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Isothermal strand displacement nucleic acid amplification |
| RE38960 |
Isothermal strand displacement nucleic acid amplification
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| Patent Drawings: | |
| Inventor: |
Dattagupta, et al. |
| Date Issued: |
January 31, 2006 |
| Application: |
10/165,281 |
| Filed: |
June 7, 2002 |
| Inventors: |
Dattagupta; Nanibhushan (San Diego, CA)
Kacian; Daniel Louis (San Diego, CA)
Spingola; Marc (Albuquerque, NM)
Stull; Paul Douglas (San Diego, CA)
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| Assignee: |
Gen-Probe Incorporated (San Diego, CA) |
| Primary Examiner: |
Sitton; Jehanne |
| Assistant Examiner: |
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| Attorney Or Agent: |
Cappellari; Charles B.Fisher; Carlos A. |
| U.S. Class: |
435/287.2; 435/6; 435/91.1; 435/91.2; 536/23.1; 536/24.33 |
| Field Of Search: |
435/6; 435/91.1; 435/91.2; 435/183; 435/194; 435/287.2; 536/23.1; 536/24.3; 536/24.32; 536/24.33 |
| International Class: |
C12P 19/34; C12M 1/34; C07H 21/04; C07H 21/02; C12Q 1/68 |
| U.S Patent Documents: |
4683195; 4683202; 4786600; 4800159; 5043272; 5108892; 5223414; 5256775; 5270184; 5384242; 5786183; 6087133 |
| Foreign Patent Documents: |
0200362; 0300796; 0329822; 0320308; 0395398; 0408295; 0427073; 0427074; 0497272; 0500224; 0543612; 8706270; 8810315; 8901050; 9102818; 9110746; WO 94/03635 |
| Other References: |
Feinburg & Vogelstein, "A Technique for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity," AnalyticalBiochemistry, 132:6-13 (1983). cited by examiner.
Murakawa et al., "Direct detection of HIV-1 RNA from AIDS and ARC patient samples," DNA, 7:287-295 (1988). cited by examiner.
Shaw et al., "Modified deoxyoligonucleotides stable to exonuclease degradation in serum," Nucleic Acids Research, 19(4):747-750 (1991). cited by examiner.
Titeeva et al., "Complexing of Single-Stranded Phage DNA with Synthetic Oligodeoxynucleotides and the Reaction of the Complexes With DNA Polymerase and Restriction Endonucleases," Bioorganicheskaya Khimiya, 12(11):1484-1491 (1986). EnglishTranslation. cited by examiner.
Walker et al., "Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system," Proc. Natl. Acad. Sci. U.S.A., 89:392-396 (1992). cited by examiner.
Walker et al., "Strand displacement amplification--an isothermal, in vitro DNA amplification technique," Nucleic Acids Research, 20:1691-1696 (1992). cited by examiner.
Chien et al., "DNA polymerase from the extreme thermophile Thermus aquaticus, " J. Bacteriology, 127:1550-1557 (1976). cited by examiner.
Kaledin et al., "Isolation and properties of DNA polymerase from extremely thermophilic bacterium Thermus aquaticus YT1," Biokhimiya, (45)4:644-651 (1980). English Translation. cited by examiner. |
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| Abstract: |
Methods for amplifying target nucleic acid sequences using a nucleic acid polymerase lacking 5' exonuclease activity and a set of oligonucleotide primers. Preferably, a primer array is used. The primer array contains two sets of primers. One set contains at least two complementary primers. The other set contains at least two sense primers. Using the described methods amplification can be carried out under essentially constant environmental conditions without the requirement for exonuclease activity or restriction endonuclease activity. |
| Claim: |
What is claimed is:
1. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of: providing the following to a samplecontaining a nucleic acid strand which includes said target sequence: a nucleic acid polymerase lacking 5' exonuclease activity; at least one nucleoside triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate; and at leasttwo different complementary primers and at least two different sense primers under primer extension conditions, wherein each said complementary primer contains a nucleotide base sequence which is designed to be complementary to a nucleic acid sequence onsaid nucleic acid strand located 3' to said target sequence, wherein said nucleotide base sequence of each said complementary primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions, wherein each saidsense primer contains a nucleotide base sequence which is designed to be analogous to a nucleic acid sequence on said nucleic acid strand located 5' to said target sequence, and wherein said nucleotide base sequence of each said sense primer is able tohybridize to a nucleic acid sequence complementary to a nucleic acid sequence on said nucleic acid strand under said conditions; and amplifying said target sequence at an essentially constant temperature and in the absence of an added restrictionendonuclease active on a product of said amplification, wherein said amplifying step is performed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.
2. The method of claim 1, wherein: said complementary primers which are adjacent to one another are separated at their closest 5' and 3' ends by a distance of 1 to 200 nucleotides; and said sense primers which are adjacent to one another areseparated at their closest 5' and 3' ends by a distance of 1 to 200 nucleotides.
3. The method of claim 1, wherein: said complementary primers which are adjacent to one another are separated at their closest 5' and 3' ends by a distance of up to 10 nucleotides; and said sense primers which are adjacent to one another areseparated at their closest 5' and 3' ends by a distance of up to 10 nucleotides.
4. The method of claim 1, wherein at least one of said complementary primers includes a 5' non-complementary region.
5. The method of claim 1, wherein said nucleic acid strand is a single strand of DNA and said polymerase is a DNA polymerase.
6. The method of claim 1, wherein said providing step further includes providing a strand-displacing agent.
7. The method of claim 6, wherein said strand-displacing agent is a helicase, a RecA protein, or a single-stranded nucleic acid binding protein.
8. The method of claim 1, wherein said conditions include dimethyl sulfoxide.
9. The method of claim 1, wherein said essentially constant temperature is at least 43.degree. C.
10. The method of claim 1, wherein said essentially constant temperature is between 50.degree. C. and 70.degree. C.
11. A method for amplifying a specific nucleic acid target sequence preferentially over non-target sequences, comprising the steps of: providing the following to a sample containing a nucleic acid strand which includes said target sequence: anucleic acid polymerase lacking 5' exonuclease activity; at least one nucleoside triphosphate selected from adenosine, thymidine, guanosine and cytidine triphosphate; and a primer array of at least five different primers comprising at least two orthree different complementary primers and at least two or three different sense primers under primer extension conditions, wherein each said complementary primer contains a nucleotide base sequence which is designed to be complementary to a nucleic acidsequence on said nucleic acid strand located 3' to said target sequence, wherein said nucleotide base sequence of each said complementary primer is able to hybridize to a nucleic acid sequence on said nucleic acid strand under said conditions whereineach said sense primer contains a nucleotide base sequence which is designed to be analogous to a nucleic acid sequence on said nucleic acid strand located 5' to said target sequence, and wherein said nucleotide base sequence of each said sense primer isable to hybridize to a nucleic acid sequence complementary to a nucleic acid sequence on said nucleic acid strand under said conditions; and amplifying said target sequence at an essentially constant temperature, wherein said amplifying step isperformed in the absence of a complementary or sense primer containing a promoter site for an RNA polymerase.
12. The method of claim 11, wherein said amplifying step is carried out in the absence of an added restriction endonuclease active on a product of said amplification.
13. The method of claim 11, wherein: said complementary primers which are adjacent to one another are separated at their closest 5' and 3' ends by a distance of 1 to 200 nucleotides; and said sense primers which are adjacent to one another areseparated at their closest 5' and 3' ends by a distance of 1 to 200 nucleotides.
14. The method of claim 11, wherein: said complementary primers which are adjacent to one another are separated at their closest 5' and 3' ends by a distance of up to 10 nucleotides; and said sense primers which are adjacent to one another areseparated at their closest 5' and 3' ends by a distance of up to 10 nucleotides.
15. The method of claim 11, wherein at least one of said complementary primers includes a 5' non-complementary region.
16. The method of claim 11, wherein said nucleic acid strand is a single strand of DNA and said polymerase is a DNA polymerase.
17. The method of claim 11, wherein said providing step further includes providing a strand-displacing agent.
18. The method of claim 17, wherein said strand-displacing agent is a helicase, a RecA protein, or a single-stranded nucleic acid binding protein.
19. The method of claim 11, wherein said conditions include dimethyl sulfoxide.
20. The method of claim 11, wherein said essentially constant temperature is at least 43.degree. C.
21. The method of claim 11, wherein said essentially constant temperature is between 50.degree. C. and 70.degree. C. |
| Description: |
FIELD OF INVENTION
This invention relates to methods for amplifying nucleic acid sequences without thermal cycling.
BACKGROUND OF THE INVENTION
This invention concerns diagnostic procedures and techniques for amplifying a nucleic acid sequence. The detection and quantitation of a specific nucleic acid sequence (i.e., a target sequence) is an increasingly important technique havingnumerous applications including identifying and classifying microorganisms, diagnosing infectious diseases, detecting and characterizing genetic abnormalities, identifying genetic changes associated with cancer, studying genetic susceptibility todisease, measuring the response to various types of treatment, identifying criminal suspects, and resolving paternity disputes.
A common method for detecting and quantitating target nucleic acid sequences is nucleic acid hybridization. Nucleic acid hybridization, typically, uses a labeled nucleic acid probe having a nucleic acid sequence complementary to the targetsequence. The probe is mixed with a sample suspected of containing the target sequence under conditions suitable for hybrid formation. The probe then hybridizes to the target sequence present in the sample. Hybridization can be detected by varioustechniques well known in the art. These techniques include selectively degrading the label present on unhybridized probe and then measuring the amount of label associated with the remaining hybridized probe (Arnold et al., PCT US88/02746).
Numerous methods are available for amplifying nucleic acid strands (i.e., nucleic acid polymers) to increase the amount of target sequence. These methods use the nucleic acid strand containing the target sequence as a template to produce acomplementary nucleic acid strand containing the target sequence. Such methods include the polymerase chain reaction method (PCR), as described by Mullis et al., (See U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159 and European Patent ApplicationNos. 863022-98.4, 86302299.2, and 87300203.4, and Methods in Enzymology, Volume 155, 1987, pp. 335-350). PCR involves the use of a pair of specific oligonucleotides as primers for the two complementary strands of the double-stranded DNA containing thetarget sequence. The primers are chosen to hybridize at the ends of each of the complementary target strands, 3' of the target sequence. Template-dependent DNA synthesis, on each strand, can then be catalyzed using a thermostable DNA polymerase in thepresence of the appropriate reagents. A thermal cycling process is required to form specific hybrids prior to synthesis and then denature the double stranded nucleic acid formed by synthesis. Repeating the cycling process geometrically amplifies thetarget sequence. A PCR method may also be used with an RNA target using RNA-dependent DNA polymerase to create a DNA template.
The PCR method has been coupled to RNA transcription by incorporating a promoter sequence into one of the primers used in the PCR reaction and then, after amplification by the PCR method, using the double-stranded DNA as a template for thetranscription of single-stranded RNA. (See, e.g., Murakawa et al., DNA 7:287-295 (1988)). Other amplification methods use multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets (See, e.g., Burg et al., WO 89/1050;Gingeras et al., WO 88/10315 (sometimes called transcription amplification system or TAS); Kacian and Fultz, EPO Application No. 89313154; Davey and Malek, EPO Application No. 88113948.9; Malek et al., WO91/02818). Urdea, WO91/10746, describe a methodthat achieves signal amplification using a T7 promoter sequence.
The ligase chain reaction (LCR) is described in European Patent Publication 320,308. This method requires at least four separate oligonucleotides, two of which hybridize to the same nucleic acid template so their respective 3' and 5' ends arejuxtaposed for ligation. The hybridized oligonucleotides are then ligated forming a complementary strand on the nucleic acid template. The double-stranded nucleic acid is then denatured, and the third and fourth oligonucleotides are hybridized with thefirst and second oligonucleotides that were joined together. The third and fourth oligonucleotides are then ligated together. Amplification is achieved by further cycles of hybridization, ligation, and denaturation. The Q.beta. replicase (Q.beta.R)method described in PCT Publication No. 87-06270 and U.S. Pat. No. 4,786,600 uses a specific RNA probe which is capable of specific transcription by a replicase enzyme. The method requires the design and synthesis of RNA probes with replicaseinitiation sites.
Amplification methods using palindromic probes are described in EPO Publication Nos. 0427073A and 0427074A. The palindromic probe forms a hairpin with a nucleic acid target sequence. The probe contains a functional promoter located in thehairpin region from which RNA transcripts are produced.
Walker et al., Proc. Natl. Acad. Sci. U.S.A., 89:392-396 (January 1992), Walker et al., Nucl. Acids Res. 20: 1691-1696 (1992), European Patent Application No. EP 0 497 272 A1, and European Patent Application No. EP 0 500 224 A2, describe anoligonucleotide-driven amplification method using a restriction endonuclease. The restriction endonuclease nicks the DNAIDNA complex to enable an extension reaction and, therefore, amplification. Becker, et al., EPO Application No. 88306717.5, describean amplification method in which a primer is hybridized to a nucleic acid sequence and the resulting duplex cleaved prior to the extension reaction and amplification.
Noonan, K. E. et al., Nucl. Acids Res. 16, 10366 (1988), describe using random hexamers to prime reverse transcriptase in a random, nonspecific manner for the synthesis of cDNA from mRNA. Amplification of the desired target is accomplishedwith PCR.
Feinberg, A. P. et al., Anal. Biochem. 132, 6 (1983), and Liang, W. et al., Nucl. Acids Res. 16,3579 (1988), describe the synthesis of DNA strands complementary to a DNA template by combining single-stranded DNA with random hexanucleotideprimers, deoxynucleoside triphosphates, buffer, the Klenow fragment of E. coli DNA polymerase I, and a labeled deoxynucleoside triphosphate.
A related process, known as Random Priming Amplification (RPA) for amplifying nucleic acid is described by Hartley, U.S. Pat. No. 5,043,272. According to Hartley, at columns 2-3, lines 63-2: The process includes the steps of primingsingle-stranded template nucleic acid strands with an excess of random oligonucleotide primers and incubating the single-stranded template nucleic acid strands and excess random oligonucleotide primers in the presence of excess amounts of an inducingagent, a strand displacement agent, and nucleoside triphosphate substrates to randomly amplify nucleic acid strands.
SUMMARY OF THE INVENTION
The present invention features methods for amplifying target nucleic acid sequences using a nucleic acid polymerase lacking 5' exonuclease activity and oligonucleotide primers. Alternatively, the method of the present invention may be performedusing a nucleic acid polymerase having 5' exonuclease activity if the oligonucleotide primers have a 5' modification which prevents or reduces nucleolytic digestion of the primers. Preferably, amplification is carried out using a primer array comprisingtwo sets of primers. One set contains at least two primers complementary to the target nucleic acid. The other set contains at least two primers of the same sense as the target nucleic acid. Using the described methods amplification can be carried outunder essentially constant environmental conditions without the requirement for exonuclease activity or restriction endonuclease activity. Preferably, amplification is carried out using a DNA template, and a DNA polymerase such as a modified Bacillusstearothermophilus (Bst) DNA polymerase which lacks 5' exonuclease activity.
The oligonucleotide members of each primer set are used as initiation points for template-dependent synthesis of a complementary nucleic acid strand, and to enhance displacement of the synthesized complementary strand. Thus, strand displacementand nucleic acid synthesis are carried out in one step. This is a surprising finding, and Applicant is unclear of the exact mechanism by which the procedure operates.
Only one set of primers is believed necessary to effectuate strand displacement and nucleic acid synthesis. A primer array is preferably used to amplify both the initial template and its complement to exponentially amplify a target sequence.
Preferably, the nucleic acid polymerase lacks 5' exonuclease activity, and the procedure is performed at higher temperatures than those at which enzymes such as the Klenow fragment of E. coli DNA polymerase I are optimally active. The reactionis more efficient at 43.degree. C. than at 37.degree. C. Optimal efficiency is obtained at temperatures between 50.degree. C. and 70.degree. C. The method is preferably used on DNA. An RNA target sequence is preferably amplified by first creating aDNA copy using, for example, reverse transcriptase. The DNA copy is then amplified by the methods described herein.
However, the present invention should be able to directly amplify an RNA sequence using an enzyme able to synthesize an RNA complementary strand on an RNA template. Such an enzyme preferably lacks 5' exonuclease activity. For example, QBreplicase used in conjunction with manganese should be suitable for this purpose.
Thus, in a first aspect, the invention features a method for amplifying a target nucleic acid sequence by contacting a nucleic acid strand containing the target sequence with a nucleic acid polymerase lacking 5' exonuclease activity (5'-exo-minuspolymerase), four different nucleotides, and at least two complementary oligonucleotide primers.
Preferably, four different deoxynucleotides and/or three of more primers are used. The amplification is carried out under primer extension conditions at essentially constant temperature.
In related aspects amplification is carried out using a nucleic acid polymerase that has 5' exonuclease activity but under conditions wherein such activity is inhibited, and amplification is carried out using at least two complementaryoligonucleotide primers in the absence of a restriction endonuclease active on the products of any extension reaction of said contacting step.
In another related aspect, the invention features a method for amplifying a target nucleic acid sequence by contacting a nucleic acid strand containing the target sequence with a nucleic acid polymerase lacking 5' exonuclease activity(5'-exo-minus polymerase), four different nucleotides, at least two complementary oligonucleotide primers, and at least two sense primers. The amplification is carried out under primer extension conditions at essentially constant temperature. Preferably, the total number of complementary primers and sense primers is more than 5.
The nucleic acid strand present prior to amplification serves as an initial nucleic acid template for the synthesis of a complementary strand containing the target sequence. The complementary strand can act as a nucleic acid template to producestrands analogous to the initial nucleic acid template. The analogous strands can act as nucleic acid templates. Thus, the resulting amplification of target sequence is geometric.
Primer extension conditions refer to conditions wherein template-dependent amplification initiated at an oligonucleotide primer can occur. Such conditions generally include provision of an appropriate buffer and, optionally, other chemicals thatmay stabilize the polymerase, or enhance primer extension reactions.
"Template" refers to a nucleic acid strand, such as DNA or RNA, which can serve as a substrate for the synthesis of a complementary nucleic acid strand.
A "target sequence" refers to a specific nucleic acid sequence contained on a single strand of an oligonucleotide or nucleic acid molecule, or the nucleic acid sequence complementary thereto.
A "complementary primer" refers to an oligonucleotide primer having a nucleic acid sequence complementary to a nucleic acid sequence present on the nucleic acid template. Complementary primers are designed to be complementary to nucleic acidsequences, on the nucleic acid template, in regions 3' to the target sequence.
By "complementary" is meant that the oligonucleotide primer can hybridize to a region of the nucleic acid template under the conditions of the contacting step so it can be extended in a primer extension reaction. Generally, such oligonucleotideshave a region of complementarity to the template of between 15 and 100 bases, preferably between about 20 and 50 bases. Preferably the region of complementarity is 100% complementary. That is, each nucleotide in the primer complementary region canhydrogen bond with a nucleotide present on the single-stranded template.
However, those of ordinary skill in the art will recognize that primers having a complementary region less than 100% complementary to the template will operate in this method. Generally, such complementarity can be as few as 15 out of 18contiguous bases.
A "sense primer" refers to an oligonucleotide analogous to a nucleic acid sequence present on the nucleic acid template. A sense primer is designed to be analogous to a region on the template 5' of the target sequence.
By "analogous" is meant that the oligonucleotide has essentially the same, or equivalent, nucleotide sequence as that in the nucleic acid template. Thus, for example, the oligonucleotide may contain uracil in place of a thymine present in thenucleic acid template. The analogous oligonucleotides are preferably sufficiently duplicative of the template so they can act as a complementary primer when hybridized to a nucleic acid that is complementary to the nucleic acid template, under primerextension conditions.
Thus, reference to complementary primers and sense primers is made with respect to a particular template. The same primer can act as a complementary primer or a sense primer depending upon whether it is analogous to, or complementary to, aparticular template.
Primer-dependent amplification of a target sequence present on a nucleic acid template is achieved in the contacting step. The degree of amplification has been found to increase with increasing numbers of primers. Examples are provided belowusing as many as 13 primers (seven sense and six complementary), and as few as two primers (two complementary primers). These examples illustrate the ability of primers to cause amplification and are not intended to limit the invention. Preferably, thecomplementary primer set and the sense primer set each contain more than two primers. In preferred embodiments, at least 4 sense primers and 4 complementary primers are used; at least 7 sense primers and 7 complementary primers are used; and at least 10sense primers and 10 complementary primers are used.
As illustrated in the FIGURE, a set of complementary primers comprise oligonucleotides complementary to the template in regions 3' of the target sequence. A set of sense primers comprise oligonucleotides analogous to the template in regions 5'of the target sequence on the same strand. The oligonucleotides are selected so a primer extension reaction is initiated on the nucleic acid template at a site different from the other oligonucleotide primers, and preferably at a site distinct from theregion in which the other oligonucleotide primers form base pairs. Preferably, the oligonucleotides in a primer set are separated at their closest 5' and 3' ends from one another by 1 to 200 bases, and more preferably, are separated by between 1 and 10bases.
By "four different nucleotides" is meant that adenosine (A), guanosine (G), cytidine (C) thymidine (T), and uridine (U) triphosphates, or an equivalent nucleotide which can be substituted for an adenosine, guanosine, cytidine, thymidine, oruridine nucleotide triphosphate, are present as substrates for nucleic acid synthesis. Preferably, deoxynucleotides are used.
By "amplifying" is meant increasing the number of target sequences.
By "contacting" is meant that the nucleic acid template, the nucleic acid polymerase, the four different nucleotides, and the oligonucleotide primer array are brought together under primer extension conditions.
By "essentially constant temperature" is meant that the user makes no attempt to raise or lower the temperature in any profound manner as is used in the polymerase chain reaction to alternatively carry out primer extension and denature theextension products. Thus, the temperature is kept below the temperature at which the template:primer extension product duplex dissociates. Preferably, the temperature is kept below 90.degree. C. More preferably, the reaction temperature is maintainedat about the optimum temperature for the reaction. Generally, such an optimum is between 42.degree. C. and 60.degree. C. and is preferably between 50.degree. C. and 70.degree. C.
In preferred embodiments, the conditions include the provision of DMSO; the essentially constant temperature is at least 43.degree. C., and most preferably between 50.degree. C. and 70.degree. C.; the conditions include a strand separationagent such as a helicase, at least one nucleotide triphosphate, selected from a protein having an activity corresponding to the RecA protein of E. coli, or a single-stranded nucleic acid binding protein; the DNA polymerase is a 5' exominus Bst DNApolymerase I or a modified Taq DNA polymerase; and at least one of the oligonucleotide primers includes a 5' non-complementary region. The RecA protein is not necessary for amplification in this invention, but rather can be used to enhance suchamplification.
By "5' exonuclease minus" is meant that the nucleic acid polymerase has essentially no 5' exonuclease activity associated with it, or has been treated such that the exonuclease activity is reduced or inactive. The preferred nucleic acidpolymerase is a 5' exonuclease minus DNA polymerase. Examples of DNA polymerases having no 5' exonuclease activity include a proteolytic fragment of DNA polymerase I from Bacillus stearothermophilus, and the DNA polymerase from Thermus aquaticus havingits domain encoding 5' exonuclease activity removed as described by Barnes, "Thermostable DNA Polymerase," PCT/US91/07084. The exonuclease minus polymerase of the Klenow fragment described by Walker, supra, may also be used.
In a related aspect, the invention features a method and means for amplifying a nucleic acid target sequence by the step of contacting the nucleic acid containing the target sequence with a nucleic acid polymerase that may have 5' exonucleaseactivity but under conditions wherein such activity is inhibited. Specifically, at least one oligonucleotide primer is modified to be resistant to nucleolytic degradation by any 5'-exonuclease present in the nucleic acid polymerase. Preferably, atleast one complementary and one sense primer is modified to be resistant to 5'-exonuclease activity. More preferably all primers are resistant to such degradation.
Thus in this aspect, the contacting step contains four different nucleotide triphosphates, at least two different complementary oligonucleotide primers and at least two different oligonucleotide sense primers, one or more of which are blocked. The reaction is carried out under oligonucleotide primer extension conditions at essentially constant temperature in the absence of a restriction endonuclease active on the products of any extension reaction of the contacting step.
In related aspects, amplification is carried out using a primer array and either a 5'-exo-minus DNA polymerase or a DNA polymerase containing 5' exonuclease activity used in conjunction with a 5'-exonuclease resistant primers.
As noted above, the present invention does not require a restriction endonuclease active on the products of any primer extension reaction resulting from the contacting step. In contrast to the description in Walker, supra, where the presence ofsuch activity appears to be essential, Applicant has surprisingly found that no restriction enzyme is needed to create a single-stranded region which can be used by oligonucleotide primers in the contacting step.
Thus, in another aspect the invention features a method for amplifying a target nucleic acid sequence by contacting a nucleic acid strand containing the target sequence with a nucleic acid polymerase lacking 5' exonuclease activity, fourdifferent nucleotides, a set of at least two complementary oligonucleotide primers, and a set of at least two sense primers. The amplification is carried out under primer extension conditions at essentially constant temperature in the absence of anyrestriction endonuclease active on the products of any extension reaction. The amplification is preferably carried out as described above.
In related aspects amplification is performed without any restriction endonuclease active on the products of any extension reaction using a nucleic acid polymerase containing 5' exonuclease activity in conjunction with a 5'-exonuclease-resistantprimer; or a single stranded DNA substrate and either a 5'-exo-minus DNA polymerase or a DNA polymerase containing 5' exonuclease activity used in conjunction with a 5'-exonuclease-resistant primer. Such amplifications are preferably carried out asdescribed above.
In this invention, amplification can be allowed to proceed for any desired length of time with product accumulating as a result of amplification of the target sequence. Where the target sequence is of analytical interest, high sensitivitydetection of the target sequence can be accomplished by amplifying the target in the manner of the present invention and then detecting the accumulated amplification product using techniques known in the art.
The present amplification method provides a number of significant benefits. The present method requires only a nucleic acid polymerase as opposed to several different enzymes, amplification of a specific nucleic acid sequence is more efficientand requires less reagent than a random amplification process, and there is no need for time-consuming and cumbersome thermal cycling.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims.
BRIEF DESCRIPTION OF THE DRAWING
The FIGURE is an example illustrating the location of primers relative to each other and a target sequence. The FIGURE shows four complementary primers ("-"), four sense ("+") primers, and the location of a target sequence (rectangular box).
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention features methods for amplifying a target nucleic acid sequence present in a nucleic acid, or a mixture of nucleic acids, by a process which includes synthesis of nucleic acid sequences in a specifically-primedtemplate-dependent manner with a nucleic acid polymerase (e.g., a DNA polymerase) and oligonucleotides primers, preferably, a primer array. A primer array is made up of a set of complementary primers and a set of sense primers. Nucleic acid synthesisis initiated at a complementary primer. Without being bound to any theory, it is believed that primer-dependent synthesis occurring from one primer causes strand displacement of a strand synthesized from a different primer.
Thus, the amplification can be performed without modifying reaction conditions such as temperature, pH, or ionic strength during the amplification procedure to alternatively carry out amplification and denature the double-stranded nucleic acidproduced during amplification. In addition, amplification can be performed without producing single-stranded substrates using enzymes having exonuclease activity or restriction endonuclease activity.
The process includes the step of priming template nucleic acid strands with a primer array of sequence-specific oligonucleotide primers, for each different target sequence to be amplified. The mixture is incubated in the presence of a nucleicacid polymerase, nucleotide substrates, and any necessary co-factors needed to amplify the target nucleic acid sequence.
As a result of the methods described herein, multiple rounds of template-dependent nucleic acid synthesis occur, concomitant with strand displacement of the previously synthesized strand. The displaced strand can act as a template in conjunctionwith the "sense primers", which now act as complementary primers.
Oligonucleotide Primer
The term "primer" refers to an oligonucleotide (whether occurring naturally as in a purified restriction digest, or produced synthetically) which can act as a point of initiation of nucleic acid synthesis on a nucleic acid template when contactedunder primer extension conditions. Appropriate conditions include the presence of nucleotide substrates, a nucleic acid polymerase (e.g., a 5'-exo-minus DNA polymerase), essentially constant temperature and a suitable pH. The nucleic acid synthesisoccurs in a template-dependent manner wherein the sequence of the newly synthesized nucleic acid strand is complementary to the sequence of the nucleic acid template.
Hybridization of the primer to the template may be inhibited by primer secondary structure arising from intramolecular or intermolecular hydrogen bonding. Thus the primer preferably lacks secondary structure and is single-stranded. A primerhaving a secondary structure which would inhibit hybridization to the template is preferably treated to denature or separate its strand(s) prior to amplification.
Preferably, the primer is an oligodeoxynucleotide and is sufficiently long to be used as a substrate for the synthesis of extension products by a nucleic acid polymerase. The appropriate primer length should take into account several factors,including temperature, structure of primer, and how the primer is used. For example, for diagnostic applications the oligonucleotide primer typically contains at least 15 nucleotides depending on the complexity of the target sequence. Otherapplications may require shorter oligonucleotide primers. It may be desirable to carry out these other application at cooler temperatures to form stable hybrid complexes with the template.
A nucleic acid polymerase as used in the invention refers to a chemical, physical, or biological agent which brings about the synthesis of either ribo- or deoxyribonucleotides into a nucleic acid polymer, or strand, in a template-dependentmanner. Examples of nucleic acid polymerases include DNA polymerases. DNA polymerases bring about nucleic acid synthesis in a template-dependent manner in a 5' to 3' direction. Because of the complementarity between the template and the synthesizedstrand, this direction would be from a 3' region on the template to a 5' region on the template. The large fragment of DNA polymerase I from Bacillus stearothermophilus (Bst DNA polymerase I) lacking 5'-3' exonucleolytic activity is a preferred DNApolymerase. Other suitable DNA polymerases include Klenow polymerase and bacteriophage T7 DNA polymerase. The use of 5' blocked primers not susceptible to nucleolytic degradation should allow the use of DNA polymerases possessing 5'-3' exonucleolyticactivity.
Complementary primers are selected to have a region complementary to the nucleic acid template. The primer sequence need not reflect the exact sequence of the template but must be able to hybridize with a template strand. Non-complementarybases can be interspersed into the primer provided the primer sequence has sufficient complementarity to hybridize with its corresponding sequence in the nucleic acid template such that the extension reaction can occur. The newly-formed extensionproduct, once displaced, can act as a template for additional nucleic acid synthesis.
Preferably, the primers contain a non-complementary 5' region. Thus, the preferred primer contains a complementary region enabling it to hybridize to the nucleic acid template, and a non-complementary region. The presence of a non-complementaryregion has been found to enhance strand displacement.
The primer sequence can include one or more of the deoxyribonucleotide bases A, T, C, or G; and/or, one or more of the ribonucleotide bases A, U, C, or G and/or one or more modified nucleotide (deoxyribonucleotide or ribonucleotide) wherein themodification does not prevent hybridization of the primer to the nucleic acid, or elongation of the primer, or strand displacement.
Primers may be modified with chemical groups to enhance their performance or to facilitate the characterization of amplification products. For example, backbone-modified oligonucleotides such as phosphorothioates or methylphosphonates which areresistant to the nucleolytic activity of certain polymerases allow the use of such enzymes. Another example of modification involves using non-nucleotide linkers (e.g., Arnold et al., EPA 88 308 766-0) which do not interfere with elongation of theprimer. Oligonucleotides containing mixtures of the desired modified and natural nucleotides may be used as well.
Primers may also contain reactive sites for enzymes such as restriction endonuclease cleavage sites or promotor sites for RNA polymerases. Examples of the use of such sites include cloning of amplification products and transcription ofamplification products after amplification. Such sites are not necessary for amplification, but are helpful for subsequent manipulations of the amplified nucleic acids.
The primers of the present invention can be prepared by methods known in the art. Preferably, the primers are synthesized by chemical methods. For example, Caruthers et al., in: Methods In Enzymology, vol. 154, p. 287 (1987), describe usingstandard phosphoramidite solid-phase chemistry to join nucleotides by phosphodiester linkages. Bhatt, U.S. Pat. No. 5,449,769 entitled "Method and reagent for sulfurization of organophosphorous compounds" filed Mar. 6, 1989, assigned to Gen-ProbeIncorporated, the assignee of the present invention, describe a procedure for synthesizing oligonucleotides containing phosphorothioate linkages. Klem et al., entitled "Improved process for the synthesis of oligomers" PCT WO92/07864, describe thesynthesis of oligonucleotides having different linkages including methylphosphonate linkages. (These references are hereby incorporated by reference herein.)
Automated synthesizers are well suited for oligonucleotide synthesis. In an automated synthesizer, one starts with a 5'-protected, immobilized (through the 3'-hydroxyl) nucleoside residue. After 5'-deprotection, a phosphite ester linkage isintroduced by reaction with a 3'-phosphoramidite, 5'-protected nucleoside residue. The phosphite linkage is then oxidized to a stable linkage and the process is cycled with the desired nucleotide residues introduced into the synthesized sequence. Theoxidative procedure can result in a typical phosphate ester or a phosphorothioate ester (Arnold et al., supra, EPA 88 308 766-0) Introduction of non-nucleotide phosphoramidite derivatives can be performed during automated synthesis. Also, thesynthesized oligonucleotide can be further modified by synthetic methods (e.g., as described in U.S. Pat. No. 4,818,681).
The 5' end of the primer can be modified to be resistant to 5'-exonuclease activity present in nucleic acid polymerase. Such modifications can be carried out by adding a non-nucleotide group to the terminal 5' nucleotide of the primer usingtechniques such as those described by Arnold et al., U.S. Pat. No. 5,585,481, entitled "Linking Reagents for Nucleotide Probes" and the CLONTECH product catalogue describing the use of label-ONTM regents, hereby incorporated by reference herein.
Test Sample
In general any source of nucleic acid, in purified or non-purified form, can be utilized as the starting nucleic acid or acids provided it contains or is suspected of containing the target nucleic acid sequence. Depending on the source andpurity of the nucleic acid, the starting nucleic acid may need to be purified prior to amplification. Purification of starting nucleic acid is preferably carried out when the nucleic acid source contains substances which inhibit amplification. Nucleicacid purification can be carried out using standard techniques.
The target sequence may be present in DNA or RNA, which may be single-stranded or double-stranded. A mixture of these nucleic acids or the nucleic acid from a previous amplification may also be utilized. The nucleic acid or mixture of nucleicacids may contain more than one target nucleic acid sequence which may be the same or different. The invention is therefore useful for simultaneously amplifying one or more different target nucleic acid sequences located on the same or different targetnucleic acid strands.
The sample suspected or known to contain the intended target nucleic acid may be obtained from a variety of sources. Such sources include a biological sample; a food or agricultural sample; an environmental sample; body fluid or exudate such asurine, blood, milk, cerebrospinal fluid, sputum, saliva, stool, lung aspirates; and throat or genital swabs.
It may be desirable to treat the test samples to obtain nucleic acids suitable for amplification. For example, in certain circumstances, it is desirable to treat the test sample to release, extract, or denature, the target nucleic acid prior toamplification. Such treatments can remove impurities which inhibit amplification, and denature the nucleic acid to minimize the secondary structure (i.e., double-stranded DNA and hairpin loops). These treatments can be carried out by means well knownin the art.
Primers are designed to hybridize to specified regions of the target nucleic acid. Two sets of primers are preferably used. One set of primers, complementary primers, are designed to hybridize to a nucleic acid sequence present on the templatenucleic acid 3' to the target sequence. Hybridization takes place based on hydrogen bonding between complementary nucleotides. The other set of primers, sense primers, are designed to be analogous to regions present on the template nucleic acid 5' tothe target sequence. The members of each primer set are co-linearly arranged as shown diagrammatically in the FIGURE.
Both sets of primers are positioned such that the target sequence, present in the template and the complement to the template, will be amplified. DNA polymerases catalyze nucleic acid synthesis in a 5' to 3' direction (with respect to thetemplate nucleotides are added from a 3' template region to a 5' template region). A complementary strand is produced from the initial template using the complementary primer. The complementary strand can act as a template for template-dependentnucleic acid synthesis.
If the sense primer is analogous to a region on the nucleic acid template 5' to the target sequence, it will be complementary to a region 3' to the target sequence on the complementary strand. The sense primer can act as a complementary primerwith respect to the complementary strand. Template-dependent nucleic acid synthesis can then occur on the complementary strand thereby producing more target sequence.
Different numbers of complementary and sense primers may be used to amplify a target sequence. It appears that as the number of primers increases, amplification increases. