |
| 10X PCR Buffer II | 1.2X | Applied Biosystems，Foster City，CA |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| DATP | 200μM | Amersham |
| DGTP | 200μM | Amersham |
| DCTP | 200μM | Amersham |
| DTTP | 200μM | Amersham |
| Amplitaq TMThe Gold archaeal dna polymerase | 0.1U/μl | Applied Biosystems，Foster City，CA |
| PCR primer (every kind) | 0.2μM |
All female mixed solutions of listing component are prepared as 1.09 * concentration of 25 μ l end reaction volumes.Female mixed solution of 23 μ l and 2 μ l genomic dna templates (5ng/ μ l) are mixed in the one PCR pipe.Negative contrast water replaces the genomic dna template.The PCR reaction cycle is as follows:
After PCR is circulated throughout, respectively get 2 μ l PCR reactant transfer in the multiple ASPE reaction as template.Following synthetic nucleic acid is used as marker allele special (TAS) primer of multiple ASPE reaction:
| Nucleic acid component | Sequence | SEQ IDNO |
| TAS primer 1 | 5′-GTGYACAYGC-c3-GCTTCATACAAACCCAC-3′ | 131 |
| The TAS primer 2 | 5′-CGAYTCTGYC-c3-GCTTCATACAAACCCAT-3′ | 132 |
| TAS primer 3 | 5′-CTAYCAAYCC-c3-CACTCTCCTCTGTAGAA-3′ | 133 |
| TAS primer 4 | 5′-GAGAYCYAAG-c3-CACTCTCCTCTGTAGAG-3′ | 134 |
| TAS primer 5 | 5′-GTTCYTGAYG-c3-GAAAATTTCTTAGTGATCCT-3′ | 135 |
| TAS primer 6 | 5′-GCYTAYCTAC-c3-AAAATTTCTTAGTGATCCC-3′ | 136 |
| TAS primer 7 | 5′-GTTAYCYTCC-c3-AGTGTTAGTTATTTGGGT-3′ | 137 |
| TAS primer 8 | 5′-CACYATACYG-c3-GTGTTAGTTATTTGGGC-3′ | 138 |
| TAS primer 9 | 5′-CYTACCYATG-c3-TAACACCAGTAAGTTGAC-3′ | 139 |
| TAS primer 10 | 5′-GYCGAYAATC-c3-TAACACCAGTAAGTTGAG-3′ | 140 |
| TAS primer 11 | 5′-GYCGTAYTTG-c3-AGAATAAGGAGAGAGCA-3′ | 141 |
| TAS primer 12 | 5′-GTYTATYCCG-c3-GAATAAGGAGAGAGCG-3′ | 142 |
| TAS primer 13 | 5′-GACAYACYTC-C3-AGAATAGTCCTTGCTATTAA-3′ | 143 |
| TAS primer 14 | 5′-GGAAYAACYG-C3-AGAATAGTCCTTGCTATTAG-3′ | 144 |
| TAS primer 15 | 5′-GATYTYCAGC-c3-AGAATGCACACTGCA-3′ | 145 |
| TAS primer 16 | 5′-GTYATYTGCG-C3-GAATGCACACTGCG-3′ | 146 |
| TAS primer 17 | 5′-GATYGTCYYG-c3-GCTAGCGGAGGCC-3′ | 147 |
| TAS primer 18 | 5′-GGYCTYATGG-c3-GCTAGCGGAGGCT-3′ | 148 |
Component in the ASPE reaction is:
| |
1 * concentration | The supplier and the place of production |
| Bis-Tris-propane pH 8.9 | 10mM | Sigma，St.Louis，MO |
| Potassium acetate | 40mM | Sigma，St.Louis，MO |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| dATP | 50μM | Amersham Pharmacia，Piscataway，NJ |
| dGTP | 50μM | Amersham Pharmacia，Piscataway，NJ |
| dCTP | 50μM | Amersham Pharmacia，Piscataway，NJ |
| dTTP | 50μM | Amersham Pharmacia，Piscataway，NJ |
| The d-isoGTP-vitamin H | 10uM | EraGen Biosciences，Inc.，Madison，WI |
| The Klentaq archaeal dna polymerase | 0.067U/μl | Ab Peptides，St.Louis，MO |
| TAS primer (every kind) | 0.067μM | EraGen Biosciences，Inc.，Madison，WI |
A kind of female mixed solution that comprises above all components is made into 1.15X.Each ASPE reaction comprises 13 μ, 1 female mixed solution, 2 μ l PCR reactants (from preceding step).The ASPE reaction cycle is as follows:
After the ASPE circulation, 5 μ l comprise 40mM Tris, and the solution of 40mM EDTA is added in the multiple ASPE reactant, to stop the activity of polysaccharase.Reactant is removed unconjugated d-isoGTP-vitamin H with the G-50 column purification.The multiple ASPE reaction solution of purifying is by being combined in Luminex then
TMAcquisition sequence on the microballon (Luminex company, Houston, Texas) untwists.The link coupled microballon is
| The microballon identity | SEQ ID NO： | |
| 1 | 2 | |
| 2 | 9 | |
| 7 | 14 | CGGXATAXAC |
| 15 | 13 | CAAXTACGXC |
| 17 | 22 | GCTGXAXATC |
| 18 | 23 | CGCAXATXAC |
| 19 | 1 | GAXGTXTGTC |
| 20 | 3 | GGXTTGXTAG |
| 21 | 4 | CTTXGXTCTC |
| 22 | 5 | CXTCAXGAAC |
| 34 | 7 | GGAXGXTAAC |
| 35 | 8 | CXGTATXGTG |
| 37 | 6 | GTAGXTAXGC |
| 38 | 10 | GATTXTCGXC |
| 45 | 28 | CXXGACXATC |
| 47 | 29 | CCATXAGXCC |
| 61 | 36 | GCXTGTXCAC |
| 62 | 37 | GXCAGAXTCG |
The link coupled microballon mixes with mixture, this mixture the storage damping fluid (10mM MOPS pH7.5,200mM NaCl, 1mM EDTA, 1%PEG8000 contains each specific microballon of equivalent in 0.05%SDS).The composition of catching hybridization reaction solution is:
| |
