CN102373288B

CN102373288B - Method and kit for sequencing target areas

Info

Publication number: CN102373288B
Application number: CN2011103919902A
Authority: CN
Inventors: 盛司潼
Original assignee: Individual
Current assignee: Wuhan Kangxinrui Gene Health Technology Co Ltd
Priority date: 2011-11-30
Filing date: 2011-11-30
Publication date: 2013-12-11
Anticipated expiration: 2031-11-30
Also published as: CN102373288A

Abstract

The invention relates to the field of genetic engineering, and provides a method and a kit for sequencing target areas. The method comprises the following steps of: A, using a specificity primer to amplify a plurality of target areas in a sample to be detected, and establishing a sequencing library based on amplified products; B, subjecting the sequencing library to monomolecular amplification to obtain a plurality of monomolecular amplification products corresponding to the plurality of target areas; and C, subjecting the plurality of monomolecular amplification products to high-throughput gene sequencing at the same time so as to obtain sequence information of the plurality of target areas. The method and the corresponding kit are used for sequencing the plurality of areas at the same time by using a high-throughput sequencing technology; the sequence information including variation of known mutations and unknown mutations in the areas can be obtained accurately; the kit has high detection sensitivity, and can be used for further detecting mainly samples at the same time.

Description

A kind of method checked order in target area and test kit

Technical field

The present invention relates to the genetically engineered field, more particularly, relate to a kind of method checked order in target area and test kit.

Background technology

Since human genome nucleotide sequence sketch and many other genome sketches appearances, and the discovery of a large amount of single nucleotide polymorphism (SNP) and little insertion/deletion in the mankind and many other biological body genome sketches, people start nucleic acid determination is become interested.Research shows, the variant nucleic acid sequence of some specific position has comprised a large amount of biological informations, may be relevant to the mankind or vegeto-animal some proterties, and these information will produce wide influence to the every aspect of medicine and agricultural etc.For instance, can be expected that, the development in this field likely realizes personalized medicine.

At present, the common methods that target area (specific position) checked order is the Sanger sequencing, can carry out zone to target area by the Sanger sequencing detects, but the Sanger sequencing can only be checked order to a certain section zone of a sample at every turn, the order-checking cost is high, sensitivity low (20%), and can not draw the precise information morphed in each mutational site.

Therefore, need to a kind ofly can carry out to a plurality of target areas of testing sample the novel method that zone is detected simultaneously, the method can accurately obtain these regional sequence informations, the variation situation that comprises each mutational site of known mutations and unknown mutation, detection sensitivity is high, can also further to a large amount of samples, be detected simultaneously.

Summary of the invention

The object of the present invention is to provide a kind of method checked order in target area and test kit, can carry out zone to a plurality of target areas of testing sample detects simultaneously, accurately obtain these regional sequence informations, the variation situation that comprises each mutational site of known mutations and unknown mutation, detection sensitivity is high, can also further to a large amount of samples, be detected simultaneously.

The present invention is achieved in that a kind of method checked order in target area, comprises the following steps:

A. utilize Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

B. sequencing library is carried out to the unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

C. described a plurality of unit molecule amplified productions are carried out to the high-throughput gene sequencing simultaneously, obtain the sequence information of described a plurality of target areas.

Wherein, described steps A comprises the following steps:

A1. utilize Auele Specific Primer, increased in a plurality of target areas in testing sample, obtain the amplified production corresponding with described a plurality of target areas;

A2. utilize joint component, the amplified production corresponding with described a plurality of target areas connected, and obtains sequencing library; Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

Wherein, steps A 2 comprises the following steps:

A21. the amplified production corresponding with described a plurality of target areas carried out to fragmentation, obtain the fragmentation product;

A22. utilize joint component, be connected with the fragmentation product, build sequencing library.

The length of the described target area of steps A sequencing library, without particular restriction, is preferably 25bp～500bp.More preferably 50bp～200bp, more preferably 70bp～130bp.

Wherein, described steps A 22 comprises the following steps:

A221. utilize the first joint to be connected with the two ends of fragmentation product, obtain the first connection product;

A222. cyclisation first connects product, obtains cyclisation product;

A223.II s type digestion with restriction enzyme cyclisation product, obtain enzyme and cut product;

A224. cut the product two ends at enzyme and connect the second joint and the 3rd joint, obtain sequencing library.

Wherein, described steps A 22 comprises the following steps:

A221 '. utilize the 4th joint to be connected with the fragmentation product, obtain the second connection product;

A222 ' .II s type digestion with restriction enzyme second connects product, must be with the endonuclease bamhi of the 4th joint;

A223 '. the endonuclease bamhi with the 4th joint is connected with the 5th joint, forms sequencing library.

Wherein, at least one joint in the described joint component of steps A 2 includes the first sequence label, in the library construction process, the sequencing library of different testing samples is carried out to mark.Described the first sequence label, be preferably the nucleic acid molecule with specific base sequence, and its base number is not limit.

Further, described the first sequence label base number is 3～20, more preferably 4～10.

Wherein, the described Auele Specific Primer of steps A and target area complete complementary or part are complementary.

Further, in Auele Specific Primer corresponding to each target area, at least one primer and this target area part complementation, 5 ' end of the primer of this part complementation includes the second sequence label, for in amplification target area process, the target area amplified production of different testing samples is carried out to mark.Described the second sequence label, be preferably the nucleic acid molecule with specific base sequence, and its base number is not limit.

Further, described the second sequence label base number is 3～20, more preferably 4～10.

Wherein, the amplification of each target area of same sample in described steps A is carried out simultaneously or part is carried out simultaneously or independently carries out respectively.

Wherein, the method for the described unit molecule amplification of step B is at least one in emulsion-based PCR, bridge-type PCR.

Wherein, the described high throughput sequencing technologies of step C is synthetic sequencing based on polysaccharase or the connection sequencing based on ligase enzyme.

Of the present inventionly also provide a kind of test kit that can be used in any sequence measurement of the present invention, the present invention is achieved in that a kind of test kit checked order in target area, comprising:

Auele Specific Primer, increased for a plurality of target areas to testing sample;

Joint component, build sequencing library for being combined with amplified production.

Wherein, described joint component adopt flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

Wherein, at least one joint in described joint component includes the first sequence label, in the library construction process, the sequencing library of different testing samples is carried out to mark.Described the first sequence label is preferably the nucleic acid molecule with specific base sequence, and its base number is not limit, and is preferably 3～20.

Wherein, described Auele Specific Primer and target area complete complementary or part are complementary.

Further, in Auele Specific Primer corresponding to each target area, at least one primer and this target area part complementation, 5 ' end of the primer of this part complementation includes the second sequence label, for in amplification target area process, the target area amplified production of different testing samples is carried out to mark.Described the second sequence label is preferably the nucleic acid molecule with specific base sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～10.

