IE83464B1 - Process for amplifying and detecting nucleic acid sequences - Google Patents

Abstract

ABSTRACT The present invention provides a process for detecting the presence or absence of at least one specific nucleic acid sequence in a sample or distinguishing between two different nucleic acid sequences in said sample, wherein each nucleic acid sequence to be detected consists of two separate strands, which process comprises amplifying the specific sequence or sequences (if present) by (a) treating the sample with one oligonucleotide primer for each of the two strands of each different specific nucleic acid sequence being detected under hybridizing conditions such that for each strand of each different sequence being detected an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be substantially complementary to each strand of each specific sequence such that the extension product synthesized from one primer, when it is separated from its complement, serves as a template for synthesis of an extension product of the other primer; (b) treating the product of step (a) under denaturing conditions to separate the primer extension products from their templates; (c) treating the product of step (b) with oligonucleotide primers such that a primer extension product is synthesized using each of the single strands produced in step (b) as a template; and detecting the thus amplified sequence or sequences (if present). PROCESS FOR AMPLIFYING AND DETECTING NUCLEIC ACID SEQUENCES E930228 wmo weuc mspeénou unoeasecnon zamcnugga inane .... ..l.:‘1.fLS

Description

PATENTS ACT 1992 930226 PROCESS FOR AMPLIFYING AND DETECTING NUCLEIC ACID SEQUENCES F. HOFFMANN—LA ROCHE AG The present invention relates to sing|e—stranded oligonucleotides allowing amplification of existing nucleic acid sequences if they are present in a test sample and detecting them if present by using a probe. More specifically, said oligonucleotides allow the production of any particular nucleic acid sequence from a given sequence of DNA or RNA in amounts which are large compared to the amount initially present so as to facilitate detection of the sequences. The DNA or RNA may be single- or double- stranded, and may be a relatively pure species or a component of a mixture of nucleic adds.The ohgonucbofides oftheinvenfiontnflme arepefifiveieacflonto acconuflbh the amplification of the desired nucleic acid sequence. the nucleic acid sequence may be only a small portion of the DNA or RNA in so that it may be difficult to detect nonisotopically labeled or end-labeled oligonucleotide probes.

For diagnostic applications in particular, target its presence using Much effort is being expended in increasing the sensitivity of the probe but little amplifying the target sequence so that it is question, detection systems, research has been conducted on present in quantities sufficient to be readily detectable using currently available methods.

Several methods have been described in the literature for the synthesis of nucleic acids de novo or from an existing sequence.

These methods are capable of producing large amounts of a given nucleic acid of completely specified sequence.

One known method for synthesizing nucleic acids de novo involves the organic of a nucleic acid from nucleoside This One type of organic synthesis is the phosphotriester synthesis derivatives. synthesis may be performed in solution or on a solid support. method, which has been utilized to prepare gene fragments -or short genes. In the phosphotriester method, oligonucleotides are prepared . which can then be joined together to form longer nucleic acids. For a description of this method, see Narang, S.A., et al., Meth. Enzymol., £33, 90 (1979) and U.S. Patent No. 4,356,270. The patent describes the synthesis and cloning of the somatostatin gene. together to form the desired nucleic acid.

Although the above processes for de novo synthesis may be utilized to synthesize long strands of nucleic acid, they are not very practical to use for the synthesis of large amounts of a nucleic Both expensive equipment and reagents, and have a low overall efficiency. acid. processes are laborious and time-consuming, require The low overall efficiency may be caused by the inefficiencies of the synthesis of the oligonucleotides and of the joining reactions. In the synthesis of a long nucleic acid, or even in the synthesis of a large amount of a shorter nucleic acid, many oligonucleotides would need to be synthesized and many joining reactions would be required.

Consequently, these methods would not be practical for synthesizing large amounts of any desired nucleic acid.

Methods also exist for producing nucleic acids in large amounts from of the These methods involve the cloning of a nucleic acid in the appropriate host appropriate vector which is used to transform the host. small amounts initial existing nucleic acid. inserted into an when the host is cultured the vector is replicated, and hence more copies of the brief description of system, where the desired nucleic acid is desired nucleic acid are produced. For a subcloning nucleic acid fragments, see Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, pp. 390- 401 (1982). See also the techniques described in U.S. Patent Nos. 4,416,988 and 4,403,036.

A third nethod for synthesizing nucleic acids, described in U.S. Patent No. 4,293,652, is a hybrid of the above-described organic synthesis and molecular cloning methods. In this process, the appropriate number of oligonucleotides to make up the desired nucleic acid sequence is organically synthesized and inserted sequentially into a vector which is amplified by growth prior to each succeeding insertion.

The present invention bears some similarity to the molecular cloning method; however, it does not involve the propagation of any organism and thereby avoids‘ the possible hazards or inconvenience which this entails. invention also does not require synthesis of nucleic acid sequences unrelated to the desired sequence, The present and thereby the present invention obviates the need for extensive purification of the product from a complicated biological mixture.

In J.Mol.Biol, Kleppe et al (1971), 341-361, discuss primer extension reactions using templates corresponding to portions of a tRNA gene, in which reactions the primers are used are complementary to substantial parts of corresponding templates and are extended therealong thereby to provide duplex DNAS.

These template copying reactions, involving simple primer extension, are termed "repair replication" by the authors. The final paragraph of the article theorises that if duplex DNA denaturation is effected in the presence of appropriate primers, two structures consisting of a full length template strand complexed to a primer could be produced upon cooling, and repair replication achieved by adding DNA polymerase. - The paragraph suggests that this process could be repeated.

However, there is no detailed explanation of the precise techniques to be carried out, nor any discussion of which primers are "appropriate", and the possibility of a problem of template renaturation (reforming a duplex) is discussed with the suggestion that, if necessary, strand separation would have to be resorted to with subsequent repair replication.

The present invention relates to single—stranded oligonucleotides allowing amplification of one or more specific nucleic acid sequences present in a nucleic acid or mixture thereof using agents for polymerization and then detecting the amplified sequence. The extension product of one oligonucleotides (primer) when hybridized to the other becomes a template for the production of the desired specific nucleic acid sequence, and vice versa, and the process is repeated as often as is necessary to produce the desired amount of the sequence. The method carried out using the oligonucleotides of the invention is expected to be more efficient than the methods described above for producing large amounts of nucleic acid from a target sequence and to produce such nucleic acid in a comparatively short period of time. The method carried out using the oligonucleotides of the invention is especially useful for amplifying rare species of nucleic acid present in a mixture of nucleic acids or effective detection of such species.

More specifically, the present invention provides a first and second single—stranded oligonucleotide allowing amplification of a specific template nucleic acid sequence contained in a single- or double- stranded nucleic acid or in a mixture of such nucleic acids, wherein (a) one oligonucleotide of said oligonucleotides contains a part which is substantially complementary to said template nucleic acid sequence in said single—stranded nucleic acid or in one strand of said double—stranded nucleic acid; (b) the other oligonucleotide of said oligonucleotides contains a part which is substantially complementary to a complement of said template nucleic acid sequence in said single—stranded nucleic acid or in said strand of said double—stranded nucleic acid; (c) said parts of oligonucleotides (a) and (b) have attached to their 5'—end a nucleotide sequence which is non—comp|ementary to said template nucleic acid sequence and which comprises a restriction site; and wherein (d) the parts of said oligonucleotides of (a) and (b) that have substantial complementarity are different and define the termini of the specific template nucleic acid sequence to be amplified.

Preferred features of the invention are set forth in claims 2 to 11.

Irish Patent No. |E62098 (842/86) discloses processes for amplifying nucleic acid sequences, and this Application is divided from Patent Application No. 843/86.

Figure 1 illustrates a 94 base pair length sequence of human B-globin desired to be amplified. The single base pair change which is associated with sickle cell anemia is depicted beneath the 94-mer.

Figure 2 illustrates a photograph of an ethidium bromide- stained polyacrylamide gel demonstrating amplification of the 94-mer contained in human wild-type DNA and in a plasmid containing a 1.9 kb fianml fragment of the normal B-globin gene (designated pBR328:HbA).

Figure 3 illustrates a photograph of an ethidium bromide- stained polyacrylamide gel demonstrating amplification of any of the specific target 94-mer pBR328:HbA, a containing a 1.9 kb gamfll fragment of the sickle cell allele of B- globin (designated pBR328:HbS), pBR328:HbA where the sequence to be amplified is cleaved with_flstII, and pBR328:HbS where the sequence to sequence present in plasmid be amplified has been treated but not cleaved with Mstll.

Figure 4 illustrates in detail the steps and products of the polymerase chain reaction for amplification of the desired 94-mer sequence of human 5-globin for three cycles using two oligonucleotide primers.

Figure 5 represents a photograph of an ethidium bromide- stained polyacrylamide gel demonstrating amplification after four cycles of a 240-mer sequence in pBR328:HbA, where the aliquots are digested with 3&2} (Lane 3), figtil (Lane 4) or fiigfl (Lane 5). Lane 1 is the molecular weight standard and Lane 2 contains the intact 240-bp product.

Figure 6 illustrates the sequence of the normal (sA) and sickle cell (55) p-globin genes in the region of the Ddel and HinfI restriction sites, where the single lines for BA mark the position of the Ddel site (CTGAG) and the ‘double bars for 5A and 53 mark the position of the HinfI site (GACTC).

Figure 7 illustrates the results of sequential digestion of normal B-globin using a 40-mer probe and Ddel followed by Hinfl restriction enzymes.

Figure 8 illustrates the results of sequential digestion of sickle B-globin using the same 40-mer probe as in Figure 7 and Ddel followed by Hinfl restriction enzymes.

Figure 9_illustrates a photograph of an ethidium bromide- stained polyacrylamide gel demonstrating the use of the same 40-mer probe as in Figure 7 to specifically characterize the beta-globin DNA which have been with the alleles present in samples of whole human subjected to amplification, hybridization probe, and sequential digestion with Ddel and Hinfl.

Figure 10 illustrates a photograph of a 6% Nusieve agarose gel visualized using ethidium bromide and UV light. This photograph demonstrates amplification of a sub-fragment of a 110-bp amplification product which sub-fragment is an inner nested set within the 110-bp fragment.

The term "oligonucleotide" as used herein in referring to primers, probes, oligomer fragments to be detected, oligomer controls and unlabeled blocking oligomers is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide.

The term "primer" or "oligonucieotides primer" as used herein refers to an oligonucleotide according to the invention whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, i.e., -in the presence of nucleotides and an agent for polymerization such as DNA polymerase and at a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may alternatively be double stranded. If double stranded, the primer is first treated to separate its strands before being used to Preferably, the primer is an products.