1 * concentration | The supplier and the place of production |
| MOPS pH7.5 | 10mM | Fisher Chemical，Fair Lawn，NJ |
| NaCl | 200mM | Fisher Chemical，Fair Lawn，NJ |
| MgCl 2 | 50mM | Sigma，St.Louis，MO |
| EDTA | 1mM | Fisher Chermical，Fair Lawn， |
| PEG8000 | ||
| 1％ | Sigma，St.Louis，MO | |
| SDS | 0.05％ | Fisher Chemical，Fair Lawn，NJ |
| Pacific herring seminal fluid DNA | 0.1mg/mL | Promega，Madison，WI |
| Mixture of microspheres | 1000 various microballoons | EraGen Biosciences，Inc.，Madison，WI |
The female mixture for preparing component listed above, reaction soln final volume are 60 μ L, 1.2 times of concentration.The mother solution of 50 μ l mixes with the multiple ASPE reaction soln of 10 μ L purifying, hybridizes at ambient temperature ten minutes.10 μ L contain streptavidin phycoerythrobilin (Phycoerethrin) (MolecularProbes, Eugene, hybridization buffer OR) (10mM MOPS pH7.5,200mM NaCl, the 50mMMgCL of 0.01mg/ml
2, 1mM EDTA, 1%PEG8000 0.05%SDS) is added into each and catches in the hybridization reaction solution, is then injected in the Luminex 100TM instrument.
Catch hybridization for each, 55 μ L reactants are joined Luminex100 by the speed of dividing with 60 μ L/
TMIn, continue reading then, when can differentiate per 50 microballoons.Fluorescent media intensity is used as the measurement that is connected in fluorescent signal on the microballoon.The result is presented on Figure 15.
The present invention is not limited to above-described special case, and further can be understood as all respects of the claim that covers claims proposition of the present invention.Various modification, being equal to and replacing and other multiple application modes, is very easy to those of ordinary skills with reference to specification sheets of the present invention.
The reporter gene oligonucleotide is connected with the locus specificity of allele-specific extension products and is captured in application in the genomic dna genotype detection on solid phase is supported microballoon
Amplification by the target nucleic acid sequence on the genome DNA sample, check, catch, we have determined the genotype of pleomorphism site on the genome.The first step, the PCR reaction, comprise a series of PCR primers: the first primer A and the second primer B, the 5 ' end of the second primer B is comprising an incomplementarity in the sequence of target, and this target sequence is meant the target sequence that contains iso-C in the junction of its assay specific sequence and incomplementarity part.Primer is designed to the area hybridization that comprises one section known pleomorphism site with mouse gene group DNA, and this section zone of increasing.Second step was that an allele specific primer extends (ASPE) reaction, comprised a series of marker allele special primers.Each marker allele special primer comprises a 5 ' flag sequence that contains non-standard Nucleotide (iso-G), the back is a C3 transcribed spacer, the back is one section 3 ' sequence again, and 3 ' sequence is designed to and hybridizes from the wherein chain in the DNA chain of front PCR step amplification.3 ' Nucleotide of the allele specific primer by every mark is determined allelic specificity.A series of marker allele special primers are designed, to be used for inquiring about the known pleomorphism site that is included in extension increasing sequence.The reporter gene oligonucleotide of ' phosphoric acid and the 1 ' biotin modification that in ASPE reaction, comprised that DAN ligase enzyme and contains 1.This reporter gene oligonucleotide is complementary to the 5 ' zone of primer B, and primer B is used to produce the amplicon of inquiry.The amplified production chain that contains this non-standard bases has the zone as the ASPE reaction template.Between the allele specific primer extended peroid, archaeal dna polymerase stops at the previous base place of template strand iso-C, stays next strand zone to be used for the hybridization of reporter gene oligonucleotide like this.Extend the ASPE primer, the complex body between template and the reporter gene oligonucleotide causes one to be suitable for the breach structure appearance that dna ligase connects.
The 3rd step, use and to contain the polynary ASPE reaction product of non-standard Nucleotide (iso-C) sequence capturing, each contain non-standard Nucleotide (iso-C) sequence all separately covalent coupling in specific luminex microballoon.The flag sequence complementation that is used to set up the label allele specific primer in acquisition sequence and the reaction of ASPE in front.Add streptavidin-phycoerythrobilin and be with extend with the allele-specific primers chain that is connected on the biotin labeling connection, so that fluorescent signal to be provided.After the hybridization, microsphere is injected into a Luminex100 device and detects the peculiar signal that links to each other with every kind of unique microsphere between acquisition sequence and flag sequence.
In this example, the single polymorphic region of mouse genome is as target position:
| Serial ID number | Target sequence | A/J | C57BL6/J | AB6F1 |
| 149 | 5′CTTCTCCCATTGCCCAGGGCACTCTCCTCT GTAGARTAGACTGATYTTTGTGGAGACATCA3′ | GG | AA | AG |
| Nucleic acid component | 5 '-3 ' sequence | SEQ ID NO： | |
| PCR primer A | PO4-CTTCTCCCATTGCCCAGG | 115 | |
| PCR | CXGCXAGXGATXTGATGTCTCCACAAAGATCAGTC | 150 | |
| |
Mouse gene group DNA; Strain: A/ | ||
| Template | |||
| 1 | Mouse gene group DNA; Strain: C57BL6/ | ||
| Template | |||
| 1 | Mouse gene group DNA; Strain: AB6F1 |
The mouse gene group DNA sample available from the Jackson laboratory (Bar Harbor, ME).All genome DNA samples 1mM MOPS pH7.5,0.1mM EDTA is diluted to 10ng/ μ l.The PCR reaction is formed:
| |
1 * concentration | The supplier and the place of production |
| 10X PCR Buffer II | 1.2X | Applied Biosystems，Foster City，CA |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| dGTP | 200μM | Promega，Madison，WI |
| dATP | 200μM | Promega，Madison，WI |
| dTTP | 200μM | Promega，Madison，WI |
| dCTP | 200μM | Promega，Madison，WI |
| Amplitaq archaeal dna polymerase Stoffel fragment | 0.2U/μl | Applied Biosystems，Foster City，CA |
| PCR primer A | 0.2μM | |
| PCR primer B | 0.2μM |
Female mixture of all listed components is prepared into 1.07 * concentration, so that the reaction final volume is 30 μ l.Female mixture of 23 μ l mixes in independent PCR tubule with the genomic dna template (5ng/ μ l) of 2 μ l.Negative control is to replace the genomic dna template by water.The thermal cycling of PCR reaction is as follows:
After the PCR circulation, (MA) 3 μ l are to remove the amplicon non-template chain that PCR primer A introduces for New EnglandBiolabs, Beverly for the lambda exonuclease of each reaction system adding 5U/ μ l.After adding lambda exonuclease, will react tubule and be heated to 37 ℃, 5 minutes, reheat to 95 ℃, 2 minutes.After the digestion, the template of 1 μ l as the ASPE reaction got in each PCR reaction like this.Following nucleotide sequence is used as marker allele specificity (TAS) primer in the ASPE reaction:
| Nucleic acid component | 5 '-3 ' sequence | Serial ID number: |
| |
CTAYCAAYCC-c3- CACTCTCCTCTGTAGAA | 151 |
| The |
GAGAYCYAAG-c3- CACTCTCCTCTGTAGAG | 152 |
| The reporter gene oligonucleotide | PO 4-YATCYCTYGCYG-vitamin H | 153 |
The component of ASPE reaction is:
| |
1 * concentration | The supplier and the place of production |
| 10X PCR Buffer II | 1.2X | Applied Biosystems，Foster City，CA |
| MgCl 2 | 2mM | Sigma，St.Louis，MO |
| DGTP | 200μM | Promega，Madison，WI |
| DATP | 200μM | Promega，Madison，WI |
| DTTP | 200μM | Promega，Madison，WI |
| DCTP | 200μM | Promega，Madison，WI |
| Amplitaq archaeal dna polymerase Stoffel fragment | 0.1U/μl | Applied Biosystems，Foster City， |
| TAS primer | ||
| 1 | 0.1μM | |
| The |
0.1μM | |
| The reporter gene oligonucleotide | 0.2μM | |
| DTT | 5mM | Fisher Scientific，Pittsburgh，PA |
| NAD | 1mM | Roche，Indianapolis，IN |
| The Taq dna ligase | 2U/μl | New England Biolabs，Beverly，MA |
The female mixture that comprises all said components is prepared as 1.11 *.Each ASPE reaction is made up of female mixture of 9 μ l and 1 μ l PCR reactant (from back).The ASPE reaction cycle is as follows:
After the ASPE circulation, reactant is coupled to the Luminex microballoon, and (TX) acquisition sequence on untwists for Luminex Corp, Austin.The coupling microsphere is:
| The microsphere identity | SEQ ID NO： | 5 '-3 ' acquisition sequence |
| 20 | 3 | GGXTTGXTAG |
| 21 | 4 | CTTXGXTCTC |
The link coupled microballon mixes with mixture, and this mixture is at storage damping fluid (10mM MOPS pH7.5,200mM NaCl, 1mM EDTA, 1%PEG8000,0.05%SDS) the specific microballon of each of moderate.The composition of catching hybridization reaction solution is:
| |
1 * concentration | The supplier and the place of production |
| MOPS pH7.5 | 10mM | Sigma，St.Louis，MO |
| NaCl | 200mM | Sigma，St.Louis，MO |
| MgCl 2 | 50mM | Sigma，St.Louis，MO |
| EDTA | 1mM | Sigma，St.Louis， |
| PEG8000 | ||
| 1％ | Sigma，St.Louis，MO | |
| SDS | 0.05％ | Sigma，St.Louis，MO |
| Pacific herring seminal fluid DNA | 0.1mg/mL | Promega，Madison，WI |
| The microballon mixture | 1000 every kind of microballons | Luminex Corp.，Austin TX |
Female mixture of above-mentioned all components is prepared into 1.2 * concentration, so that the reaction final volume is 60 μ l.This female mixture of 50 μ l adds in each ASPE reaction system, and room temperature hybridization 10 minutes.(0.075mg/ml is in 10mM MOPS pH 7.5, NaCl 200mM, MgCl to catch hybridization system adding 10 μ l streptavidin-phycoerythrobilin solution to each before injecting Luminex 100 devices
250mM, EDTA1mM, PEG 8,000 1%, SDS 0.05) (Molecular Probes, Eugene, OR).
Catch hybridization for each, get 45 μ l reaction mixtures and inject Luminex100, continue reading then, when can differentiate per 100 microballoons with the speed of 60 μ l/min.Fluorescent media intensity (MFI) is used as the measurement that is connected in fluorescent signal on the microballoon.The result is presented on Figure 17.
In above-mentioned example, the non-standard bases of primer B is positioned at 5 ' end incomplementarity sequence and analyte distinguished sequence joining region, is designed to be used for stoping the extension of enzymatic in label and analyte distinguished sequence junction.Can envision clearly, other suitable joints also can be used to stop polymeric enzyme reaction, comprise, for example, 2-O-methyl base is as 2 '-O-methyl ribonucleotides.
Sequence table
<110>Grenier，Jennifer
Marshall，David
Prudent，James
Richmond，Craig
Roesch，Eric
Scherrer，Christopher
Sherrill，Christopher
Ptacin，Jerod
＜120〉solid support assay systems and the method for use non-standard bases
<130>031403-9012
<140>09/977,615
<141>2001-10-15
<150>60/240,397
<151>2000-10-14
<150>60/282,831
<151>2001-04-10
<150>09/861,292
<151>2001-0518
<150>60/293,259
<151>2001-05-22
<160>156
<170>PatentIn version 3.0
<210>1
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>1
<210>2
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>2
<210>3
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7).(7)
＜223〉n represents isoguanine
<400>3
<210>4
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>4
<210>5
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>5
<210>6
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>6
<210>7
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>7
<210>8
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>8
<210>9
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(10)..(10)
＜223〉n represents isoguanine
<400>9
<210>10
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>10
<210>11
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>11
<210>12
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>12
<210>13
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>13
<210>14
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>14
<210>15
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>15
<210>16
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>16
<210>17
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>17
<210>18
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>18
<210>19
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>19
<210>20
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>20
<210>21
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>21
<210>22
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>22
<210>23
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>23
<210>24
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>24
<210>25
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>25
<210>26
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>26
<210>27
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>27
<210>28
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>28
<210>29
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>29
<210>30
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>30
<210>31
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>31
<210>32
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>32
<210>33
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<40)0>33
<210>34
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>34
<210>35
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>35
<210>36
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>36
<210>37
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>37
<210>38
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>38
<210>39
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>39
<210>40
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>40
<210>41
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>41
<210>42
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>42
<210>43
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>43
<210>44
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>44
<210>45
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>45
<210>46
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>46
<210>47
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>47
<210>48
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>48
<210>49
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>49
<210>50
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>50
<210>51
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>51
<210>52
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>52
<210>53
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>53
<210>54
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>54
<210>55
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>55
<210>56
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>56
<210>57
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>57
<210>58
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>58
<210>59
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>59
<210>60
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>60
<210>61
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜＜223〉n represents isoguanine
<400>61
<210>62
<<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>62
<210>63
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>63
<210>64
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>64
<210>65
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>65
<210>66
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine.
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine.
<400>66
<210>67
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>67
<210>68
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>68
<210>69
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>69
<210>70
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>70
<210>71
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>71
<210>72
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>72
<210>73
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>73
<210>74
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>74
<210>75
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>75
<210>76
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>76
<210>77
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>77
<210>78
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>78
<210>79
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>79
<210>80
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>80
<210>81
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>81
<210>82
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>82
<210>83
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9).
＜223〉n represents isoguanine
<400>83
<210>84
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>84
<210>85
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>85
<210>86
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>86
<210>87
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>87
<210>88
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>88
<210>89
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>89
<210>90
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3).(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>90
<210>91
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>91
<210>92
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<400>92
<210>93
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>93
<210>94
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(9)..(9)
＜223〉n represents isoguanine
<400>94
<210>95
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>95
<210>96
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(6)..(6)
＜223〉n represents isoguanine
<400>96
<210>97
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>97
<210>98
<211>9
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(4)..(4)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(7)..(7)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8)..(8)
＜223〉n represents isoguanine
<400>98
<210>99
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<400>99
<210>100
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<400>100
<210>101
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(3)..(3)
＜223〉n represents isoguanine
<400>101
<210>102
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<400>102
<210>103
<211>10
<212>DNA
＜213〉synthetic oligonucleotide
<400>103
<210>104
<211>155
<212>DNA
＜213〉synthetic oligonucleotide
<400>104
agaaacaacc atctaatccc acactaaaat tcaaggctcc acagacgaaa cagtgaagaa 60
taattgttca gcatactaac caactgatta catatttacc atactcaggt ttgtgcttca 120
tacaaaccca ctagtccggc gctccctgtt agatg 155
<210>105
<211>63
<212>DNA
＜213〉synthetic oligonucleotide
<400>105
cttctcccat tgcccagggc actctcctct gtagaagtag actgatcttt tgtggagaca 60
tca 63
<210>106
<211>68
<212>DNA
＜213〉synthetic oligonucleotide
<400>106
agtgcctgct acctgtcagg tgaaaatttc ttagtgatcc ctaagctcaa tgggtgcygg 60
cttgcagg 68
<210>107
<211>73
<212>DNA
＜213〉synthetic oligonucleotide
<400>107
ggttggaatg tttgcacatg cagtgttagt tatttgggct gataactact tagcttatct 60
agcctggtcc agc 73
<210>108
<211>81
<212>DNA
＜213〉synthetic oligonucleotide
<400>108
ctgatctgac ctcagactgt tgtgctaaca gatataacac cagtaagttg acgtcaaata 60
ctgcaggaag tagagccttg c 81
<210>109
<211>90
<212>DNA
＜213〉synthetic oligonucleotide
<400>109
gactgctgga gagctgaggg aggctgtgga gaataaggag agagcagtag tctcgtgccc 60
tgccctgccc atactgagca gccaagacac 90
<210>110
<211>97
<212>DNA
＜213〉synthetic oligonucleotide
<400>110
ggactgtcca aakggatctc aaggagaata gtccttgcta ttaaggagta taaaggcata 60
aaagaggtca taggggacaa ccatgaccaa gaagttg 97
<210>111
<211>108
<212>DNA
＜213〉synthetic oligonucleotide
<400>111
ccttcctgca ytccacagta taaacacaga atgcacactg caggtcgttg tatttgtgtt 60
cgatgtgaat taaagatgct ttggctaagc caggagatga taatactg 108
<210>112
<211>130
<212>DNA
＜213〉synthetic oligonucleotide
<400>112
cacatacacc atgtcagcca tcagcgcaaa gccttcgagt ttcagctgtg agatgaaggc 60
ttggagaagc acgttgatct gcaaagaagc aaaggagcta gcggaggcct ggtcactgac 120
<210>113
<211>18
<212>DNA
＜213〉synthetic oligonucleotide
<400>113
catctaacag ggagcgcc 18
<210>114
<211>23
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>114
ngaaacaacc atctaatccc aca 23
<210>115
<211>18
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>115
nttctcccat tgcccagg 18
<210>116
<211>23
<212>DNA
＜213〉synthetic oligonucleotide
<400>116
tgatgtctcc acaaagatca gtc 23
<210>117
<211>19
<212>DNA
＜213〉synthetic oligonucleotide
<400>117
agtgcctgct acctgtcag 19
<210>118
<211>16
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>118
nctgcaagcc agcacc 16
<210>119
<211>21
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>119
ngttggaatg tttgcacatg c 21
<210>120
<211>21
<212>DNA
＜213〉synthetic oligonucleotide
<400>120
gctggaccag gctagataag c 21
<210>121
<211>22
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>121
ntgatctgac ctcagactgt tg 22
<210>122
<211>19
<212>DNA
＜213〉synthetic oligonucleotide
<400>122
gcaaggctct acttcctgc 19
<210>123
<211>19
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>123
nactgctgga gagctgagg 19
<210>124
<211>21
<212>DNA
＜213〉synthetic oligonucleotide
<400>124
gtgtcttggc tgctcagtat g 21
<210>125
<211>20
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>125
ngactgtcca aagggatctc 20
<210>126
<211>22
<212>DNA
＜213〉synthetic oligonucleotide
<400>126
caacttcttg gtcatggttg tc 22
<210>127
<211>19
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents indodicarbocyanine 3-1-O-(2-cyanoethyl)-(N, N-di-isopropyl)-phosphoramidit
<400>127
nccttcctgc aytccacag 19
<210>128
<211>25
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6-FAM) mark
<400>128
nagtattatc atctcctggc ttagc 25
<210>129
<211>19
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(1)..(1)
＜223〉n represents the deoxythymidylic acid with 6-carboxyl fluorescent yellow (6FAM) mark
<400>129
nacatacacc atgtcagcc 19
<210>130
<211>17
<212>DNA
＜213〉synthetic oligonucleotide
<400>130
tgagcagtcg gtcagtg 17
<210>131
<2111>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>131
gtgyacaygc gcttcataca aacccac 27
<210>132
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>132
cgaytctgyc gcttcataca aacccat 27
<210>133
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>133
ctaycaaycc cactctcctc tgtagaa 27
<210>134
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>134
gagaycyaag cactctcctc tgtagag 27
<210>135
<211>30
<212>DNA
＜213〉synthetic oligonucleotide
<400>135
gttcytgayg gaaaatttct tagtgatcct 30
<210>136
<211>29
<212>DNA
＜213〉synthetic oligonucleotide
<400>136
gcytayctac aaaatttctt agtgatccc 29
<210>137
<211>28
<212>DNA
＜213〉synthetic oligonucleotide
<400>137
gttaycytcc agtgttagtt atttgggt 28
<210>138
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>138
cacyatacyg gtgttagtta tttgggc 27
<210>139
<211>28
<212>DNA
＜213〉synthetic oligonucleotide
<400>139
cytaccyatg taacaccagt aagttgac 28
<210>140
<211>28
<212>DNA
＜213〉synthetic oligonucleotide
<400>140
gycgayaatc taacaccagt aagttgag 28
<210>141
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>141
gycgtayttg agaataagga gagagca 27
<210>142
<211>26
<212>DNA
＜213〉synthetic oligonucleotide
<400>142
gtytatyccg gaataaggag agagcg 26
<210>143
<211>30
<212>DNA
＜213〉synthetic oligonucleotide
<400>143
gacayacytc agaatagtcc ttgctattaa 30
<210>144
<211>30
<212>DNA
＜213〉synthetic oligonucleotide
<400>144
ggaayaacyg agaatagtcc ttgctattag 30
<210>145
<211>25
<212>DNA
＜213〉synthetic oligonucleotide
<400>145
gatytycagc agaatgcaca ctgca 25
<210>146
<211>24
<212>DNA
＜213〉synthetic oligonucleotide
<400>146
gtyatytgcg gaatgcacac tgcg 24
<210>147
<211>23
<212>DNA
＜213〉synthetic oligonucleotide
<400>147
<210>148
<211>23
<212>DNA
＜213〉synthetic oligonucleotide
<400>148
ggyctyatgg gctagcggag gct 23
<210>149
<211>61
<212>DNA
＜213〉synthetic oligonucleotide
<400>149
cttctcccat tgcccagggc actctcctct gtagartaga ctgatytttg tggagacatc 60
a 61
<210>150
<211>35
<212>DNA
＜213〉synthetic oligonucleotide
<220>
＜221〉base of Xiu Shiing
<222>(2)..(2)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(5)..(5)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(8).(8)
＜223〉n represents isoguanine
<220>
＜221〉base of Xiu Shiing
<222>(12)..(12)
＜223〉n represents isoguanine
<400>150
cngcnagnga tntgatgtct ccacaaagat cagtc 35
<210>151
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>151
ctaycaaycc cactctcctc tgtagaa 27
<210>152
<211>27
<212>DNA
＜213〉synthetic oligonucleotide
<400>152
gagaycyaag cactctcctc tgtagag 27
<210>153
<211>12
<212>DNA
＜213〉synthetic oligonucleotide
<400>153
yatcyctygc yg 12
<210>154
<211>18
<212>DNA
＜213〉synthetic oligonucleotide
<400>154
agaacccttt cctcttcc 18
<210>155
<211>47
<212>DNA
＜213〉synthetic oligonucleotide
<400>155
aagaaccctt tcctcttccg atgcaggata cttaacaata aatattt 47
<210>156
<211>39
<212>DNA
＜213〉synthetic oligonucleotide
<400>156
gcagacagga yaaatattta ttgttaagta tcctgcatc 39
Claims (10)
1. method of analyzing the target oligonucleotide comprises that step is as follows:
(a) assay that comprises the assay distinguished sequence is contacted with second primer with first primer, first primer comprise a flag sequence and with the assay first sequence complementary sequence, second primer comprises and assay second sequence complementary sequence and the non-standard bases;
(b) enzymatic extends first and second primers to form the target oligonucleotide and second oligonucleotide, and its hit one of oligonucleotide and second oligonucleotide contain the assay distinguished sequence, and another contains and assay distinguished sequence complementary sequence; With on first primer that extends, mix the complementary non-standard bases of a mark with respect to the position of the second primer non-standard bases;
(c) under hybridization conditions, the oligonucleotide of catching that is coupled to support is contacted with the target oligonucleotide with hybridization target oligonucleotide, the target oligonucleotide comprises the complementary sequence of flag sequence and assay distinguished sequence or assay distinguished sequence, catch oligonucleotide and comprise a part recognition sequence, the flag sequence complementation of this molecular recognition sequence and target oligonucleotide; With
(d), detect target oligonucleotide and the hybridization of catching oligonucleotide based on the signal that detects from the complementary non-standard bases of described mark.
2. the process of claim 1 wherein that (c) at room temperature carries out.
3. the process of claim 1 wherein and do not wash after (c) thereupon.
4. the method for at least two target oligonucleotides of an analysis, it comprises that step is as follows:
(a) assay that comprises the assay distinguished sequence is contacted with second primer with first primer, second primer comprises, from 5 ' to 3 ' order, and incomplementarity is in the sequence of assay distinguished sequence, non-standard bases and assay distinguished sequence;
(b) primer of enzymatic extension (a) is to form extension products;
(c) extension products of second primer hybridization in the primer of at least one mark and the step (b), the primer of mark comprises that from 5 ' to 3 ' in proper order, comprised at least one non-standard bases 5 ' flag sequence and with the second primer extension product complementary, 3 ' sequence;
(d) enzymatic extends the labeled primer of step (c) to mix and the second primer complementary sequence;
(e) hybridizing with the sequence complementary report oligonucleotide of incomplementarity second primer assay distinguished sequence in step (a) and the extension products of step (b);
(f) making the hybridization product of step (e) catch oligonucleotide with at least one under suitable hybridization conditions contacts, described catch oligonucleotide comprise at least one non-standard Nucleotide and with the support coupling so that the hybridization product of step (e) and at least one are caught oligonucleotide hybridization; With
(g) by detecting signal, detect the hybridization of step (f) from the report oligonucleotide.
5. the method for claim 4, wherein (f) at room temperature carries out.
6. the method for claim 4 is not wherein washed after (f) thereupon.
7. the method for claim 4 further comprises with ligase enzyme the extension products of (d) is covalently bound with report oligonucleotide (e).
8. method of analyzing the target oligonucleotide, it comprises that step is as follows:
(a) assay that comprises the assay distinguished sequence is contacted with second primer with first primer, second primer comprises, from 5 ' to 3 ' order, and incomplementarity is in the sequence of assay distinguished sequence, non-standard bases and assay distinguished sequence;
(b) enzymatic extends primer, and to form the target oligonucleotide and second oligonucleotide, its hit one of oligonucleotide and second oligonucleotide contain the assay distinguished sequence, and another one contains and assay distinguished sequence complementary sequence;
(c) make the extension products hybridization of second primer in the allele-specific primers of mark and the step (b), the allele-specific primers of mark comprises, from 5 ' to 3 ' in proper order, 5 ' the flag sequence that has comprised non-standard bases, connecting joint and with the second primer extension product complementary, 3 ' sequence;
(d) enzymatic extends the allele-specific primers of step (c), to mix and the second primer complementary sequence;
(e) hybridizing with the sequence complementary report oligonucleotide of incomplementarity second primer assay distinguished sequence in step (a) and the extension products in the step (d);
(f) make and catch oligonucleotide with the support link coupled and contact with the hybridization product of step (e); With
(g) examining report oligonucleotide and the hybridization of catching oligonucleotide.
9. the method for claim 8, wherein (f) at room temperature carries out.
10. the method for claim 8 is not wherein washed after (f) thereupon.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US24039700P | 2000-10-14 | 2000-10-14 | |
| US60/240,397 | 2000-10-14 | ||
| US60/282,831 | 2001-04-10 | ||
| US09/861,292 | 2001-05-18 | ||
| US60/293,259 | 2001-05-22 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1697881A CN1697881A (en) | 2005-11-16 |
| CN100385013C true CN100385013C (en) | 2008-04-30 |
Family
ID=35350137
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB018205151A Expired - Lifetime CN100385013C (en) | 2000-10-14 | 2001-10-15 | Solid support assay systems and methods utilizing non-standard bases |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100385013C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2865764B1 (en) * | 2012-06-20 | 2018-09-05 | Toray Industries, Inc. | Method for detecting nucleic acid and use of a nucleic acid detection kit |
| US9587268B2 (en) * | 2014-01-29 | 2017-03-07 | Agilent Technologies Inc. | Fast hybridization for next generation sequencing target enrichment |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996031622A1 (en) * | 1995-04-07 | 1996-10-10 | Oxford Gene Technology Limited | Detecting dna sequence variations |
| EP0742287A2 (en) * | 1995-05-10 | 1996-11-13 | McGall, Glenn H. | Modified nucleic acid probes |
| US5605794A (en) * | 1991-09-06 | 1997-02-25 | Boehringer Mannheim Gmbh | Method of detecting variant nucleic acids |
| WO1997046711A1 (en) * | 1996-06-06 | 1997-12-11 | Wisconsin Alumni Research Foundation | Artificial mismatch hybridization |
| WO1998014610A2 (en) * | 1996-10-04 | 1998-04-09 | The Perkin-Elmer Corporation | Multiplex polynucleotide capture methods and compositions |
| US6037120A (en) * | 1995-10-12 | 2000-03-14 | Benner; Steven Albert | Recognition of oligonucleotides containing non-standard base pairs |
-
2001
- 2001-10-15 CN CNB018205151A patent/CN100385013C/en not_active Expired - Lifetime
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5605794A (en) * | 1991-09-06 | 1997-02-25 | Boehringer Mannheim Gmbh | Method of detecting variant nucleic acids |
| WO1996031622A1 (en) * | 1995-04-07 | 1996-10-10 | Oxford Gene Technology Limited | Detecting dna sequence variations |
| EP0742287A2 (en) * | 1995-05-10 | 1996-11-13 | McGall, Glenn H. | Modified nucleic acid probes |
| US6037120A (en) * | 1995-10-12 | 2000-03-14 | Benner; Steven Albert | Recognition of oligonucleotides containing non-standard base pairs |
| WO1997046711A1 (en) * | 1996-06-06 | 1997-12-11 | Wisconsin Alumni Research Foundation | Artificial mismatch hybridization |
| WO1998014610A2 (en) * | 1996-10-04 | 1998-04-09 | The Perkin-Elmer Corporation | Multiplex polynucleotide capture methods and compositions |
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| Publication number | Publication date |
|---|---|
| CN1697881A (en) | 2005-11-16 |
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