Compared with prior art, method of the present invention and test kit carry out degree of depth order-checking by high throughput sequencing technologies to a plurality of target areas of testing sample simultaneously, accurately draw the sequence information of these target areas, the variation situation that comprises each mutational site of known mutations and unknown mutation, accurately draw the frequency morphed in each mutational site of each sample, detection sensitivity is high, and can further to a large amount of samples, carry out the multizone order-checking simultaneously.

The accompanying drawing explanation

Fig. 1 be in one embodiment of the invention checked order in target area method flow diagram;

Fig. 2 is the joint schematic diagram of the single protruding terminus joint in one embodiment of the invention;

Fig. 3 is the structural representation of the two protruding terminus joints in one embodiment of the invention;

Fig. 4 is the structural representation of the joint with loop-stem structure in one embodiment of the invention;

Fig. 5 is the structural representation of the y splice in one embodiment of the invention;

Fig. 6 is the structural representation of the T end y splice in one embodiment of the invention;

Fig. 7 is the structural representation of the y splice in another embodiment of the present invention;

Fig. 8 is the structural representation of the two deoxidation y splices in one embodiment of the invention;

Fig. 9 utilizes fragmentation product and joint component to build the method flow diagram of sequencing library in one embodiment of the invention;

Figure 10 utilizes fragmentation product and joint component to build the method flow diagram of sequencing library in another embodiment of the present invention;

Figure 11 utilizes fragmentation product and joint component to build the method flow diagram of sequencing library in another embodiment of the present invention.

Embodiment

In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.

Target area of the present invention, for the arbitrary sequence on any genome, can be selected as required, include but not limited to the internal sequence of gene, the regulation and control zone, outside of gene, the internal sequence of described gene, include but not limited to the intron zone, exon region of gene, the zone of simultaneously containing intron and exon.

Fig. 1 shows a kind of method flow checked order in target area of the present invention, and the method comprises the following steps:

S1. utilize Auele Specific Primer, increased in a plurality of target areas in testing sample, and build sequencing library based on amplified production;

S2. sequencing library is carried out to the unit molecule amplification, obtain a plurality of unit molecule amplified productions corresponding with described a plurality of target areas;

S3. described a plurality of unit molecule amplified productions are carried out to the high-throughput gene sequencing simultaneously, obtain the sequence information of described a plurality of target areas.

Present method is carried out degree of depth order-checking by high throughput sequencing technologies to the target area of testing sample, the method can be detected a plurality of target areas of testing sample simultaneously, accurately obtain these regional sequence informations, the variation situation that comprises each mutational site of known mutations and unknown mutation, detection sensitivity is high, can accurately draw the frequency morphed in each mutational site of each sample.In addition, by controlling the size of each target area amplified production, can remove the fragmentation step from, reduce experimental procedure, improve conventional efficient, reduce cost.

By the sequence information of present method gained, can be used for various scientific researches, include but not limited to crowd's sequential analysis, gene functional research, protein function research.

It should be noted that:

Testing sample described in step S1 is the sample that can extract the arbitrary form of nucleic acid, includes but not limited to: whole blood, serum, blood plasma and tissue sample; Described tissue sample includes but not limited to: paraffin-embedded tissue, flesh tissue and frozen section.

In step S1 gained sequencing library, there is multiple sequencing library molecule, sequencing library is carried out to the unit molecule amplification, refer to, by the multiple library molecule in sequencing library, form with denier (even unit molecule) is spatially isolated (but these library molecules still belong to same reaction system on the whole), and realizes amplification in space separately, to promote the signal of various molecules in follow-up sequencing reaction.

In prior art, the Sanger sequencing technologies can only be checked order to a certain section zone of a sample at every turn, realize the order-checking to a plurality of target areas, can only be by repeatedly reacting and realize.And in the present invention, each molecule in sequencing library is after the unit molecule amplification, each sequencing library molecule all forms unit molecule copy array, each unit molecule copy array when carrying out the high-throughput gene sequencing in different positions, make the hybridization between sequencing primer and unit molecule copy array, and the extension under the enzyme effect can carry out simultaneously, do not interfere with each other each other.Therefore, can carry out sequencing reaction to a large amount of (millions of up to ten million, even more) unit molecule copy arrays simultaneously simultaneously, then by gathering corresponding signal, and then obtain required sequence information, and the sensitivity of order-checking be higher than Sanger.

Wherein, the described Auele Specific Primer of step S1 and target area complete complementary or part are complementary.

Further, in every pair of primer for target area, at least one primer and target area part complementation, and 5 ' end of this primer is with the second sequence label.This second sequence label, in amplification target area process, carry out mark to the target area amplified production of different testing samples.

This second sequence label, be preferably the nucleic acid molecule with particular sequence, and its base number is not limit.The base number of this second sequence label is preferably 3～20, and more preferably 4～10.

In addition, described Auele Specific Primer also can, with other marker, include but not limited to: biotin labeling, poly histidine mark, antigen, antibody, thus make the purifying of target area amplified production very convenient.

In addition, the amplification in the different target zone of same testing sample can be carried out simultaneously or independently carry out respectively or part is carried out simultaneously.In concrete experimentation, can select as required above-mentioned any scheme to carry out.

If increased respectively in each target area respectively, can guarantee in step S1 to be consistent for the molecule number of the target area amplified production of establishing target zone sequencing library by the amount of measuring amplified production, can not cause because of amplification step the different target zone copy number difference of same testing sample, and then affect follow-up sequencing reaction result.

Certainly, when the size of each target area close, when GC content is also close, by rational design primer, utilize multiple PCR technique, step S1 can increase to a plurality of target areas simultaneously, and guarantees that the amplification efficiency between each target area keeps basically identical, so just can effectively improve conventional efficient, reduce the cost of reaction.

When testing sample has when a plurality of, the amplification of the target area of different samples must be carried out respectively.

In one embodiment, the specific implementation process of step S1 is:

S11. utilize Auele Specific Primer, increased in a plurality of target areas in testing sample, obtain the amplified production corresponding with described a plurality of target areas;

S12. utilize joint component, the amplified production corresponding with described a plurality of target areas connected, and obtains sequencing library; Described joint component adopts flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

It should be noted that:

In step S11, to the amplification of a plurality of target areas in same testing sample, can carry out simultaneously or independently carry out respectively or partly carry out simultaneously.Can be according to practical situation, as: the annealing temperature of specific amplification primer, the size of the target area of amplification, GC content, the quantity of the target area of amplification etc., adjust accordingly.The amplification of the target area of different testing samples must be carried out respectively.

In step S12, the mode of connection of described joint component and amplified production, can adopt various ways to realize, comprises that joint component directly is connected with amplified production, or connected after amplified production is processed again.

Joint component in step S12, for building sequencing library, can comprise one or more joints.

Wherein, at least one joint in the described joint component of step S12 includes the first sequence label, and this first sequence label in the library construction process, carries out mark to the sequencing library of different testing samples.Like this, after obtaining respectively the target area sequencing library, the target area sequencing library of different testing samples may be combined in same reaction system, carries out the unit molecule amplified reaction, and then carries out high-flux sequence simultaneously.Improve the efficiency of sequencing reaction, reduced the cost of sample detection.