The primer must be sufficiently long to prepare extension oligodeoxyribonucleotide. prime the synthesis of extension products in the presence of the agent for polymerization. The exact lengths of the primers will depend on including temperature and source of primer. For the complexity of the target sequence, the many factors, example, depending on oligonucleotide primer typically contains 15-25 or more nucleotides, although generally Short primer molecules stable it may contain fewer nucleotides. require cooler temperatures to form sufficiently hybrid complexes with template.

The primers herein are selected to be "substantially" complementary to the different strands of each specific sequence to be amplified. This that the complementary to hybridize with their respective strands. means primers must be sufficiently Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non—complementary nucleotide fragment may be attached to the 5' end of the primer, with the remainder of the primer complementary to the strand. Alternatively, non- sequence being complementary bases or longer sequences can be interspersed into the primer, provided that the complementarity with the sequence of the strand to be amplified to hybridize therewith and thereby form a template for synthesis of the extension product of the other primer. primer sequence has sufficient As used herein, the terms "restriction endonucleases", and "restriction enzymes" refer to bacterial enzymes each of which cut double-stranded DNA at or near a specific nucleotide sequence.

As used herein, the term "DNA polymorphism" refers to the condition in which two or more different nucleotide sequences can exist at a particular site in DNA.

The term "restriction fragment length polymorphism" ("RFLP") refers to the differences among individuals in the lengths of restriction fragments formed by digestion with a particular restriction endonuclease.

The present invention is directed to a first and second single-stranded oligonucleotide allowing amplification of any one or more desired specific nucleic acid sequences suspected of being in a nucleic acid. Because large amounts of a specific sequence may be produced by this process, the present invention may be used for improving the efficiency of cloning DNA or messenger RNA and for amplifying a target sequence to facilitate detection thereof.

In general, the process which can be performed using the oligonucleotides of the present invention involves a chain reaction for producing, in exponential quantities relative to the number of reaction steps involved, at least one specific nucleic acid sequence given (a) that the ends of the required sequence are known in sufficient detail that oligonucleotides can be synthesized which will hybridize to them, and (b) that a small amount of the sequence is available to initiate the chain reaction. The product of the chain reaction will be a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed.

Any source of nucleic acid, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it is suspected of containing the specific nucleic acid sequence DNA or RNA, including messenger RNA, which DNA or RNA may be single stranded or double stranded. a DNA-RNA hybrid which contains one desired. Thus, the process may employ, for example, In addition, strand of each may be utilized. A mixture of any of these nucleic acids may also be employed, or the nucleic acids produced from a previous amplification reaction herein using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified may be only a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; -11.. it may be a minor fraction of a complex mixture, such as a portion of the p-globin gene contained in whole human DNA or a portion of nucleic acid sequence due to a particular microorganism which organism might constitute only a very minor fraction of a particular biological sample. The starting nucleic acid may contain more than one desired specific nucleic acid sequence which may be the same or different.

Therefore, the present oligonucleotides are useful not only for producing large amounts of one specific nucleic acid sequence, but also for amplifying simultaneously more than one different specific nucleic acid sequence located on the same or different nucleic acid molecules.

The nucleic acid or acids may be obtained from any source, for example, from plasmids such as pBR322, from cloned DNA or RNA, or from natural DNA or RNA from any source, including bacteria, yeast, DNA or RNA may be extracted from blood, tissue material such as chorionic villi viruses, and higher organisms such as plants or animals. or amniotic cells by a variety of techniques such as that described by Maniatis et al., Molecular Cloning (1982), 280-281.

Any specific nucleic acid sequence can be produced by the present oligonucleotides. It is only necessary that a sufficient number of bases at both ends of the sequence be known in sufficient detail so that two oligonucleotide primers can be prepared which will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer, when it is separated from its template (complement), can serve as a template for extension of the other primer into a nucleic acid of defined length. of the primers for the target nucleic acid sequence, and thus the greater the It will be understood that the word primer The greater the knowledge about the bases at both ends sequence, the greater can be the specificity of the efficiency of the process. as used hereinafter may refer to more than one primer, particularly in the case where there is some ambiguity in the information regarding the terminal sequence(s) of the fragment to be amplified. For instance, in the case where a nucleic acid sequence is_1'nferred from protein sequence information a collection of primers containing sequences representing all possible codon variations based on _12_ degeneracy of the genetic code will be used for each strand. One primer from this collection will be homologous with the end of the desired sequence to be amplified.

The oligonucleotide primers may be prepared using any suitable method, such as, for example, the phosphotriester and phosphodiester methods described above, or automated embodiments thereof. In one such automated embodiment diethylphosphoramidites are used as starting materials and may be synthesized as described by Beaucage et al., Tetrahedron Letters (1981), 22:1859-1862. One method for synthesizing oligonucleotides on a modified solid support is described in U.S. ?atent No. 4,458,066. It is also possible to use a isolated frmn a biological source (such as a primer which has been restriction endonuclease digest).

The specific nucleic acid sequence is produced by using the nucleic acid containing that sequence as a template. If the nucleic acid contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template, either as a simultaneously with the synthesis of the primer separate step or extension products. This strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic One physical method of separating the strands of the nucleic it is completely (>99%) means. acid involves heating the nucleic acid.until denatured. Typical heat denaturation may involve temperatures ranging from about 80 to 105°C for times ranging from about 1 to 10 minutes.

Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or the enzyme RecA, which has helicase activity and in the presence of riboATP is known to denature DNA. The reaction conditions suitable for separating the strands of nucleic reviewed in C. Radding, Ann. Rev. Genetics, }Ji:405-37 (1982).

If the original nucleic acid containing the sequence to be amplified is single stranded, its complement is synthesized by adding one or two oligonucleotide primers thereto. If an appropriate single CSH- -13.. in the an agent for polymerization and the four nucleotides below. The will be complementary to the single-stranded nucleic acid and will hybridize length is added, a primer extension product is synthesized presence of the primer, primer described product partially with the nucleic acid strand to form a duplex of unequal strands that may then be separated into single strands as described above to produce two single separated complementary strands.

Alternatively, two appropriate primers may be added to the single- stranded nucleic acid and the reaction carried out.

If the original nucleic acid constitutes the sequence to be amplified, the primer extension product(s) produced will be completely complementary to the strands of the original nucleic acid and will hybridize therewith to form a duplex of equal length strands to be separated into single-stranded molecules. when the complementary strands of the nucleic acid or acids whether the nucleic acid was originally double or the strands are ready to be used as a template for are separated, single stranded, the synthesis of additional nucleic acid strands. This synthesis can be performed using any suitable method. Generally it occurs in a buffered aqueous solution, preferably at a pH of 7-9, most preferably about 8. about 100021 primer:template, and for genomic nucleic acid, usually about 106:1 primer:template) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process herein is used for diagnostic applications, so that the amount of primer relative to the amount of complementary Preferably, a molar excess (for cloned nucleic acid, usually strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process. coli bstable enzymes, which will The deoxyribonucleoside triphosphates dATP, dCTP, dGTP and TTP are also added to the synthesis mixture in adequate amounts and the resulting solution is heated to about 90-100°C for from about 1 to minutes, preferably from 1 to 4 minutes. After this heating period the solution is allowed to cool to from 20—40°C, which is preferable for the primer hybridization. To the cooled mixture is added an agent and the in the art. for polymerization, reaction is allowed to occur under conditions known This synthesis reaction may occur at from room temperature up to a temperature above which the agent for polymerization no longer functions efficiently. Thus, for example, if DNA polymerase is used as the agent for polymerization, the temperature is generally no greater than about 45°C. Preferably an amount of dimethylsulfoxide (DMSO) is present which is effective in detection of the 35-40°C. Most preferably, 5-10% by volume DMSO is present and the temperature is 35- 40°C. are over 110 base pair fragments, such as the HLA DQ-a or -5 genes, an (e.g., 10% by of DMSO is added to the amplification mixture, and the carried at 35-40°C, to obtain detectable results or to enable cloning. signal or the temperature is For certain applications, where the sequences to be amplified effective amount volume) reaction is The agent for polymerization may be any compound or system function to accomplish the synthesis of primer extension Suitable enzymes for this which will products, including enzymes. purpose include, for example, E. coli DNA polymerase I, Klenow fragment of TLL available DNA polymerases, reverse transcriptase, and other enzymes, including heat- facilitate combination of the nucleotides DNA polymerase 1, T4 DNA polymerase, other in the proper manner to form the primer extension products which are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3' end of each primer and proceed in the 5' template strand, until synthesis terminates, producing molecules of different however, which initiate synthesis at the 5‘ end and proceed in the other direction, using the same process as described above. direction along the lengths. There may be agents, The newly synthesized strand and its complementary nucleic acid strand form a double-stranded molecule which is used in the succeeding steps of the process. In the next step, the strands of the double-stranded molecule are separated using any of the procedures described above to provide single-stranded molecules.

New nucleic acid is synthesized on the single—stranded molecules. Additional inducing agent, nucleotides and primers may be added if necessary for the reaction to proceed under the conditions prescribed above. Again, the synthesis will be initiated at one end proceed along the single After of the oligonucleotide primers and will strands of the template to produce additional nucleic acid. this step, half of the extension product will consist of the specific nucleic acid sequence bounded by the two primers., The steps of strand separation and extension product synthesis can be repeated as often as needed to produce the desired quantity of the specific nucleic acid sequence. As will be described in further detail below, the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. when it is desired to produce more than one specific nucleic acid sequence from the first nucleic acid or mixture of nucleic acids, of different if two different sequences are to be produced, four primers are utilized. the appropriate number oligonucleotide primers are nucleic acid Two of the utilized. For example, specific primers are specific for one of the specific nucleic acid sequences and the other two primers are specific for the second specific nucleic acid sequence. In this manner, each of the two different specific sequences can be produced exponentially by the present process.

The present invention can be performed in a step-wise fashion where after each step new reagents are added, or simultaneously, where all reagents are added at the initial step, or partially step-wise and partially simultaneous, where fresh reagent is added after a steps. If a method of strand separation, such as heat, is employed which will inactivate the agent given number of for polymerization, as in the case of a heat-labile enzyme, then it is necessary to replenish the agent for polymerization after every strand separation step. The simultaneous method may be utilized when a number of purified components, including an enzymatic means such as In the simultaneous in addition to the the strand- helicase, is used for the strand separation step. procedure, the reaction mixture may contain, nucleic acid strand(s) containing the desired sequence, separating enzyme (e.g., helicase), an appropriate energy source for the strand-separating enzyme, such as rATP, the four nucleotides, the oligonucleotide primers in molar excess, and the inducing agent, e.g., If heat denaturation in a—simultaneous process, a heat-stable inducing agent Klenow fragment of E. coli DNA polymerase I. is used for such as a thermostable polymerase may be employed which will operate at an elevated temperature, preferably 65-90°C depending on the inducing agent, at which temperature the nucleic acid will consist of single and double strands in equilibrium. For smaller lengths of nucleic acid, lower temperatures of about 50°C may be employed. The upper temperature will depend on the temperature at which the enzyme will degrade or the temperature above which an insufficient level of Such a heat-stable enzyme is -651 will S. Kaledin et al., Biokhimiya, fgi, will hybridization occur. described, e.g., by A. (1980). Each step of the sequentially notwithstanding the initial presence of all the reagents. Additional After the appropriate length of primer process occur materials may be added as necessary. time has passed to produce the desired amount of the specific nucleic acid sequence, the reaction may be halted by inactivating the enzymes in any known manner or separating the components of the reaction.