This first sequence label is preferably the nucleic acid molecule with specific base sequence, and its base number is not limit.Further, the base number of described the first sequence label is 3～20, like this, and at every turn at least can be to 4 ³individual sample detects simultaneously.After considering various situations, as: the length of the specificity of label, the cost of joint, joint etc., the base number of the first sequence label is preferably 4～10.By the combination of the second label and the first label, invention of the present invention can detect at least 4 at every turn ³* 4 ³individual sample, i.e. 4096 samples.

The modification mode of joint has multiple, includes but not limited to: by biotinylation or methylate, or simultaneously by biotinylation with methylate.In one embodiment, this joint is by biotinylation, and is connected the separation and purification that has the sequencing library that is beneficial to structure of vitamin H with not biotinylated fragmentation product.In another embodiment, this joint is methylated, and be connected with unmethylated fragmentation product, then connect product with the digestion with restriction enzyme that only cuts methylate DNA, because only have the connection product successfully connected to be cut, so only have successfully the connector connected cut, thereby guarantee that enzyme cuts the unicity of product.

The structure formation of joint also has multiple, includes but not limited to: flat end fitting, protruding terminus joint, with joint and the y splice of loop-stem structure.Build in the sequencing library process and can use one or more joints.Wherein, the protruding terminus joint, all can effectively prevent that with the joint of loop-stem structure and y splice a plurality of joints are from the generation that connects phenomenon in connection procedure.Above-mentioned joint form for joint, below will provide a plurality of embodiment.

In the first embodiment, joint component adopts flat end fitting, and this joint is the nucleic acid molecule of double-stranded complete complementary.

In a second embodiment, joint component adopts the protruding terminus joint, and this joint is double chain acid molecule, and this double chain acid molecule at least comprises a protruding terminus.The base number of this protruding terminus, without concrete restriction, is preferably 1～10 base.According to the structure of this double chain acid molecule, the protruding terminus joint can be divided into two classes, is respectively single protruding terminus joint, two protruding terminus joint.

Single protruding terminus joint as shown in Figure 2, the one end is flat end, the other end is protruding terminus.Wherein the single protruding terminus joint with y splice can prevent that joint is from connecting.In order to prevent that single protruding terminus joint that an end is flat end from connecting certainly, can 3 ' OH on flat end be modified (including but not limited to amino sealing hydroxyl), maybe 5 ' phosphate group on flat end is removed.

Two protruding terminus joints as shown in Figure 3, it contains two protruding terminuses, and these two protruding terminuses can be on a nucleotide chain, and (Fig. 3 is a) or on different nucleotide chains (Fig. 3 b).When these two protruding terminuses are on different nucleic acid chains, they are not complementary each other, in case occur that when connecting joint is from connecting.

In the 3rd embodiment, joint component adopts the joint with loop-stem structure, as shown in Figure 4.This joint is single stranded nucleic acid molecule, this single stranded nucleic acid molecule comprises that (Fig. 4 a) for 1 ,Jing Huan district 2, the first complementary pairing district and the second complementary pairing district 3, the first complementary pairing district 1 can with the second complementary pairing district 3 complementary pairings, and the complementary pairing district that they form comprises at least one restriction enzyme enzyme recognition site, and cut recognition site by this enzyme, specific endonuclease capable Jiang Jinghuan cuts in district or excision, thereby single stranded nucleic acid molecule is become to double chain acid molecule, so that follow-up operation.As shown in Figure 4 b, also can be with protruding terminus 4 with the joint of loop-stem structure, this protruding terminus can be positioned at 5 ' end or the 3 ' end of single stranded nucleic acid molecule.The existence of protruding terminus 4 can further prevent that joint is from the generation that connects phenomenon.This protruding terminus is preferably T.

In the 4th embodiment, joint component adopts y splice, and as shown in Figure 5, this joint is double chain acid molecule, comprises collochore and crotch region, and two strands of described crotch region respectively comprise at least one amplimer binding site.Preferably, the collochore of described y splice comprises at least one restriction enzyme enzyme recognition site, and this enzyme is cut recognition site can cut the formation end by enzyme in building the storehouse process, so that carry out follow-up operation.

The bifurcation design of this y splice can avoid a plurality of joints from connecting the appearance of phenomenon building the storehouse process; The amplimer binding site comprised on described crotch region, can be directly used in conjunction with amplimer, carries out amplified reaction.

Wherein, every chain of the crotch region of described y splice contains N Nucleotide; Preferably, 9≤N≤30.Wherein, the Nucleotide logarithm of the collochore complementary pairing of described y splice is not limit; Preferably, the Nucleotide logarithm of complementary pairing is 7～15, preferred, and the Nucleotide logarithm of complementary pairing is 9～13.

Wherein, 3 ' end of the collochore of described y splice is protruding terminus or flat end.Preferably, 3 ' end of the collochore of described y splice is protruding terminus, this protruding terminus can with the sticky end complementary pairing of described fragmentation product, improved joint efficiency, be beneficial to build carrying out smoothly of sequencing library reaction.

Preferably, described y splice is T end y splice, and 3 ' end of the collochore of this joint is protruding terminus, and last base of protruding terminus is T; The T end y splice shown in Fig. 6 for example, in figure, N is any in A, T, C, G base.

Preferably, 3 ' end of the collochore of described y splice is protruding terminus, and the Nucleotide of protruding terminus comprises universal base.Wherein, the base number of protruding terminus, without particular restriction, is preferably 1～4.The y splice shown in Fig. 7 for example, in figure, N is any in A, T, C, G base, X is universal base.

Preferably, described y splice is two deoxidation joints, and 3 ' end of the collochore of this joint is flat end, and last Nucleotide of 3 ' end is the Nucleotide with two deoxidation bases; Two deoxidation y splices shown in Fig. 8 for example, in figure, N is any in A, T, C, G base, dd means that this last Nucleotide of 3 ' end is for the cytidylic acid(CMP) with two deoxidation bases.

It should be noted that above-mentioned joint component is part embodiment, not in order to limit the scope of the invention.

Implementation about step S12:

In one embodiment, step S12 adopts joint component and the direct-connected mode of amplified production, constructs sequencing library.

In another embodiment, when the target area amplified production is larger or the target area sequencing library need built hour, after amplified production being carried out to the fragmentation processing, the fragmentation product is connected with joint component again, build sequencing library, as shown in Figure 9, step S12 comprises the following steps:

S121. the amplified production corresponding with described a plurality of target areas carried out to fragmentation, obtain the fragmentation product;

S122. utilize joint component, be connected with the fragmentation product, build sequencing library.

By fragmentation step S121, the target area amplified production is become to less fragmentation product, thereby contribute to the further degree of depth order-checking to target area.In addition, can also process by fragmentation, different amplified productions is become to the similar fragmentation product of length, can contribute to follow-up unified order-checking.

It should be noted that:

In step S121, the method for described fragmentation target area amplified production has multiple, includes but not limited to: ultrasonic method, spray method, chemical shearing method and enzyme cutting method.Can, according to practical situation, adopt the method adapted to be tested.

Described fragmentation also can comprise the separation and purification of fragmentation product and end modified step after processing.According to the fragment length needs of order-checking, the nucleic acid fragment obtained for fragmentation, carry out the separation and purification of purpose nucleic acid fragment, and separation method can adopt common method, as gel electrophoresis, saccharose gradient or cesium chloride gradient sedimentation, column chromatography for separation etc.According to used fragmentation method, further end modified to the purpose nucleic acid fragment of gained, include but not limited to: phosphorylation or dephosphorylation, end-filling and end add A, so that follow-up joint component connects.Above-mentioned purpose nucleic acid fragment length is not limit, and is preferably 25bp～500bp, more preferably 30bp～200bp, more preferably 40～100bp.

The length of the described fragmentation product of step S121 is not limit, and is preferably 25bp～500bp, more preferably 30bp～200bp, more preferably 40bp～100bp.Under prerequisite in realization to the order-checking of target area, along with shortening of the target area fragment length contained in the sequencing library molecule of target area, the degree of depth of the order-checking to target area of high throughput sequencing technologies is deepened; And the order-checking degree of depth is darker, more to the order-checking number of times of each base position of target area, sequencing result is more accurate, just sensitiveer to the detection of a small amount of sudden change in sample; So just can effectively prevent because on the low side with the ratio of target area of sudden change in sample, and cause the absolute value of order-checking signal of this sudden change on the low side, the inaccurate phenomenon of sequencing result occurs.

In order to realize the restriction to target area sequencing library size, can, after step S121 or S122, to fragmentation product or target area sequencing library, carry out separation and purification.The method of separation and purification has multiple, includes but not limited to: gel method, saccharose gradient or cesium chloride gradient sedimentation and column chromatography for separation.Can, according to practical situation, adopt the method adapted to be tested.

According to above-mentioned joint component, for step S122, below will to this step, be further detailed by a plurality of embodiment and accompanying drawing.

In one embodiment of the invention, directly at the two ends of fragmentation product, connect joint and form sequencing library.

Described joint can adopt above-mentioned flat end fitting, protruding terminus joint, with the joint of loop-stem structure and at least one in y splice.

In another embodiment of the present invention, as shown in figure 10, step S122 specifically can be realized by following steps:

S1221. utilize the first joint to be connected with the two ends of fragmentation product, obtain the first connection product;

S1222. cyclisation first connects product, obtains cyclisation product;

S1223.II s type digestion with restriction enzyme cyclisation product, obtain enzyme and cut product;

S1224. cut the product two ends at enzyme and connect the second joint and the 3rd joint, obtain sequencing library.

In step S1221, described the first joint can adopt flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, and described the first joint includes II s type digestion with restriction enzyme recognition site, described II s type restriction enzyme is the restriction enzyme of cleavage site outside recognition sequence, include but not limited to: Acu I, Alw I, Bbs I, BbV I, Bcc I, BceA I, BciV I, BfuA I, Bmr I, Bpm I, BpuE I, Bsa I, BseM II, BseR I, Bsg I, BsmA I, BsmB I, BsmF I, BspCN I, BspM I, BspQ I, BtgZ I, Ear I, Eci I, EcoP15 I, Fau I, Fok I, Hga I, Hph I, HpyAV, Mbo II, Mly I, Mme I, Mnl I, NmeAIII, Ple I, Sap I, SfaN I and TspDT I, be preferably Acu I, Bsg I, EcoP15 I or Mme I.

When described the first joint is y splice, this II s type digestion with restriction enzyme recognition site is positioned at collochore; When described the first joint is the joint with loop-stem structure, the distance between restriction enzyme enzyme recognition site and loop-stem structure, than the near distance between II s type digestion with restriction enzyme recognition site and loop-stem structure.

If the fragmentation product is repaired through the end repair enzyme, and end adds the A reaction, and described the first joint is preferably the y splice with the T end.If the fragmentation product is just repaired through the end repair enzyme, by the end-filling of fragmentation product, preferably two deoxidation joints of described the first joint.

In step S1222, cyclisation first connects product multiple implementation.

In one embodiment of this invention, step S1222 comprises the following steps:

S12221. utilize enzyme to cut primer pair the first connection product and increased, obtain amplified production;

S12222. amplified production is carried out to enzyme and cut, make amplified production form sticky end, and self loop changes into cyclisation product.

3 ' the end that described enzyme is cut primer is connected respectively two terminal portions complementations of product with first, 5 ' end all contains the restriction enzyme enzyme recognition site.The amplified production that the amplification of process step S12221 forms, two end all contains the restriction enzyme enzyme recognition site, then, under the effect of corresponding enzyme, makes the two ends of amplified production form sticky end, and these two sticky end complementations, can carry out recirculation.

In another embodiment of the present invention, described the first joint includes 2 enzymes and cuts recognition site, one of them is the restriction enzyme enzyme recognition site, for making first of step S1222 formation connect the two ends of product under the effect of corresponding enzyme, form sticky end, and complementary between them, can carry out recirculation.Another is II s type digestion with restriction enzyme recognition site, at step S1223, utilizes the enzyme identification cyclisation product of this restriction enzyme site of identification, carries out enzyme and cuts, and then obtain enzyme and cut product.

Should illustrate, above two embodiment only realize that for two kinds in the present invention cyclisation first connects the embodiment of product, does not do any concrete restriction for protection scope of the present invention.

In step S1223, the enzyme that utilization can be identified on the first joint is cut recognition site, and cutting cyclisation product (DNA) but the enzyme that do not cut the first joint carries out enzyme cuts.Enzyme on described the first joint is cut recognition site and included but not limited to: Mme I enzyme cuts that recognition site, Acu I enzyme are cut recognition site, Bsg I enzyme is cut recognition site.

In step S1224, described the second joint can be flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, can be with biotin labeling.Described the 3rd joint can be flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice.The second joint and the 3rd joint can be identical or different.Preferably, described the second joint is identical with the 3rd joint, is y splice.Preferred, described y splice is T end y splice or two deoxidation y splice.

It should be noted that, between step S1222 and S1223, also can comprise step S1222A: the rolling circle amplification cyclisation product obtains the rolling circle amplification product.By step S1222A, can guarantee that follow-up enzyme cuts step S1223 enough starting material are arranged.

Or, also can comprise step S1221A between step S1221 and S1222: utilize amplimer to be connected product to first and increased, obtain amplified production.Described amplimer is connected respectively the joint sequence complementation at product two ends with first.By step S1221A, can guarantee that follow-up cyclisation step has enough starting material.

This programme can avoid carrying out rolling circle amplification after step S1222, and carry out common pcr amplification with the step S1221A before step S1222, replaces, and can effectively reduce follow-up enzyme to cut in step the consumption of II s type restriction enzyme.

Wherein, described the first joint is preferably y splice.

Wherein, the collochore of described y splice can comprise at least one enzyme and cuts recognition site.Described enzyme is cut recognition site and be can be common restriction enzyme enzyme recognition site, also can be II s type digestion with restriction enzyme recognition site.

Wherein, the described amplimer of step S1221A is preferably the biotinylation primer, is conducive to the recovery purifying of amplified production.

Wherein, the described amplimer of step S1221A is cut recognition site with at least one specific enzymes.

If on amplimer with specific enzymes to cut recognition site be the uridylic base, in step S1222, utilize uridylic specificity excision reagent to carry out enzyme and cut, and then connected cyclisation.

If it is the restriction enzyme enzyme recognition site that specific enzymes that amplimer is with is cut recognition site, in step S1222, utilizes corresponding restriction enzyme to carry out enzyme and cut, and then connected cyclisation.

In another embodiment of the present invention, as shown in figure 11, step S122 specifically can be realized by following steps:

S1221 '. utilize the 4th joint to be connected with the fragmentation product, obtain the second connection product;

S1222 ' .II s type digestion with restriction enzyme second connects product, must be with the endonuclease bamhi of the 4th joint;

S1223 '. the endonuclease bamhi with the 4th joint is connected with the 5th joint, forms sequencing library.

It should be noted that:

In step S1221 ', described the 4th joint can adopt flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, and described the 4th joint includes II s type digestion with restriction enzyme recognition site; When described the 4th joint is y splice, this II s type digestion with restriction enzyme recognition site is positioned at collochore; When described the 4th joint is the joint with loop-stem structure, the distance between restriction enzyme enzyme recognition site and loop-stem structure, than the near distance between II s type digestion with restriction enzyme recognition site and loop-stem structure.

In step S1222 ', the enzyme that utilization can be identified on the 4th joint is cut recognition site, and cutting cyclisation product (DNA) but the enzyme that do not cut the 4th joint carries out enzyme cuts.Enzyme on described the 4th joint is cut recognition site and included but not limited to: Mme I enzyme cuts that recognition site, Acu I enzyme are cut recognition site, Bsg I enzyme is cut recognition site.If the fragmentation product is repaired through the end repair enzyme, and end adds the A reaction, and described the 4th joint is preferably the y splice with the T end.If the just process end repair enzyme reparation of fragmentation product, the preferably two deoxidation joints of described the 4th joint.

In step S1223 ', described the 5th joint can be flat end fitting, protruding terminus joint, a kind of with in the joint of loop-stem structure and y splice, can be with biotin labeling.

In another embodiment of the present invention, step S122 specifically comprises the following steps:

S1221 ". directly at the two ends of fragmentation product, connect the joint with loop-stem structure, form the fragmentation product with loop-stem structure;

S1222 ". utilize restriction enzyme, the fragmentation product Jing Huan district with loop-stem structure is cut or excision, thereby form sequencing library.

The technical program utilization, with the joint of loop-stem structure, has prevented that a plurality of joints are from the generation that connects phenomenon.

Very few when the quantity of the sequencing library formed by above-mentioned several schemes, while being unfavorable for follow-up unit molecule amplification step, can also carry out following steps:

Utilize the primer with the shank complementation at sequencing library two ends, target area, take the sequencing library of gained as masterplate is increased, obtain the sequencing library after amplification.This step can meet the requirement of subsequent experimental to the amount of target sequencing library.

Wherein, the described unit molecule amplification of step S2 refers to the molecule in the sequencing library of target area, form with denier (even unit molecule) is spatially isolated (but these library molecules still belong to same reaction system on the whole), and realize amplification in space separately, to promote the signal of various molecules in follow-up sequencing reaction.The method of described unit molecule amplification includes but not limited to: emulsion-based PCR (Emulsion PCR, EPCR), bridge-type PCR.

The characteristics of described EPCR maximum are can form the huge independent reaction space of number to carry out DNA cloning.Primary process is that the aqueous solution that will comprise all reacted constituents of PCR is injected into the mineral oil surface of high speed rotating before PCR reaction, and aqueous solution moment forms the numerous little water droplet wrapped up by mineral oil.These little water droplets have just formed independently PCR reaction compartment.Under perfect condition, each little water droplet is only containing a DNA profiling (target area sequencing library molecule) and a magnetic bead, contain the primer complementary with the consensus sequence (being introduced by joint component) of target area sequencing library molecule on magnetic bead, after the PCR reaction, magnetic bead surfaces just is fixed with the DNA profiling amplified production in the same source of copy huge amount.The concrete steps of EPCR can be with reference to PCR amplification from single DNA molecules on magnetic beads in emulsion:application for high-throughput screening of transcription factor targets, Takaaki Kojima, Yoshiaki Takei, Miharu Ohtsuka et al, Nucleic Acids Research, 2005, Vol.33, No.17; Dual primer emulsion PCR for nextgeneration DNA sequencing, Ming Yan Xu, Anthony D.Aragon, Monica R.Mascarenas et al, BioTechniques 48:409-412 (May 2010); BEAMing:single-molecule PCR on microparticles in water-in-oil emulsions, Frank Diehl, Meng Li, Yiping He, nature methods, Vol.3, No.7, July 2006 etc.

The ultimate principle of described bridge-type PCR is, the primer of bridge-type PCR is fixed on solid phase carrier, in the PCR process, pcr amplification product can be fixed on solid phase carrier, and pcr amplification product can with solid phase carrier on the primer complementary pairing, Cheng Qiaozhuan, then the primer of complementary pairing be take and increased as template with the amplified production of its Cheng Qiao.The amount added by controlling original template, after bridge-type PCR react, amplified production form with cluster bunch on solid phase carrier exists, and the amplified production of every cluster be the DNA profiling amplified production of originating together.The principle that it is concrete and embodiment can be with reference to Publication about Document: CN20061009879.X, US6227604.

As previously mentioned, in prior art, the Sanger sequencing technologies, due to the technical limitation of self, can only be checked order to a certain section zone of a sample at every turn.For disposable realization, to detecting when a plurality of zone in sample, the present invention takes the high-throughput gene order surveying method on sequence measurement.It is more convenient sensitive that the relative Sanger sequencing of high-throughput gene sequencing detects sequence information, each molecule in sequencing library is after the unit molecule amplification, each sequencing library molecule all forms unit molecule copy array, each unit molecule copy array when carrying out the high-throughput gene sequencing in different positions, make the hybridization between sequencing primer and unit molecule copy array, and the extension under the enzyme effect can carry out simultaneously, do not interfere with each other each other.Therefore, can carry out sequencing reaction to a large amount of (millions of up to ten million, even more) unit molecule copy arrays, then by gathering corresponding signal simultaneously simultaneously, and then obtain accurately required sequence information, and the sensitivity of order-checking is higher than Sanger.Especially the amplified production of a plurality of target areas carried out to the fragmentation processing, being equivalent to has increased the order-checking number of times of each base of the target area molecule of identical sequence, can further improve the sensitivity of order-checking.

Wherein, the described high throughput sequencing technologies of step S3 includes but not limited to: the synthetic sequencing based on polysaccharase, the connection sequencing based on ligase enzyme.

Synthetic sequencing based on polysaccharase is based on band can remove that the Nucleotide of mark carries out.In each building-up reactions, each template strand can only extend once at the most, and the roughly flow process of the synthetic sequencing based on polysaccharase is as follows:

A. sequencing primer is combined in (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) on the total known array of unit molecule amplified production by complementary pairing, under the effect of archaeal dna polymerase, the Nucleotide that can remove mark with band carries out the single-basic extension building-up reactions, collect the marking signal that this time adds Nucleotide, can obtain the base sequence information with the next bit of the unit molecule amplified production (being fixed on primer-solid phase carrier mixture) of sequencing primer 3 ' least significant end base complementrity.

B. excision can be removed mark, then under the effect of archaeal dna polymerase, can remove the Nucleotide of mark with band and proceed the single-basic extension building-up reactions, collection adds the marking signal of Nucleotide, can obtain the base sequence information of lower two with the unit molecule amplified production of sequencing primer 3 ' terminal bases complementation.

Repeat the b step, until can not proceed building-up reactions, thus obtain the full sequence information of unit molecule amplified production.

Connection sequencing based on ligase enzyme all is based on is with fluorescently-labeled oligonucleotide probe to carry out.Wherein a kind of connection sequencing of ligase enzyme is based on that specific position carries out with fluorescently-labeled oligonucleotide probe, this oligonucleotide probe is with n base, a bit strip from its 5 ' terminal number has fluorescent mark, the specific fluorescent mark of different base pairs wherein, because 3 ' end of this oligonucleotide probe has carried out specific modification, between oligonucleotide probe, can not directly interconnect, each ligation, each unit molecule amplified production can only connect an oligonucleotide probe.This roughly flow process that connects sequencing is as follows:

A. sequencing primer is combined on the total known array of unit molecule amplified production on (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) by complementary pairing, utilize above-mentioned oligonucleotide probe, under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then gather fluorescent signal, can obtain a bit base sequence information after 3 ' end of the known array total with the unit molecule amplified production.

B. excise the fluorescent mark on oligonucleotide, under the effect of ligase enzyme, take above-mentioned oligonucleotide probe as raw material, proceed ligation, then gather fluorescent signal, thereby obtain the base sequence information of 2a position after 3 ' end of the total known array of unit molecule amplified production.

Repeat the B step, until can not proceed ligation, thereby obtain the base sequence information of a, 2a after 3 ' end of the total known array of unit molecule amplified production, 3a, 4a...... position.

Then sequencing primer and oligonucleotide probe sex change from the unit molecule amplified production of connecting thereof are eluted, use the primer of comparing the few base of 3 ' end with sequencing primer before instead and repeat above-mentioned reaction, thereby obtain the base sequence information of a-1,2a-1 after 3 ' end of the total known array of unit molecule amplified production, 3a-1,4a-1...... position.Repeat this step, finally obtain a-(a-1), 2a-(a-1) after 3 ' end of the total known array of unit molecule amplified production, 3a-(a-1), 4a-(a-1) ... the base sequence information of position, thereby obtain the full sequence information of single-stranded amplification product.

The connection sequencing of another kind of ligase enzyme equally also is based on fluorescently-labeled oligonucleotide probe carries out, this oligonucleotide probe is with n base, be divided into h (h≤n) group, the Different Alkali basic sequence of the corresponding same specific position of different fluorescent marks of same group of oligonucleotide probe, the difference between on the same group is not: the specific position difference that different fluorescent marks are corresponding, because 3 ' end of this oligonucleotide probe has carried out specific modification, between oligonucleotide probe, can not directly interconnect, each ligation, each unit molecule amplified production can only connect an oligonucleotide probe.This roughly flow process that connects sequencing is as follows:

A. sequencing primer is combined on the total known array of unit molecule amplified production on (this unit molecule amplified production is fixed on primer-solid phase carrier mixture) by complementary pairing, (the base position that fluorescent mark is corresponding is x to utilize in above-mentioned oligonucleotide probe one group, x≤h), under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then gather fluorescent signal, can obtain x bit base sequence information after 3 ' end of the known array total with single-stranded amplification product, sequencing primer and oligonucleotide probe sex change from the unit molecule amplified production of connecting thereof are eluted.

B. then again sequencing primer is combined on the unit molecule amplified production, (the base position that fluorescent mark is corresponding is y to use the oligonucleotide probe group different from a step instead, y≤h), under the effect of ligase enzyme, nucleic acid probe is connected with above-mentioned oligonucleotide chain, then gather fluorescent signal, can obtain y bit base sequence information after 3 ' end of the known array total with single-stranded amplification product, sequencing primer and oligonucleotide probe sex change from the unit molecule amplified production of connecting thereof are eluted.

C. repeating step b, until h group oligonucleotide probe carried out a ligation respectively, thereby obtain after 3 ' end of the total known array of unit molecule amplified production the 1st, the base sequence information of 2......, h position.

Use the primer of comparing 3 ' end many one or each universal base with sequencing primer before instead and reacted by above-mentioned principle, can extend the total known array of the unit molecule amplified production of acquisition 3 ' terminal bases sequence read long.

This connection order-checking ratio juris and specific embodiments based on ligase enzyme can be with reference to CN200710170507.1.

The exons 19,21 that detects the EGFR gene of take is example, the present invention proposes an embodiment, is designed for the Auele Specific Primer of amplification EGFR gene extron 19,21: 19F (SEQ ID NO:1), 19R (SEQID NO:2), 21F (SEQ ID NO:3), 21R (SEQ ID NO:4).

One, the extraction of testing sample DNA

Utilize nucleic acid extraction kit common on market to extract respectively the DNA of whole blood sample (1 to 10), serum sample (11 to 20), paraffin organization sample (21 to 30), and do respectively corresponding mark.

Two, the amplification of target area

The amplification of the EGFR gene extron 19,21 of same sample is carried out in same reaction system, and reaction system is as follows:

19F(10μM) 2μL；

19R(10μM) 2μL；

21F(10μM) 2μL；

21R(10μM) 2μL；

DNTP (each 2.5mM) 4 μ L;

Testing sample DNA 20ng;

Ex Taq(5U/μL) 0.25μL；

10×Ex Taq Buffer 5μL；

ddH ₂O up to 50μL。

The PCR reaction conditions is as follows:

95℃3min；

94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Repeat 25 circulations;

72℃7min。

Utilize PCR cleaning agents box, respectively the amplified production of each sample cleaned, remove primer and the dNTP of not amplification, reclaim amplified production.

Three, establishing target zone sequencing library

This step can have multiple embodiments, in one embodiment of the present of invention, comprises following two steps:

1. the fragmentation of amplified production

Utilize ultrasonic method to carry out the fragmentation processing.Concrete operations are: the amplified production (approximately 50 μ L) after the clean recovery of the PCR of each sample are added in the TE buffer of 400 μ L, and ultrasonic 4s under the 430W power condition, interval 20s, 5 times repeatedly, obtain the fragmentation product.Utilize 1% sepharose to carry out separation and purification to the fragmentation product of each sample, cut glue and reclaim the fragmentation product of size between 40bp to 100bp.

2. structure sequencing library

Before the sequencing library of establishing target zone, need to cut fragmentation product that glue reclaims carry out respectively end modified so that the connection of joint component.In the present embodiment, the end modified of fragmentation product that cuts the glue recovery comprised to phosphorylation, end-filling and end add the A reaction.

Be implemented as follows:

1) phosphorylation and end-filling reaction

System is:

Cut the fragmentation product 20 μ L (about 2000ng) that glue reclaims;

10mM dNTP 1.5μL；

T4DNA polysaccharase (5U/ μ L) 1 μ L;

Klenow archaeal dna polymerase 0.1 μ L;

T4 polynucleotide kinase (10U/ μ L) 0.5 μ L;

10m MATP 1.5μL；

10 * T4 ligase enzyme damping fluid, 10 μ L;

Add ddH ₂o to 100 μ L.

Reaction conditions is: hatch 20min for 20 ℃.After finishing, reaction utilize the recovery test kit to carry out the purifying recovery.

2) end adds the A tail

Reaction system is:

Recovery product 60 μ L (about 1000ng) after phosphorylation and end-filling;

Klenow damping fluid (NEB Buffer2) 10 μ L;

10mM dATP 2μL；

Klenow enzyme (3 ' to 5 ' exo minus, 10U/ μ L) 1 μ L;

Add ddH ₂o to 100 μ L.

Reaction conditions is: hatch 30min for 37 ℃.Reaction utilizes the purification kit purifying to reclaim after finishing.

3) jointing 1

In the present embodiment, employing T end y splice as shown in Figure 6 is as joint 1, and the T end y splice used of same sample is identical, the T end y splice difference of different samples, difference is the sequence label difference, and the sequence label that different samples are corresponding is as shown in the table.

Under the effect of T4 ligase enzyme, above-mentioned T end y splice respectively with add the A tail after the product that reclaims of purifying be connected, form the fragment of belt lacing 1.Linked system is:

Add the product 50 μ L (about 500ng) that purifying reclaims after the A tail;

Joint 12 μ L (about 3000ng);

10mM ATP 5μL；

T4DNA ligase enzyme (10U/ μ L) 1 μ L;

10 * T4 ligase enzyme damping fluid, 10 μ L;

Add ddH ₂o to 100 μ L.

Reaction conditions is: 16 ℃ hatch 4h more than.Reaction utilizes the purification kit purifying to reclaim after finishing.

4) fragment of pcr amplification belt lacing 1

Amplification system is:

The about 300ng of the fragment of belt lacing 1;

10×Ex Taq Buffer 100μL；

Primer F(100μM，SEQ ID NO：11) 10μL；

Primer R(100μM，SEQ ID NO：12) 10μL；

Ex Taq(5U/μL) 7.5μL；

ddH ₂O up to 1000μL。

The PCR reaction conditions is as follows:

95℃3min；

94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 30s; Repeat 25 circulations;

72℃7min。

Utilize PCR cleaning agents box, respectively the amplified production of each sample cleaned, remove primer and the dNTP of not amplification, reclaim the amplified production of the fragment of belt lacing 1.

5) II s type digestion with restriction enzyme

Utilize the amplified production of the fragment of the belt lacing 1 after Acu I enzyme cuts back to close, reaction system is as follows:

The amplified production 60 μ L (3～5 μ g) of the fragment of the belt lacing 1 after recovery;

10×NEB Buffer 4 8μL；

Acu I(NEB) 2μL(10U)；

SAM (3.2mM) 1 μ L (final concentration 50 μ M);

ddH ₂O up to 80μL。

Reaction conditions: hatch 1h for 37 ℃.Reaction utilizes the purification kit purifying to reclaim enzyme and cuts product after finishing.

6) jointing 2

Utilization protruding terminus y splice as shown in Figure 7, as joint 2, is cut product with the enzyme reclaimed and is connected, and obtains sequencing library, and linked system is as follows:

The enzyme reclaimed is cut product 2 μ g;

Joint 21 μ L (10pM);

10T4DNA ligase enzyme (3U/ μ L) 1 μ L;

10 * T4 ligase enzyme damping fluid, 2 μ L;

Add ddH ₂o to 100 μ L.

Condition of contact, hatch 2h for 14 ℃.Reaction utilizes the purification kit purifying to reclaim after finishing, then sex change forms strand, obtains sequencing library.

Four, the target area sequencing library is carried out to the unit molecule amplification

Measure (three) step and obtain 30 target area sequencing libraries concentration separately, then, by mixing with isoconcentration, then carry out the unit molecule amplification, obtain the unit molecule amplified production, the method for described unit molecule amplification can adopt EPCR or bridge-type PCR.

Be preferably the EPCR amplification, the unit molecule amplimer is preferably: SEQ ID NO:5 and SEQ ID NO:6.

Five, the unit molecule amplified production is carried out to high-flux sequence

Described sequence measurement can adopt the synthetic sequencing based on synthetic enzyme, also can adopt the connection sequencing based on ligase enzyme.Sequencing result is carried out to bioinformatic analysis, can obtain the exons 19 of EGFR gene of above-mentioned 30 samples and 21 sequence information.The analytical sequence result is known, and except the exons 19 of the EGFR gene of sample 23 is undergone mutation (sequence information is shown in SEQ ID NO:8, and mutation rate is 6.2%), each exon of other sample is wild-type.

It should be noted that the present embodiment is a specific embodiment of the present invention, to the present invention, without any restriction effect, for example the Auele Specific Primer of EGFR gene can be replaced with other the primer of process appropriate design; In addition, a plurality of steps in the present embodiment all can be replaced with reference to aforesaid method, do not repeat them here.

For the sensitivity of the method checked order in target area of the present invention, the present invention adopts following embodiment to be verified.

By conventional design, build the plasmid that contains respectively EGFR gene extron 19 wild-type sequences (SEQ IDNO:7) and saltant type sequence (SEQ ID NO:8); The plasmid that contains EGFR gene extron 21 wild-type sequences (SEQ ID NO:9) and saltant type sequence (SEQ ID NO:10).

Then prepare mutation rate and be respectively 20%, 10%, 5%, 3%, 1%, 0% plasmid mixed solution.The above-mentioned plasmid mixed solution that contains 1000 copy plasmids of take is template, and then the method by reference above-described embodiment is detected.Detected result is as shown in the table:

Result shows, the lowest detection of the method checked order in target area of the present invention is limited to 3% left and right, 5% and more than detected result and practical situation basically identical.

A kind of test kit checked order in target area comprises:

This test kit is for the target area order-checking, can carry out degree of depth order-checking to a plurality of target areas of sample simultaneously, accurately draw the sequence information of these target areas, the variation situation that comprises each mutational site of known mutations and position sudden change, accurately draw the frequency morphed in each mutational site of each sample, detection sensitivity is high, can, for the large-scale crowd examination of target area, determine the ratio of the range gene type of this target area.

Wherein, described joint component is for establishing target zone sequencing library, comprises at least one or multiple joint.

Wherein, described joint component can adopt various ways, as: flat end fitting, protruding terminus joint, the joint with loop-stem structure, at least one in y splice.

Wherein, described joint can be methylated or biotinylation, or simultaneously by biotinylation with methylate.

Wherein, at least one joint in described joint component includes the first sequence label, in the library construction process, the sequencing library of different testing samples is carried out to mark.Described the first sequence label is preferably the nucleic acid molecule with specific base sequence, and its base number is not limit, and is preferably 3～20, and more preferably 4～20.

Wherein, described Auele Specific Primer and target area are complementary or partly complementary.

Further, in Auele Specific Primer corresponding to each target area, at least one primer and this target area part complementation, 5 ' end of this primer includes the second sequence label, for in amplification target area process, the target area amplified production of different testing samples is carried out to mark.Described the second sequence label is preferably the nucleic acid molecule with specific base sequence, and its base length is not limit.

Further, the base number of described the second sequence label is 3～20, more preferably 4～10.

Wherein, described test kit can also comprise pcr amplification reagent, and described pcr amplification reagent comprises PCR enzyme, dNTP, PCR damping fluid, Mg ²⁺solution.

Wherein, described test kit also can comprise the enzyme of cutting recognition site for identifying enzyme on joint component.

It should be noted that by method of the present invention or test kit and detect the gene order information obtained, can be used for various scientific researches, include but not limited to crowd's sequential analysis, gene functional research, protein function research.For example, in conjunction with statistical study of follow-up further molecular biology test, clinical trial, clinical observation and integrated data etc., thereby realize multiple scientific research purpose, include but not limited to: the genotype distribution of some specific gene of certain specific crowd, the mutation type of each gene and the relation of relevant physiological activity etc.

The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.

Claims

1. the method checked order in target area, is characterized in that, comprises the following steps:

C. described a plurality of unit molecule amplified productions are carried out to the high-throughput gene sequencing simultaneously, obtain the sequence information of described a plurality of target areas;

Described steps A comprises the following steps:

A2. utilize joint component, the amplified production corresponding with described a plurality of target areas connected, and obtains sequencing library;

Described joint component comprises at least one y splice; Described y splice is double chain acid molecule, comprises collochore and crotch region; Two strands of described crotch region respectively comprise at least one primer binding site; Described collochore comprises at least one restriction enzyme enzyme recognition site, and 3 ' end of described collochore is flat end or protruding terminus; When 3 ' end of described collochore is flat end, last Nucleotide of flat end is the Nucleotide with two deoxidation bases;

At least one joint in described joint component includes the first sequence label, in the library construction process, the sequencing library of different testing samples is carried out to mark;

In Auele Specific Primer corresponding to each target area, have a primer and this target area part complementation at least, 5 ' end of the primer of this part complementation includes the second sequence label, in amplification target area process, the target area amplified production of different testing samples is carried out to mark.

2. the method checked order in target area according to claim 1, is characterized in that, in described y splice, 3 ' end of the collochore of at least one y splice is protruding terminus, and this protruding terminus is T.

3. the method checked order in target area according to claim 1, is characterized in that, described steps A 2 comprises the following steps:

A22. utilize joint component, be connected with described fragmentation product, build sequencing library.

4. the method checked order in target area according to claim 3, is characterized in that, described steps A 22 comprises the following steps:

A222. cyclisation first connects product, obtains cyclisation product;

A223. II s type digestion with restriction enzyme cyclisation product, obtain enzyme and cut product;

5. the method checked order in target area according to claim 3, is characterized in that, described steps A 22 comprises the following steps:

A222 '. II s type digestion with restriction enzyme second connects product, must be with the endonuclease bamhi of the 4th joint;

6. according to the described method checked order in target area of any one in claim 1 to 5, it is characterized in that, described Auele Specific Primer is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

7. the method checked order in target area according to claim 5, it is characterized in that, described the 4th joint is the y splice be comprised of SEQ ID NO:13 and SEQ ID NO:14, reverse complemental between 5 '-NNNN-3 ' in 5 '-NNNN-3 ' in SEQ ID NO:13 and SEQ ID NO:14, described the 5th joint is the y splice be comprised of SEQ ID NO:15 and SEQ ID NO:16, reverse complemental between the 5 '-NNN-3 ' in the 5 '-NNN-3 ' in SEQ ID NO:15 and SEQ ID NO:16.

8. the method checked order in target area according to claim 5, is characterized in that, the primer that the amplification of unit molecule described in step B is used is SEQ ID NO:5 and SEQ ID NO:6.

9. the test kit checked order in target area, is characterized in that, comprising:

Joint component, build sequencing library for being combined with amplified production;

Described joint component comprises at least one y splice; Described y splice is from double chain acid molecule, comprises collochore and crotch region; Two strands of described crotch region respectively comprise at least one primer binding site; Described collochore comprises at least one restriction enzyme enzyme recognition site, and 3 ' end of described collochore is flat end or protruding terminus; When 3 ' end of described collochore is flat end, last Nucleotide of flat end is the Nucleotide with two deoxidation bases;

10. the test kit checked order in target area according to claim 9, is characterized in that, in described y splice, 3 ' end of the collochore of at least one y splice is protruding terminus, and this protruding terminus is T.

11. according to claim 9, the 10 described test kits that checked order in target area, it is characterized in that, described Auele Specific Primer is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

12. according to claim 9, the 10 described test kits that checked order in target area, it is characterized in that, described joint component comprises the 4th joint and the 5th joint, described the 4th joint is the y splice be comprised of SEQ ID NO:13 and SEQ ID NO:14, reverse complemental between 5 '-NNNN-3 ' in 5 '-NNNN-3 ' in SEQ ID NO:13 and SEQ ID NO:14, described the 5th joint is the y splice be comprised of SEQ ID NO:15 and SEQ ID NO:16, reverse complemental between 5 '-NNN-3 ' in 5 '-NNN-3 ' in SEQ ID NO:15 and SEQ ID NO:16.

13., according to claim 9, the 10 described test kits that checked order in target area, it is characterized in that, also comprise the unit molecule amplimer, described unit molecule amplimer is SEQ ID NO:5 and SEQ ID NO:6.