The process which can be carried out using the oligonucleotides of the present invention may be conducted continuously. in one embodiment of an automated process, the reaction may be cycled through a denaturing region, a reagent addition region, and a reaction region. In another embodiment, the enzyme used for the synthesis of primer extension products can be immobilized in a column.

The other reaction components can be continuously circulated by a pump through the column and a heating coil thus the nucleic acids produced can be repeatedly denatured without inactivating the in series, enzyme.

The present invention is demonstrated diagrammatically below where double-stranded DNA sequence [5] comprised of complementary strands [ST] and [S'] is utilized as the nucleic acid. During the first and each subsequent reaction cycle extension of each oligonucleotide primer on the original template will length which containing the desired produce one new ssDNA molecule product of indefinite terminates with only one of the primers. These products, hereafter referred to as "long products," will accumulate in a linear fashion; that is, proportional to the number of cycles. the amount present after any number of cycles will be The long'products thus produced will act as templates for one or the other of the oligonucleotide primers during subsequent cycles and will produce molecules of the desired sequence [S+] or [S'] These molecules will also function as templates for one or the other of the oligonucleotide primers, producing further [S+] and [S'], and thus a chain reaction can be sustained which will result in the accumulation of [S] at an exponential rate relative to the number of cycles.

By-products formed by oligonucleotide hybridizations other than those intended are not self-catalytic (except in rare instances) and thus accumulate at a linear rate.

The specific sequence to be amplified, [S], can be depicted diagrammatically as: [S+] 5' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCC 3' [S'] 3' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' The appropriate oligonucleotide primers would be: Primer 1: GGGGGGGGGG Primer 2: AAAAAAAAAA so that if DNA containing [S] ....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz.... ....zzzzzzzzzzzzzzzZTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz.... is separated into single strands and its single strands are hybridized to Primers 1 and 2, the following extension reactions can be catalyzed _]_8_ by DNA polymerase in the presence of the four deoxyribonucleoside triphosphates: 3| 5| extends( GGGGGGGGGG Primer 1 ....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz.... original template strand+ original template strand’ ....zzzzzzzzzzzzzzzZTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz....

AAAAAAAAAA————————————j> extends | 3| Primer 2 On denaturation of the two duplexes formed, the products are: ' 5' ....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG newly synthesized long product 1 ' 3' ....zzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCZZZzzzzzzzzzzzzz.... original template strand 3! 5| ....zzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGzzzzzzzzzzzzzzzz.... original template strand‘ | 3| AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz.... newly synthesized long product 2 If these four strands are allowed to rehybridize with Primers 1 and 2 in the following reactions: next cycle, agent for polymerization will catalyze the Primer 2 5' AAAAAAAAAA-————————————————€> extends to here '....zzzzzzzzzzzzzzzzZZTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5‘ newly synthesized long product 1 GGGGGGGGGG 5' Primer 1 extends ( Primer 2 5' AAAAAAAAAA ;} extends '....zzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYGGGGGGGGGGzzzzzzzzzz....5‘ original template strand" extends to here é———-——-——————————GGGGGGGGGG 5' ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz..3‘ newly synthesized long product 2 Primer 1 If the strands of the above four duplexes are separated, the following strands are found: ' 5' AAAAAAAAAAXXXXXXXXXXQCCCCCCCCC 3' newly synthesized [S J ‘....zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5‘ first cycle synthesized long product 1 '....zzzzzzzzzzzzzzzzzzzTTTTTTTTTTYYYYYYYYYYGGGGGGGGGG S‘ newly synthesized long product 1 '....zzzzzzzzzzzzzzzzzzzAAAAAAAAAAXXXXXXXXXXCCCCCCCCCCZzzzzzzzz....3‘ original template strand ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzzz...3‘ newly synthesized long product 2 '..zzzzzzzzzzzzzzZTTTTTTTTTTYYYYYYYYYYGGGGGGGGGGZzzzzzzzzzzzzzzz...5‘ original template strand‘ ' TTTTTTTTTTYYYYYYYYYYGGGGGGGGGG 5' newly synthesized [S’] ' AAAAAAAAAAXXXXXXXXXXCCCCCCCCCCzzzzzzzzzzzzzzz...3' first cycle synthesized long product 2 It is that each strand which terminates with the oligonucleotide sequence of one primer and the complementary sequence of the other is the specific nucleic acid sequence [3] that is desired seen to be produced.

The steps of this process can be repeated indefinitely, being limited only by the amount of Primers 1 and 2, the agent for polymerization and nucleotides present. For detection, the number of cycles used is that required to produce a detectable signal, an amount which will depend, e.g., on the nature of the sample. For example, if the sample is pure or diluted, fewer cycles may be required than if it is a complex mixture. If the sample is human genomic DNA, preferably the number of cycles is from about 10-30.

The amount of original nucleic acid remains constant in the entire process, because it is not replicated. The amount of the long products increases linearly because they are produced only from the original nucleic acid. The amount of the specific sequence increases Thus, the This is relative amounts specific will become the illustrated exponentially. sequence predominant species. in the following table, which indicates the of the species theoretically present after n cycles, assuming 100% efficiency at each cycle: Number of Double Strands After 0 to n Cycles Long Specific Cycle Number Template Products Sequence [8] 0 1 - - 1 1 1 O 2 1 2 1 3 1 3 4 1 5 26 1 10 1013 1 15 32,752 1 20 1,048,555 n 1 n (2"—n-1) when a single-stranded nucleic acid is utilized as the template, only one long product is formed per cycle.

The nucleic acid sequence for insertion into a suitable expression oligonucleotides herein may be utilized to clone a particular vector. The vector may then be used to transform an appropriate host _21_ organism to produce the gene product of the sequence by standard methods of recombinant DNA technology.

Normally, such cloning would either involve direct ligation into a vector or the addition of oligonucleotide linkers followed by restriction enzyme cleavage. Both of these methods involve, however, the inefficient blunt-end ligation reaction. Also, neither technique would control for the orientation or multiplicity of insertion of the amplified product into the cloning vector.

The amplification process which can be carried out using the oligonucleotides herein may yield a mixture of nucleic acids, resulting from the original template nucleic acid, the expected target amplified products, and various background non-target The amplified product can also be a mixture if the original DNA heterozygous diploid genome or when products. contains multiple target such as in a template sequences, there is a family of related genes.

The primers herein may be modified to assist the rapid and specific cloning of the mixture of DNAS produced by the amplification reaction. In such modification the same or different restriction ends of the primers to result in when cut sites are incorporated at the 5‘ restriction sites at the two ends of the amplified product. with the appropriate enzymes, the amplified product can then be easily inserted into plasmid or vial vectors and cloned. This cloning allows the analysis or expression of individual amplified products, not a mixture.

Although the same restriction site can be used for both primers, the use of different sites allows the insertion of the product into the vector with a specific orientation and suppresses multiple insertions as well as insertions arising from amplifications based on only one of the two primers. The specific orientation is useful when into sequencing vectors, when when the cloned cloning single-strand single-strand hybridization probes are used, or product is being expressed.

One method to prepare the primers is to choose a primer sequence which differs minimally from the target sequence. Regions in —22v which each of the primers is to be located are screened for homology to restriction sites appropriate to the desired vector. For example, the target sequence "CAGTATCCGA..." differs by only one base from one containing a gamhl site. A primer sequence is chosen to match the target exactly at its 3' end, and to contain the altered sequence and restriction site near its 5’ end (for example, "CAGgATCCGA...", where the letter with the This minimally altered sequence will not interfere with lower case symbolizes a mismatch target sequence). the ability of the primer to hybridize to the original target sequence and to initiate polymerization. After the first amplification cycle the primer is copied, becomes the target, and matches exactly with new primers. After the amplification process, the products are cleaved with the appropriate restriction enzymes, optionally separated from inhibitors of ligation such as the nucleotide triphosphates and salts by passing over a desalting column or molecular weight chromatography and inserted by into a cloning vector such as column, ligation bacteriophage M13. The gene may then be sequenced and/or expressed using well known techniques.

The second method for preparing the primers involves taking the 3' end of the primers from the target sequence and adding the to the 5‘ For the above example, a iflfldlli site could be added to make the sequence "cgaagcttCAGTATCCGA...", The added bases would not contribute to the hybridization in but would match in desired restriction site(s) end of the primer. where lower case letters are as described above. the cycles. 'The final amplified products are then cut with restriction first cycle of amplification, subsequent enzyme(s) and cloned and expressed as described above. The gene being amplified may be, for example, human beta-hemoglobin or the human HLA DQ, DR or DP—a and -5 genes. complementary to the DNA sequence which is being amplified. It is only necessary that they be able to hybridize to ;the sequence to be extended by the polymerase enzyme or by of a sufficiently well whatever other inducing agent is employed. The ‘product -237 polymerase chain reaction wherein the primers employed are not exactly complementary to the original template will contain the sequence of the primer rather than the template, thereby introducing an in vitro mutation. In further cycles this mutation will be amplified with an undiminished efficiency because no further mispaired primings are required. The mutant thus produced may be inserted into an appropriate vector by standard molecular biological techniques and might confer mutant properties on this vector such as the potential for production of an altered protein.

The process of making an altered DNA sequence as described above could be repeated on the altered DNA using different primers so as to induce further sequence changes. In this way a series of mutated could gradually be produced wherein ’each new addition to the series could differ from the last in a minor way, but sequences from the original DNA source sequence in an increasingly major way.

In this feasible in a single step due to the inability of a very seriously manner changes could be made ultimately which were not mismatched primer to function.

In addition, the primer can contain as part of its sequence a non-complementary sequence provided that a sufficient amount of the primer contains a sequence which is complementary to the strand to be amplified. For example, a nucleotide sequence which is not complementary to the template sequence (such as, e.g., a promoter, linker, coding sequence, etc.) may be attached at the 5‘ end of one or both of the primers, and thereby appended to the product of the amplification process. After the extension primer is added, sufficient cycles are run to -achieve the desired amount of new template containing the non-complementary nucleotide insert. This allows production of large quantities of the combined fragments in a relatively short period of time (e.g., two hours or less) using a simple technique.

Moreover, the oligonucleotides herein may be used to synthesize a nucleic acid fragment from an existing nucleic acid fragment which is shorter than its product (called the core segment) _using certain _24.. primers the 3' ends of which are complementary to or substantially complementary to ,the 3' ends of the single strands produced by separating the strands of the original shorter nucleic acid fragments, and the 5' ends of which primers contain sequence information to be appended to the core segment. This process comprises: (a) treating the strands of said existing fragment with two oligonucleotide under conditions such that an extension product of each primer is synthesized which is complementary to each primers nucleic acid strand, wherein said primers are selected so as to be substantially complementary to the 3' end of each strand of said existing fragment such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer, and wherein contains, at its 5' end, a sequence of nucleotides which are not complementary to said existing fragment and which correspond to the two ends of the nucleic acid fragment being each primer synthesized; (b) separating the primer extension products from the templates on which they were synthesized to produce single—stranded molecules; and (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions such that a product is synthesized using each of the single step (b) double-stranded primer extension strands produced in as a template so as to produce two intermediate nucleic acid molecules, into each of which has been incorporated the nucleotide sequence present in the 5‘ end of one of the oligonucleotide primers, and two full-length double- which has been stranded nucleic acid molecules, into each of incorporated the nucleotide sequence present in the 5' ends of both of the oligonucleotide primers; (d) repeating steps (b) and (c) for a sufficient number of times to produce the full-length double-stranded molecules_ in an effective amount; - (e) treating the strands of the product of step (d) with two primers so as to lengthen the product of step (d) on both ends; and (f) repeating steps (a)-(d) using the product of step (d) as the core fragment and two oligonucleotide primers which are complementary or substantially complementary to the 3' ends of the single strands produced by separating the strands of the product of step (d).

Steps (b) and (c) repeated as often as usually at least 5 times, to produce the required amount of the full- are necessary, length double-stranded product to synthesize the final product (i.e., In addition, the core segment may be obtained The produced in step (d) may be purified before a new cycle of extension the effective amount). amplification cycle. product as the product of a previous and amplification, or used directly by employing the reaction mixture containing the product.

If the 3‘ ends of the primers are not exactly complementary to the 3' ends of the single strands of the original shorter nucleic acid, the core fragment of the product will not be exactly the same as the acid. sequence information resident in the original shorter nucleic Therefore, mutants of the original nucleic acid may be made by using primers which are substantially complementary at their 3' ends to the 3' ends of the single strands of the original shorter nucleic acid.

If restriction site linkers are incorporated into the primers, then the amplified double-stranded products can be digested with the appropriate restriction enzymes and ligated directly into an M13 The M13 plaques containing the specific amplified target sequences can be identified lift filters with a probe specific for the vector for rapid cloning and sequencing. by hybridizing plaque target sequence.

The oligonucleotides herein may also be used to enable detection and/or characterization of specific nucleic acid sequences associated with infectious diseases, genetic disorders or cellular disorders such as cancer, Amplification is useful when the amount of nucleic acid available for analysis is very small, as, for example, e.g., oncogenes. diagnosis of sickle cell anemia using DNA obtained Amplification is to be done on a in the prenatal from fetal cells. is particularly useful if such an analysis small sample using non-radioactive detection techniques which may be inherently insensitive, or where radioactive techniques are being employed but where rapid detection is desirable.

For purposes of this invention genetic diseases may include specific deletions and/or mutations in genomic DNA from any organism, such as, e.g., sickle cell anemia, cystic fibrosis, a-thalassemia, @- thalassemia, and the like. Sickle cell anemia can be readily detected via oligomer restriction analysis or a RFLP-like analysis following amplification of the appropriate DNA sequence by the present method. a-Thalassemia can be detected by the absence of a sequence, and B- detected by the polymorphic linked to a thalassemia can be presence of a restriction site closely mutation which causes the disease.

All of these genetic diseases may be detected by amplifying the appropriate sequence and analyzing it by Southern blots without using radioactive probes. In such a process, for example, a small sample of DNA from, e.g., amniotic fluid containing a very low level of the desired sequence is amplified, cut with a restriction enzyme, and analyzed via a Southern blotting technique. The use of non- radioactive probes is facilitated by the high level of the amplified signal.

In another embodiment a small sample of DNA may be amplified to a convenient level and then a further cycle of extension reactions performed wherein nucleotide derivatives which are readily detectable (such as 32P-labeled or biotin labeled nucleoside triphosphates) are DNA product, analyzed by restriction and electrophoretic separation or any other An example of this technique in a model system is incorporated directly into the final which may be appropriate method. demonstrated in Figure 5. -globin DNA.

In a further embodiment, demonstrated in a model system in Figure 3, the nucleic acid may be exposed to a particular restriction endonuclease prior to amplification. Since a sequence which has been cut cannot be amplified, the appearance of an amplified fragment, despite prior restriction of the DNA sample, implies the absence of a site for the endonuclease within the amplified sequence. The presence or absence of an amplified sequence can be detected by an appropriate method.

Sickle cell anemia is a hemoglobin Figure 6 illustrates the sequences of of their polymorphism, where the single bars mark the location of a Ddel site normal and sickle cell genes in the region present only in the normal gene and where the double bars mark the location of a Hinfl site which is non-polymorphic and thus present in both the normal process of oligomer restriction of normal B-globin DNA using a probe labeled and sickle cell alleles. Figure 7 illustrates the spanning both restriction sites and where the asterisk appears. (The probe is preferably labeled at the end which is fewer base pairs from the restriction site than the other end of the probe.) The DNA, amplified as provided herein, is denatured and annealed to the labeled probe. The amplification may be carried out (35-40°C) in the sulfoxide to minimize formation of secondary structure. 353; cleaves the DNA at the reformed Ddg} site and generates a labeled Under the conditions used in the test the octamer is short at elevated temperatures presence of dimethyl The enzyme octamer. enough to dissociate from the duplex. The subsequent addition of the enzyme_flinfI has no effect on the now single-stranded octamer. Figure 8 illustrates the same process applied to the sickle cell allele of 5- The enzyme_QggI cannot cleave the duplex formed by the amplified DNA and the labeled probe because of the A-A base pair mismatch. The enzyme Hinfl, however, does restrict the hybrid and a labeled trimer is produced. In practice the method can diagnose the DNA of an as being either homozygous for the wild type, homozygous for the sickle type or a heterozygous carrier of the sickle individual cell trait, since a specific signal is associated with the presence of either allele. Use of this above-described method to amplify the pertinent sequence allows for a rapid analysis of a single copy gene using a probe with only a single 32P label.

Various infectious diseases can be diagnosed by the presence samples of specific DNA sequences characteristic of the These include bacteria, such as Salmonella, in clinical causative microorganism. and parasites, such as U.S.

Patent 4,358,535 issued to Falkow describes the use of specific DNA Chlamydia, Neisseria; viruses, such as the hepatitis viruses, the Plasmodium responsible for malaria. hybridization probes for the diagnosis of infectious diseases. A problem inherent in the Falkow procedure is that a relatively small number of pathogenic organisms may be present in a clinical sample and the DNA fraction sample. of immobilization and hybridization detection of the DNA samples could greatly improve the sensitivity and specificity of these procedures. from these DNA sequences extracted of the total may the to from an infected patient small in constitute only a very Specific amplification suspected prior use of DNA probes for the diagnosis of infectious be if non- radioactively labeled probes could be employed as described in EP 63,879 to ward. In this procedure biotin-containing DNA probes are detected by chromogenic enzymes Routine clinical diseases would simplified considerably linked to avidin or biotin-specific This type of detection is convenient, but relatively insensitive. The combination of specific DNA amplification by the present method and the use of stably labeled probes could provide the antibodies. convenience and sensitivity required to make the Falkow and ward procedures useful in a routine clinical setting.

In addition, the probe may be a biotinylated probe in which the biotin is attached to a spacer arm of the formula: -29..

I where‘ Y is O, NH or N-CHO, x is a number from 1 to 4, and y is a number from 2 to 4. The spacer arm is in turn attached to a psoralen moiety of the formula: The psoralen moiety intercalates into and crosslinks a "gapped circle" probe as described by Courage-Tebbe et al., Biochim. Biophys. Acta, Q1 (1982) the gapped circle spans the region contained in the primers. -5, wherein the single-stranded hybridization region of The oligonucleolides herein can also be utilized to produce sufficient quantities of DNA from a single copy human gene such that detection by a simple non- specific DNA stain such as ethidium bromide can be employed so as to make a DNA diagnosis directly.

In addition to detecting infectious diseases and pathological abnormalities in the genome of organisms, the oligonucleotides herein can also be used to detect DNA polymorphism which may not be associated with any pathological state.

The following examples are offered by way of illustration and are not intended to limit the invention in any manner. In these examples all percentages are by weight if for solids and by volume if and all otherwise noted. for liquids, temperatures are in degrees Celsius unless EXAMPLE 1 A 25 base pair sequence having the nucleotide sequence ' CCTCGGCACCGTCACCCTGGATGCT 3' 3' GGAGCCGTGGCAGTGGGACCTACGA 5' contained on a 47 base pair FokI restriction fragment of pBR322 obtainable from ATCC was prepared as follows. A Fokl digest of pBR330 containing the 47-bp fragment was produced by digesting pBR322 with fog} in accordance with the conditions suggested by the supplier, New England Biolabs Inc. The utilized were 5‘ d(CCTCGGCACCG) 3' and 5' d(AGCATCCAGGGTG) 3', and were prepared using conventional techniques. The following ingredients were added to 33 pl of buffer which consisted of 25 mM potassium phosphate, 10 mM magnesium chloride and 100 mM sodium chloride at pH 7.5: 2433 pmoles of each of the primers described above, 2.4 pmoles of the 595} digest of pBR322, 12 nmoles of dATP, 22 nmoles of dCTP, 19 nmoles of dGTP and nmoles of TTP. primers which were The mixture was heated to 85°C for five minutes and allowed to cool to ambient temperature. Five units of the Klenow fragment of E. coli DNA polymerase I were added and the temperature was maintained for 15 minutes. After that time, the mixture was again heated to 85°C for five minutes and allowed to cool. Five units of the Klenow fragment were again added and the reaction was carried out for 15 minutes. The heating, cooling and synthesis steps were repeated eleven more times.

After the final repetition, a 5 pl aliquot was removed from This was heated to 85°C for three minutes and of a-P33 — the reaction mixture. allowed to cool to ambient temperature. 12.5 undies deoxycytidine triphosphate and 5 units of Klenow fragment were added The labeled products were examined by polyacrylamide gel The fog} digest was labeled in a similar fashion and served as a control The only heavily labeled band visible and the reaction was allowed to proceed for 15 minutes. electrophoresis. and molecular weight markers. after the 13 cycles was the intended 25 base pair sequence.

EXAMPLE 2 The desired sequence to be amplified was a 94 base pair sequence contained within the human beta-globin gene and spanning the The sequence has the MstII site involved in sickle cell anemia. nucleotide sequence shown in Figure 1. _3]_.

. Synthesis of Primers The following two oligodeoxyribonucleotide primers were prepared by the method described below: ' CACAGGGCAGTAACG 3' Primer A and 5' TTTGCTTCTGACACA 3' Primer B Automated Synthesis Procedures: The diethylphosphoramidites, synthesized according to Beaucage and Caruthers (Tetrahedron Letters (1981) 22:1859—1862) were sequentially condensed to a nucleoside derivatized controlled pore glass support using a Biosearch SAM-1. The procedure included detritylation with trichloroacetic acid in dichloromethane, condensation using benzotriazole as activating proton donor, and capping with acetic anhydride and dimethylaminopyridine in tetrahydrofuran and pyridine.

Cycle time was approximately 30 minutes. Yields at each step were essentially quantitative and were determined by collection and spectroscopic examination of the dimethoxytrityl alcohol released during detritylation.

Oligodeoxyribonucleotide Deprotection and Purification Procedures: The solid support was removed from the column and exposed to 1 ml concentrated ammonium hydroxide at room temperature for four hours in a closed tube. The support was then removed by filtration and the the. protected oligodeoxynucleotide was brought to 55°C for five hours. removed and the residue was applied to a preparative polyacrylamide gel. Electrophoresis was carried out at 30 volts/cm for 90 minutes after which the band containing the product was identified by UV The band was excised and eluted solution containing partially Anmonia was shadowing of a fluorescent plate. with 1 ml distilled water overnight at 4°C. to an Altech RP18 column and eluted with a acetonitrile in 1% ammonium acetate buffer at pH 6.0. monitored by UV absorbance at 260 nm and the appropriate fraction collected, quantitated by UV fixed volume and evaporated to dryness at room temperature in a vacuum centrifuge.

This solution was applied 7-13% gradient of The elution was absorbance in a ._32..

Oligodeoxyribonucleotides: Test 3% labeled with labeled compounds Characterization of of the purified oligonucleotides were aliquots polynucleotide kinase and 7-32P—ATP. The 14-20% polyacrylamide gels at 50 This Base composition was determined by WEN? examined by autoradiography of after electrophoresis for 45 minutes volts/cm. procedure verifies the molecular weight. digestion of the oligodeoxyribonucleotide to nucleosides by alkaline phosphatase and subsequent use of venom diesterase and bacterial separation and quantitation of the derived nucleosides using a reverse phase HPLC column and a 10% acetonitrile, 1% ammonium acetate mobile phase.

. Source of DNA A. Extraction of whole Human wild-Type DNA Human genomic DNA homozygous for nomnal 5—globin was extracted from the cell line Molt4 (obtained from Human Genetic Mutant Cell Repository and identified as GM2219c) using the technique described by Stetler et al., Proc. Nat. Acad. Sci. (1982), 79:5966— 5970.

B. Construction of Cloned Globin Genes May 25, 1984.

This fragment, which includes the first and second exons, was added.

Each recombinant plasmid was transformed into and propagated in E. coli MM294 (ATCC No. 39,607).

C. Digestion of Cloned Globin Genes with MstII bovine serum III. Polymerase Chain Reaction To 100 pl of buffer consisting of 60 mM sodium acetate, 30 mM Tris acetate and 10 mM magnesium acetate at pH 8.0 was added 2 pl of a solution containing 100 picomoles of Primer A (of the sequence d(CACAGGGCACTAACG)), 100 picomoles of Primer B (of the sequence d(TTTGCTTCTGACACA)) and 1000 picomoles each of dATP, dCTP, dGTP and TTP. was added: In addition, one of the following sources of DNA described above pg whole human wild-type DNA (Reaction 1) 0.1 picomole pBR328:HbA (Reaction II) .1 picomole pBR328:HbS (Reaction III) .1 picomole pBR328:HbA/MstII (Reaction IV) 0.1 picomole pBR328:HbS/MstII (Reaction V) No target DNA (Reaction VI) Each resulting solution was heated to 100°C for four minutes and allowed to cool to room temperature for two minutes, whereupon 1 pl containing four units of Klenow fragment of E. coli DNA polymerase Each reaction was allowed to proceed for 10 minutes, after which the cycle of adding the primers, nucieotides and DNA, heating, cooling, adding polymerase, and reacting was repeated nineteen times for Reaction I and four times for Reactions II-VI. -34..

Four microliter aliquots of Reactions I and II removed before the first cycle and after the last cycle of each reaction were applied to a 12% polyacrylamide gel 0.089 M in Tris-borate buffer at pH 8.3 and 2.5 mM in EDTA. for four hours, transferred to a nylon membrane serving as solid phase support and probed with a 5'-32P-labeled 40 bp synthetic fragment, prepared by standard techniques, of the sequence The gel was electrophoresed at 25 volts/cm ‘d(TCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAG)3' in 30% formamide, 3 x SSPE, 5 x Denhardt's, 5% sodium dodecyl sulfate at pH 7.4. for Reactions 1 and 11.

Figure 2 is an autoradiograph of the probed nylon membrane Lane 1 is 0.1 picomole of a 58—bp synthetic is complementary to the above fragment control one strand of which probe. Lane 2 is 4 pl of Reaction I prior to the first amplification cycle. Lane 3 is 4 pl of Reaction I after the 20th amplification cycle. Lane 4 is 4 pl of Reaction 11 after five amplification cycles. Lane 5 is a molecular weight standard consisting of a jig} (New England Biolabs) digest of pBR322 (New England Biolabs) labeled with alpha-32P-dNTPs and polymerase. Lane 3 shows that after twenty cycles the reaction mixture I contained a large amount of the specific weight and no other detectable sequence of the proper molecular products. Reaction mixture 11 after five cycles also contained this product, as well as the starting nucleic acid and other products, as shown by Lane 4.

To 5.0 pl aliquots of Reactions II-VI after the fourth cycle were added 5 pmoles of each primer described above. The solutions were heated to 100°C for four minutes and allowed to equilibrate to room temperature. Three pmoles each of alpha-32P-dATP, alpha-32P- dCTP, alpha-32P-dGTP and alpha-32P—TTP and four units of Klenow fragment were added. The reaction, in a final volume of 10 pl and at the salt concentrations given above, was allowed to proceed for 10 minutes. The polymerase activity was terminated by heating for 20 minutes at 60°C. a 12% polyacrylamide gel 0.089 M in Tris-borate buffer at pH-8.3 and 2.5 mM in EDTA. The gel was electrophoresed at 25 volts/cm for four hours after which autoradiography was performed.

Four pl aliquots of Reactions II-VI were loaded onto _35_ Figure 3 is an autoradiograph of the electrophoresis. Lane 1 is a molecular weight standard, Lane 2 is Reaction II, Lane 3 is Reaction III, Lane 4 is Reaction IV and Lane 5 is Reaction V. Another sequence Figure 4 illustrates the chain reaction for three cycles in ammifying the 94—bp sequence. PCO1 and PCO2 are Primers A and B.

The numbers on the right indicate the cycles, whereas the numbers on the left indicates the cycle number in which a particular molecule was produced.

EXAMPLE 3 This example illustrates amplification of a 110 bp sequence spanning the allelic MstII site in the human hemoglobin gene.

A total of 1.0 microgram whole human DNA, 100 picomoles d(ACACAACTGTGTTCACTAGC) and 100 picomoles d(CAACTTCATCCACGTTCACC) the primers having been prepared by the technique of Example 2, were dissolved in 100 pl of a solution which was: 1.5 mM in each of the four deoxyribonucleoside triphosphates mM in Tris acetate buffer at pH 7.9 60 mM in sodium acetate mM in magnesium acetate .25 mM in dithiothreitol _36_ The solution was heated to 100°C for one minute and brought rapidly to 25°C for one minute, after which was added 2.5 units Klenow fragment of DNA polymerase. The polymerase reaction was allowed to proceed for two minutes at 25°C, after which the cycle of heating, cooling, adding Klenow, and reacting was repeated as often as desired. with a 70% efficiency at each cycle, 15 cycles resulted in the synthesis of 1.4 femtomoles of the desired 110 bp fragment of the B—globin gene.

EXAMPLE 4 This example illustrates amplification of a 240 bp sequence spanning the allelic Mstll site in the human hemoglobin gene. This sequence contains Ncol, Hinfl and MstII restriction sites.

To 100 pl of a mixture of 60 mM sodium acetate, 30 mM Tris acetate and 10 mM magnesium acetate at pH 8.0 containing 0.1 pmole pBR328:HbA was added 2 pl of Solution A containing: pmoles d(GGTTGGCCAATCTACTCCCAGG) primer 100 pmoles d(TAACCTTGATACCAACCTGCCC) primer 1000 pmoles each of dATP, dCTP, dGTP and TTP The two primers were prepared by the technique described in Example 2. The solution was heated to 100°C for four minutes and allowed to cool in ambient air for two minutes, after which was added 1 pl containing coli DNA polymerase. The reaction was allowed to proceed for 10 minutes after adding four units Klenow fragment of E. cooling, polymerase, and reacting was repeated three times. To a 5.0 pl aliquot of the reactions was added 5 picomoles of each oligonucleotide The solution was heated to 100°C for four after which 3 deoxyribonucleoside which the cycle of solution A addition, heating, primer described above. minutes and allowed to come to ambient temperature, each of the alpha-32P-labeled triphosphates and 4 units Klenow fragment were added. in a final volume of 10 pl and at the salt concentrations given above, was allowed to proceed for 10 minutes. The polymerase activity was picomoles ‘The reaction, terminated by heating for 20 minutes at 60°C. Two pl aliquots were digested with Iggy, fgtjl, or lfinfl and loaded onto a 12% polyacrylamide gel 0.089 M in Tris-borate buffer at pH 8.3 and 2.5 mM in EDTA. The gel was electrophoresed at 25 volts/cm for four hours and autoradiography was Figure 5 illustrates the autoradiograph of the electrophoresis, where Lane 1 is the molecular weight Lane 2 (240 bp intact), Lane 3 is digestion with [Q31 (131 and 109 bp), Lane 4 is digestion with fistll (149 and 91 bp), and Lane 5 is digestion with liinfl (144 and 96 bp). amplification of the 240 bp sequence. performed. standard, is without digestion with enzyme The autoradiograph is consistent with the EXAMPLE 5 This example illustrates use of the process herein to detect sickle cell anemia by sequential digestion.

Synthesis and Phosphorylation of Oligodeoxyribonucleotides A labeled DNA probe, R306, of the sequence: ' *CTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGG 3' where * indicates the label, and an unlabeled blocking oligomer, RS10, of the sequence ' GACAGAGGTCACCTCTTCAGACGGCAATGACGGGACACCC 5' with RSO6 were synthesized The probe RS06 was labeled by contacting five pmole thereof with 4 units of T4 polynucleotide kinase (New England Biolabs) and 50 pmole y'32P-ATP (New England Nuclear, about 7200 Ci/mmole) in a 40 pl reaction volume containing 70 mM Jris buffer (pH 7.6), 10 mM MgCl2, 1.5 mM spermine, and 2.5 mM dithiothreitol for 90 minutes at 37°C. The total volume was then adjusted to 100 pl with 25 mM EDTA and purified according to the procedure of Maniatis et al., Molecular Cloning (1982), 464-465 over a 1 ml Bio Gel P-4 spin dialysis column from BioRad equilibrated with Tris-EDTA (TE) buffer (10 mM Tris buffer, 0.1 mM EDTA, pH 8.0).

The labeled probe was further purified by electrophoresis on a 18% which has three base pair mismatches according to the procedures provided in Example 2(1) _33_ polyacrylamide gel (19:1 acrylamide:BIS, BioRad) in Tris-boric acid- EDTA (TBE) buffer (89 mM Tris, 89 mM boric acid, 2.5 mM EDTA, pH 8.3) for 500 vhr. After localization by autoradiography, the portion of the gel containing the labeled probe was excised, crushed and eluted into 0.2 nfl TE buffer overnight at 4°C. TCA precipitation of the reaction product indicated that the specific activity was 4.9 Ci/mmole and the final concentration was 20 pmole/ml.

The unlabeled RS10 used at a concentration of 200 pmole/ml. blocking oligomer was Isolation of Human Genomic DNA from Cell Lines High molecular weight genomic DNA was isolated from the lymphoid cell lines Molt4, SC—1 and GM2064 using essentially the method of Stetler et al., PNAS (1982), 723 5966-5970 (for Molt4) and Maniatis et al., Molecular Cloning (1982), 280-281.

Molt4 (Human Mutant Cell GM2219C) is a T cell line homozygous for normal 5—globin, and SC-1, deposited with ATCC on March 19, 1985, is an EBV—transformed B cell line homozygous for the GM2064 (Human Mutant Cell Repository, GM2064) was for hereditary Repository, sickle cell allele. originally isolated from an individual homozygous persistance of fetal hemoglobin (HPFH) and contains no beta- or delta- globin gene sequences. All cell with 10% fetal calf serum. lines were maintained in RPMI-1640 Isolation of Human Genomic DNA from Clinical Blood Samples A clinical blood sample designated CH12 from a known sickle cell carrier (AS) was obtained from Dr. Bertram Lubin of Children's Hospital in Oakland, California. Genomic DNA was prepared from the buffy coat fraction, which is composed primarily of peripheral blood lymphocytes, using a modification of the procedure described by Nunberg et al., Proc. Nat. Acad. Sci., 153 5553-5556 (1978).

The cells were resuspended in 5 ml Tris-EDTA-Nacl (TEN) buffer (10 mm Tris buffer pH 8, 1 mM EDTA, 10 mM Nacl) and adjusted to 0.2 mg/ml- proteinase K, 0.5% SDS, and incubated overnight at 37°C.

Sodium perchlorate was then added to 0.7 M and the lysate gently shaken for 1-2 hours at room temperature. The lysate was extracted with 30 ml phenol/chloroform (1:1), then with 30 ml chloroform, and followed by ethanol precipitation of the nucleic acids. The pellet was resuspended of TE buffer and RNase A added to 0.005 After digestion for one hour at 37°C, the DNA was extracted with chloroform, and ethanol precipitated. ml TE buffer and the concentration was determined by absorbance at 260 in 2 ml mg/ml. each volumes of phenol, phenol/chloroform, and once equal The DNA was resuspended in 0.5 flfll.

Polymerase Chain Reaction to Amplify Selectively 5-Globin Sequences Two micrograms of genomic DNA was amplified in an initial 100 pl reaction volume containing 10 mM Tris buffer (pH 7.5), 50 mM NaCl, 10 mM MgCl2, 150 d(CACAGGGCACTAACG), and d(CTTTGCTTCTGACACA) prevent evaporation.

Primer A of the of the mineral oil to pmole of sequence Primer B pmole of sequence and overlayed with about 100 pl Each DNA sample underwent 15 cycles of amplification where one cycle is composed of three steps: 1) Denature in a heat block set at 95°C for two minutes.

) Transfer immediately to a heat block set at 30°C for two minutes to allow primers and genomic DNA to anneal.

) Add 2 pl of a solution containing 5 units of the Klenow fragment of E. coli DNA polymerase I (New England Biolabs), 1 nmole each of dATP, dCTP, dGTP and TTP, in a buffer composed of 10 mM Tris (pH 7.5), 50 mM Nacl, 10 mM MgCl2, and 4 inM dithiothreitol. This extension reaction was allowed to proceed for 10 minutes at 30°C.

After the final reaction was terminated by heating at 95°C for two minutes. cycle, the The mineral oil was extracted with .2 ml of chloroform and discarded. The final reaction volume was 130 Hybridization/Digestion of Amplified Genomic DNA with Probes and Ddel/Hinfl of the amplified genomic DNA was of TE Ten microliters (containing the pre-amplification equivalent Forty-five microliters ethanol precipitated and resuspended in an equal volume buffer. of 154 ng of genomic DNA) was dispensed into a 1.5 ml Microfuge tube and 20 pl of TE buffer to a final overlayed with mineral oil and denatured at 95°C for 10 minutes. Ten volume of 30 pl. The sample was microliters of 0.6 M NaCl containing 0.02 pmole of labeled R506 probe was added to the tube, mixed gently, and immediately transferred to a 56°C heat block for one hour. Four microliters of unlabeled R310 added and the continued for an additional 10 minutes at the same temperature. microliters of 60 mM MgCl2/0.1% BSA and 1 pl of Eye} (10 units, New England Biolabs) were added and the reannealed DNA was digested for 30 at 56°C. (10 units, was then added and incubated for another 30 minutes. The blocking oligomer (0.8 pmole) was hybridization Five minutes New England Biolabs) reaction was stopped by the addition of 4 pl 75 mM EDTA and 6 pl One microliter of Hinfl tracking dye to a final volume of 61 pl.

The mineral oil was extracted with 0.2 ml chloroform, and 18 pl of the reaction mixture (45 ng genomic DNA) was loaded onto a 30% (1921, Bio Rad) in a SE200 The gel was electrophoresed at approximately 300 volts for polyacrylamide mini-gel Hoeffer apparatus. one hour until the bromphenol blue dye front migrated to 3.0 cm off- origin. The top 1.5 cm of the gel was removed and the remaining gel was exposed for four days with one intensification screen at -70°C.

Discussion of Photograph (Figure 9) Each lane contains 45 ng of amplified genomic DNA. Lane A contains Molt4 DNA; Lane B, CH12; Lane C, SC-1; and Lane 0, GM2064.

Molt4 represents the genotype of a normal individual with two copies of the BA gene per cell (AA), CH12 is a clinical sample from a sickle cell carrier with one BA and_ one B5 gene per cell (AS), and SC-1 represents the genotype of a sickle cell individual with two copies of the B5 gene per cell (55). GM2064, which contains no beta— or delta- globin sequences, is present as a negative control.

As seen in the photograph, the Egg}-cleaved, BA-specific octamer is present only in those DNA's containing the BA gene (Lanes A and B) and the jfinfl-cleaved, B5-specific trimer is present only in those DNA's containing the B5 gene (Lanes B and C). The presence of both trimer and octamer (Lane B) is diagnostic for a sickle cell carrier and is distinguishable from a normal individual (Lane A) with only octamer and a sickle cell afflicted individual (Lane C) with only trimer.

As a comparison, repeating the experiment described above using that the increased the sensitivity of detection by at least 1000 fold. non-amplified genomic DNA revealed amplification EXAMPLE 6 This illustrates direct detection of a totally unpurified single copy gene in whole human DNA on gels without the example need for a labeled probe.

Using the described in Example 3, a 110-bp fragment from a sequence in the first exon of the beta-globin gene was amplified from 10 micrograms of whole human DNA after 20 cycles. This technique -bp fragment produced after 20 cycles was easily visualized on gels stained with ethidium bromide.

The sequence was not amplified when it was first cut with the restriction enzyme Qggl unless, as in the beta-globin 5 allele, the sequence does not contain the restriction site recognized by the enzyme.

EXAMPLE 7 A. A total of 100 fmoles pBR328 containing a 1.9 kb insert from the human beta-globin A allele, 50 nmoles each alpha-32P-dNTP at 500 Ci/mole, and 1 nmole of each of the primers used in Example 3 were dissolved in a solution containing 100 LA 30 nM Tris-acetate at pH 7.9, 60 mM sodium acetate, 100 mM dithiothreitol, and 10 mM magnesium acetate. This solution was brought to 100°C for two minutes and cooled to 25°C for one minute. A total of 1 pl containing 4.5 units Klenow fregment of E. coli DNA polymerase I and 0.09 units inorganic pyrophosphatase was added to pyrophosphate in the reaction mixture, and the reaction was allowed to proceed for two minutes at 25°C, after which the cycle of heating, Ten-pl prevent the possible. build-up of cooling, adding enzyme, and reacting was repeated nine times. were removed and added to 1 pl 600 mM EDTA after each synthesis cycle. Each was analyzed on a 14% polyacrylamide gel in 90 mM Tris—borate and 2.5 mM EDTA at pH 8.3 and 24 volts/cm for 2.5 hours. The completed gel was soaked for 20 minutes in the same buffer with the addition of 0.5 pg/ml washed with the original buffer, and photographed in UV light using a red filter. aliquots ethidium bromide, The 110-bp fragment produced was excised from the gel under light and the 399 counted An attempt to fit the data to an equation of the form: ultraviolet incorporated by Cerenkov radiation. pmoles/10 pl = 0.01 [(1+y)N-yN-1], where N represents the number of cycles and y the fractional yield per cycle, was optimal with y = .619. This indicates that a significant amplification is occurring.

. The above experiment was repeated except that 100 nmoles of each dNTP was added to a 100 pl employed, and aliquots were not removed at each cycle. radiolabel was After 10 cycles the reaction was terminated by boiling for two minutes and reaction, no rehybridization was performed at 57°C for one hour. The sequence of the 110-bp product was confirmed by subjecting 8 pl aliquots to restriction analysis by addition of 1 pl bovine serum albumin (25 mg/ml) and 1 pl of the appropriate restriction enzyme (flinfI,_flnlI, ggggi, jggj) and by reaction at 37°C for 15 hours. PAGE was performed as described above.

EXAMPLE 8 This example illustrates the use of different primers to amplify various fragments of pBR328 and 322. —43—.

A. The experiment described in Example 7A was repeated except using the following primers: d(TTTGCTTCTGACACAACTGTGTTCACTAGC) and d(GCCTCACCACCAACTTCATCCACGTTCACC) to produce a 130-bp fragment of pBR328. in Example 7A was except using the following primers: d(GGTTGGCCAATCTACTCCCAGG) and d(TGGTCTCCTTAAACCTGTCTTG) to produce a 262-bp fragment of pRR328. The reaction time was 20 minutes per cycle.

B. The experiment described repeated C. The experiment described in Example 88 was repeated except that 100 fmoles of an fgtjl digest of pBR328 containing a 1.9 kb insert from .the human beta-globin S allele was used as initial This plasmid was cleaved several times by ffitll but not addition, template. inside the sequence to be amplified. In the primers employed were as follows: d(GGTTGGCCAATCTACTCCCAGG) and d(TAACCTTGATACCAACCTGCCC) to produce a 240-bp fragment.

D. The experiment described in Example 78 was repeated except that 100 fmoles of an _flruj digest of pBR322 was used as template, 200 nmoles of each dNTP were used in the 100 pl reaction, and the primers were: d(TAGGCGTATCACGAGGCCCT) and d(CTTCCCCATCGGTGATGTCG) to produce a 500-bp fragment from pBR322. Reaction times were 20 minutes per cycle at 37°C. Final rehybridization was 15 hours at °C. Electrophoresis was on a 4% agarose gel.

EXAMPLE 9 This example illustrates the invention process wherein an in vitro mutation is introduced into the amplified segment.

A. A total of 100 fmoles of pBR322 linearized with NruI, 1 nmole each of the primers: d(CGCATTAAAGCTTATCGATG) and d(TAGGCGTATCACGAGGCCCT) _44- designed to produce a 75—bp fragment, 100 nmole each dNTP, in 100 pl 40 mM Tris at pH 8, 20 mM in Mgclz, 5 mM in dithiothreitol, and 5 mg/ml bovine serum albumin were combined. The mixture was brought to 100°C for one minute, cooled for 0.5 minutes in a water bath at 23°C, whereupon 4.5 and 0.09 units pyrophosphatase were added, and a reaction was allowed to proceed for The cycle of heating, cooling, adding enzymes, and units Klenow fragment inorganic three minutes. reacting was repeated nine times. The tenth reaction cycle was terminated by freezing and an 8-pl aliquot of the reaction mixture was applied to a 4% agarose gel visualized with ethidium bromide.

B. The experiment described in Example 9A was repeated except that the oligonucleotide primers employed were: d(CGCATTAAAGCTTATCGATG) and d(AATTAATACGACTCACTATAGGGAGATAGGCGTATCACGAGGCCCT).

These 101-bp nucleotides of which (in the second listed primer) are not present in pBR322. These nucleotides represent the sequence of the T7 promoter, which was appended to the 75-bp sequence from pBR322 by using the primer with 20 complementary bases and a 26-base 5' extension. The primers are designed to produce a fragment, 26 procedure required less than two hours and produced two picomoles of the relatively pure 101-bp fragment from 100 fmoles of pBR322.

The T7 promoter can be used to initiate RNA transcription.

T7 polymerase may be added to the 101-bp fragment to produce single- stranded RNA.

C. The experiment described in Example 8D was repeated except that the oligonucleotide primers employed were as follows: d(TAGGCGTATCACGAGGCCCT) and d(CCAGCAAGACGTAGCCCAGC) to produce a 1000-bp fragment from pBR322.

D. The experiment described in Example 9C was repeated except that the oligonucleotide primers employed were as follows: d(TAGGCGTATCACGAGGCCCT) and _ - d(AATTAATACGACTCACTATAGGGAGATAGGCGTATCACGAGGCCCT) so as to produce a 1026-bp fragment, 26 nucleotides of which (in the second listed primer) are not present in pBR322 and represent the T7 promoter described above. The promoter has been inserted adjacent to a 1000-bp fragment from pBR322.

The results indicate that a primer which is not a perfect match to the template sequence but which is nonetheless able to hybridize sufficiently to be enzymatically extended produces a long product which contains the sequence of the primer rather than the corresponding sequence of the original template. The long product serves as a template for the second primer to introduce an in vitro this because no further mispaired primings are mutation. In further cycles mutation is amplified with an undiminished efficiency, In this case, a primer which carries a non-complementary its 5' product adjacent to the template sequence being copied. required. extension on end was used to insert a new sequence in the EXAMPLE 10 This example illustrates employing nested sets of primers to decrease the background in the amplification of single copy genes. whole human DNA homozygous for the wild-type 5-globin allele was subjected to twenty cycles of amplification as follows: A total of 10 pg DNA, 200 picomoles each of the primers: d(ACACAACTGTGTTCACTAGC) and d(CAACTTCATCCACGTTCACC) and 100 nanomoles each dNTP in 100 pl of 30 mM Tris-acetate pH 7.9, 60 mM sodium acetate, 10 mM dithiothreitol, and 10 mM magnesium acetate were heated to 100°C for one minute, cooled to 25°C for one minute, The cycle A ten-pl removed from the reaction mixture and subjected to a and treated with 2 units Klenow fragment for two minutes. of heating, cooling and adding Klenow was repeated 19 times. aliquot was further ten cycles of amplification using each of the primers: d(CAGACACCATGGTGCACCTGACTCCTG) and d(CCCCACAGGGCAGTAACGGCAGACTTCTCC), - ’ -46.. which amplify a 58-bp fragment contained within the 110-bp fragment produced above. This amplification was accomplished by diluting the 10-pl aliquot into 90 pl of the fresh Tris-acetate buffer described above containing 100 nanomoles each dNTP Reaction conditions were as above. final ten cycles of and 200 pmoles of each primer.

After ten cycles a 10-pl aliquot (corresponding to 100 nanograms of the original DNA) was applied to a 6% Nusieve (FMC Corp.) agarose gel and visualized using ethidium bromide.

Figure 10 illustrates this gel illuminated with UV light and photographed through a red filter as is known in the art. Lane 1 is molecular weight markers. Lane 2 is an aliquot of the reaction described above. Lane 3 is an aliquot of a reaction identical to that described above, except that the original wild—type DNA was cleaved with Ddel prior to amplification. Lane 4 is an aliquot of a reaction identical to that described above, except that human DNA homozygous for the treated with Ddel amplification (the sickle allele does not contain a Ddel site in the sickle betaglobin allele was prior to fragment being amplified here). Lane 5 is an aliquot of a reaction to that described above, except that salmon sperm DNA was substituted for human DNA. Lane 6 identical to that described above, except that the aliquot was treated with Egg} after amplification (Dggj should convert the 58-bp wild-type product into 27-and 31-bp fragments). Lane 7 is an aliquot of the treated with _Qde} (the 58-bp sickle product contains no Ddel site). identical is an aliquot of a reaction Lane 4 material after amplification Detection of a 58-bp fragment representative of a single- copy gene from one microgram of human DNA using only ethidium bromide staining of an agarose gel requires an amplification of about 500,000- fold. sets of oligonucleotide primers herein. accomplished by using the two nested The first set amplifies the 110-bp fragment and the inner nested set amplifies a sub-fragment of this product up to the level of convenient detection shown in Figure 10.

This contained within This was procedure of using primers amplifying a smaller sequence the’ amplification process and contained in the extension products of the sequence being amplified in the previous locus resorting to EXAMPLE 11 The present process is expected to be useful in detecting, patient DNA associated with an specific infectious e.g., hybridization probe spanning the desired amplified sequence and using in U.S. 4,358,535, gggyj. The biotinylated hybridization probe may be prepared by intercalation and irradiation of a partially double-stranded DNA with a 4'-methylene substituted ,5‘trimethylpsoralen attached to biotin via a spacer arm of the in a sample, a sequence such as, using a biotinylated disease Chlamydia the process described formula: H I —Y-(CH2)2—0—[CH2)X0]y—CH2CH2-N- NH or N-CHO, x is a number from 1 to 4, and y is a Detection of the biotinyl groups on the probe may where Y is 0, number from 2 to 4. streptavidin-acid phosphatase complex be accomplished using a commercially obtainable from Enzo Biochem Inc. using the detection procedures suggested by the manufacturer in its -brochure. The hybridized probe is seen as a spot of precipitated stain due to the binding of the and the subsequent reaction catalyzed by acid phosphatase, which produces a precipitable dye. detection complex, EXAMPLE 12 In this example, the process of Example 7 was basically used to amplify a 119 base pair fragment on the human 5-hemoglobin gene using the primers: '-CTTCTGCagCAACTGTGTTCACTAGC-3' (GH18) ' ‘-CACaAgCTTCATCCACGTTCACC-3' (GH19) .48.. where lower case Tetters denote mismatches from wiid-type sequence to create restriction enzyme sites.

Tabie I iiiustrates a diagram of the primers GH18 and GH19 which are The fuii scheme is shown in Tabie I. used for cioning and sequencing a 119-base pair fragment of the human 3-giobin gene and which are designed to contain internai restriction sites. The start codon ATG is GH18 is a 26-base oiigonucieotide compiementary to the negative strand and contains an underiined. internal Pstl site. GH19 is a 23-base oiigonucieotide compiementary recognition by DNA The boxed sequences indicate the restriction enzyme to the pius strand and contains an internai Hindlli prepared as described in previous exampies. :_ 3....

Amplification and Cloning After twenty cycles of amplification of 1 microgram of human genomic DNA isolated from the cell line Molt 4 as described in Example 2, 1/14th of the reaction product was hybridized to the labeled 3- RS06, of the sequence 5‘- glovin specific oligonucleotide probe, CTGACTCCTGAGGAGAAGTCTGCCGTTACTGCCCTGTGGG-3' restriction. treated with lgygl restriction digestion conditions as described above, to produce an 8- using the methods described above for oligomer Following solution hybridization, the reaction mixture was under base pair oligonucleotide. The amount of this 8-base pair product is to the products were amplified product produced. The a 30% proportional amount of digestion resolved on polyacrylamide gel and visualized by autoradiography. that the amplification was comparable in efficiency to that of amplification with PCO3 (5'—ACACAACTGTGTTCACTAGC-3') and PC04 (5'- CCACTTGCACCTACTTCAAC-3'), which are complementary to the negative and positive strands, respectively, of the wild-type 5-globin.

Analysis of the autoradiogram revealed primers which is publicly available from Boehringer-Mannheim. the sample was The entire ligation mixture was transformed into ii; coli strain JM103, which is publicly available from BRL in Bethesda, MD. followed for strain is The procedure preparing the transformed described in Messing, J. (1981) Third Cleveland Symposiimn on Macro- molecules:Recombinant DNA, ed. A. Walton, Elsevier, Nnsterdam, 143- 153.

The transformation mixture was plated onto x-gal media for screening via plaque hybridization with nylon filters. The filters were probed with a B-globin-specific oligonucleotide probe R524 of the '—CCCACAGGGCAGTAACGGCAGACTTCTCCTCAGGAGTCAG-3' to determine The filters were then reprobed with sequence the number of B—globin inserts. the primer PCO4 to determine the total number of inserts.

Plating and Screening ’ Table II summarizes the plating and plaque hybridization data. The filters were probed with the primer PCO4 to determine the percentage of inserts resulting from amplification and cloning; 1206 clear plaques (90% of total number of clear plaques) hybridized to the primer. Fifteen plaques hybridized to the B-globin specific probe RS24. primer-positive plaques is approximately 1%.

The percentage of B-globin positive plaques among the amplified TABLE 11 Blue No B-Globin Plate No. Plaques Inserts* 1nserts** Inserts 1 28 25 246 1 2 29 18 222 2 3 11 25 180 0 4 24 20 192 S 22 27 185 5 6 39 21 181 3 TOTAL 158 132 1206 15 % of plaques containing amplified sequences which contain B-globin insert = 15/1206 x 100 = 1.24% % of total plaques which contain 5-globin insert = ca.1% /1496 x 100 = % of total plaques which contain amplified sequences = 1206/1496 x 100 = 0.8% * Clear plaques which do not hybridize to primer PCO4 ** Clear plaques which hybridize to primer PCO4 Restriction Enzyme and Southern Blot Analysis DNA from phage DNA minipreparation of three B—globin positive and two B-globin negative (but PCO4 primer positive) plaques were analyzed by restriction enzyme analysis. MstII digestion of DNA from M13 clones containing the amplified 5-globin fragment 'should generate a characteristic 283 base-pair fragment. Following MstII , digestion, the three 5-globin positive clones all produced the predicted 283 base pair fragment, while the two clones which were positive only with the primer produced larger fragments. the 6—globin probe.

Seguence Analysis Ten 5-globin positive clones which were shown by restriction enzyme analysis to contain the 5—globin insert were sequenced using the M13—dideoxy sequencing method. Of the ten clones, nine were identical to the B-globin wild-type sequence. The other clone was identical to the o-globin gene which had been shown to be amplified to only a small degree by the B-globin primers.

In conclusion, the modified linker primers were nearly as efficient as the The primers were able to facilitate insertion of amplified unmodified primers in amplifying the 5-globin sequence.

DNA into cloning vectors. Due to the amplification of other segments of the genome, only 1% of the clones contained hemoglobin sequences.

Nine of the ten clones were found to be identical to the published 5-globin showing that the technique amplifies genomic DNA with high fidelity. with the published b—globin sequence, confirming that the primers are the p-globin sequence homology with 6—globin. sequence, One clone was found to be identical significant specific for gene despite their having when cloning was carried out with a 267 base pair fragment only when °C) _in the of the effective dimethylsulfoxide was cloning was (10% by B-globin gene, present volume at amplification procedure.

Restriction site-modified primers were also used to amplify and clone and partially sequence the human N-ras oncogene and to clone 240-base pair segments of the HLA DQ-a and DQ-B genes. All of these amplifications were carried out in the presence of 10% by volume dimethylsulfoxide at 37°C. The primers for amplifying HLA DQ—a and DQ-5 genes were much more specific for their intended targets than were the 5-globin and DR-3 primers, which, rather than giving a discrete band on an ethidium bromide stained agarose gel, produced only a smear. In addition, the HLA DQ—a primers produced up to 20% of clones, with amplified inserts which contained the desired HLA target 1% of the 5—globin contained the target The HLA DQ—a and DQ-B gene cloning was only effective when fragment, whereas clones sequence. the DMSO was present and the temperature was elevated.

EXAMPLE 13 This example illustrates the use of the process herein to gene of 494 oligonucleotides of 74 base pairs each. prepare the TNF base pairs starting from two PRIMERS The primers employed were prepared by the method described in Example 2 and are identified below, each being 74 mers.

(TNIO) 5'-CCTCGTCTACTCCCAGGTCCTCTTCAAGGGCCAAGGCTGCCCCGACTATGTGCTCCTCA- CCCACACCGTCAGCC-3' (TN11) 5'-GGCAGGGGCTCTTGACGGCAGAGAGGAGGTTGACCTTCTCCTGGTAGGAGATGGCGAAG— CGGCTGACGGTGTGG-3' (LL09) 5'—CCTGGCCAATGGCATGGATCTGAAAGATAACCAGCTGGTGGTGCCAGCAGATGGCCTGT- ACCTCGTCTACTCCC-3' (LL12) 5'-CTCCCTGATAGATGGGCTCATACCAGGGCTTGAGCTCAGCCCCCTCTGGGGTGTCCTTC- GGGCAGGGGCTCTTG-3' (TNO8) 5'-TGTAGCAAACCATCAAGTTGAGGAGCAGCTCGAGTGGCTGAGCCAGCGGGCCAATGCCC- TCCTGGCCAATGGCA-3' (TN13) 5'-GATACTTGGGCAGATTGACCTCAGCGCTGAGTTGGTCACCCTTCTCChGCTGGAAGACC- CCTCCCTGATAGATG—3' (LL07) 5'-CCTTAAGCTTATGCTCAGATCATCTTCTCAAAACTCGAGTGACAAGCCTGTAGCCCATP- TTGTAGCAAACCATC-3' (TN14) 5'-GCTCGGATCCTTACAGGGCAATGACTCCAAAGTAGACCTGCCCAGACTCGGCAAAGTCG- » AGATACTTGGGCAGA—3' OVERALL PROCEDURE . Ten cycles of the protocol indicated below were carried out using primers TNIO and TN11, which interact as shown in the diagram below, step (a).

II. A total of 2 pl of the reaction mixture from Part I above added to the primers LL09 and LL12. carried out for 15 cycles, so that the primers would interact with The protocol described below product of Part I as shown in the diagram below, step (b).

III. A total of 2 pl of the reaction mixture from Part II above added to the primers TN08 and TNI3. carried out for 15 cycles, so that the primers would interact with The protocol described below product of Part II as shown in the diagram below, step (C).

IV. A total of 2 pl of the reaction mixture from Part III above added to the primers LL07 and LL14. carried out for 15 cycles, so that the primers would interact with product of Part III as shown in the diagram below, step (d).

The protocol described below PROTOCOL Each reaction contained 100 pl of: 2 mM of each of dATP, dCTP, DGTP and TTP pM of each of the primers used at that step x (30 nM Tris-acetate, 60 mM acetate, 10 mM Mg-acetate, 2.5 mM dithiothreitol) Each cycle constituted: polymerase buffer, was was the was was the was was the ) 1 min. in boiling water 2) 1 min. cooling at room temperature ) add 1 pl (5 units) of the Kl enow fragment of DNA polymerase For the next ) allow the polymerization reaction to proceed for 2 min. cycle start again at step 1.

DIAGRAM a) '- TN10 —?-—-——9 <——*:- 5' TN11 __————%xxxxxxx product from Part I xxxxxxxxxx (--1‘ b) ' LL09~————> xxxxxxxxxx(———————5‘ TN11 'TN10 -———) xxxxxxxxxx L %——————5' LL12 ' LL09 ————9 xxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxx (-j—— 5' TN11 intermediate state + ' TN10 ——% xxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxx %—-—-- 5' LL12 only the sequence between 5' of LL09 and 5' of LL12 will be full length. The strands that con- tain TN10 and TN11 have non- growing 5' ends. Thus...

' LL09 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx xxxxxxxxxxxxxxxxxxxxxxxxxxxxx<————-————————S‘ LL12 This is the product of Part II c) 'TN08 xxxxxxxxxxxxxxxxxxxxxxxxxxxx4————~———————- 5' LL12 + ' LL09 xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx e———————-5' TN13 same intermediate schema as (b) ' LL07 ‘V ——*—-——9XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXX?-—-—-—‘ ' TN14 (TNF gene) Deposit of Materials The cell line SC-1 (CTCC #0082) was deposited on March 19, 1985 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 USA, with ATCC Accession No.

CRL#8756. The deposit of SC-1 was made pursuant to a contract between the ATCC and the this patent Cetus Corporation. The ATCC availability of the progeny of this cell issuance of the U.S. patent describing and identifying the deposit or the publications or upon the laying open to the public of any U.S. or first, and for assignee of application, with permanent contract provides for line to the public on the assignee of the present application has agreed that if the cell line on deposit should die or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced on notification with a viable culture of the same cell line.

In gummary, the present invention is seen to provide a process for detecting sequences in nucleic acids by first amplifying one or more specific nucleic acid sequences using a chain reaction in which primer extension products are produced which can subsequently act as templates for further primer extension reactions. The process in detecting nucleic acid sequences which are Also, the amplification is especially useful initially present in only very small amounts. process can be used for molecular cloning.

Claims (1)

1.Claims A first and second single-stranded oligonucleotide allowing amplification of a specific template nucleic acid sequence contained in a single- or double- stranded nucleic acid or in a mixture of such nucleic acids, wherein ‘ (a) one oligonucleotide of said oligonucleotides contains a part which is substantially complementary to said template nucleic acid sequence in said single-stranded nucleic acid or in one strand of said double-stranded nucleic acid; (b) the other oligonucieotide of said oligonucleotides contains a part which is substantially complementary to a complement of said template nucleic acid sequence in said single-stranded nucleic acid or in §_aic_l strand of said double-stranded nucleic acid; . (c) said parts of oligonucleotides (a) and (b) have attached to their 5‘-end a nucleotide sequence which is non-complementary to said template nucleic A acid sequence and which comprises a restriction site; and wherein (d) the parts of said oligonucleotides of (a) and (b) that have substantial complementarity are different and define the termini of the specific template nucleic acid sequence to be amplified. The oligonucleotides according to claim 1, wherein said specific template nucleic acid sequence is contained within a larger sequence. The oligonucleotides according to claim 1 or 2, wherein said nucleic acid is DNA or RNA, including messenger RNA, which DNA or RNA may be single-stranded or double-stranded, or is a DNA—RNA hybrid. The oligonucleotides according to any one of claims 1 to 3, wherein said nucleic; acid is genomic DNA. The oligonucleotides according to any one of claims 1 to 4, wherein the parts of the oligonucleotides that have substantial complementarity contain at least about 15 to 25 nucleotides. The oligonucleotides according to any one of claims 1 to 5, wherein at least one of said oligonucleotides is a detectable oligonucleotide. Use of the oligonucleotides according to any one of claims ‘l to 6 for amplifying a specific template nucleic acid sequence contained in a single- or double- stranded nucleic acid or in a mixture of such nucleic acids. The use according to claim 7 for enabling amplification, detection and/or characterization of specific template nucleic acid sequences associated with infectious diseases, genetic disorders or cellular disorders. The use according to claim 8, wherein said infectious disease is caused by bacteria, viruses or protozoan parasites. The use according to claim 8, wherein said genetic disorder is caused by specific deletions and/or mutationsin genomic DNA. The use according to claim 8, wherein said cellular disorder is cancer. F. R. KELLY & CO., AGENTS FOR THE APPLICANTS.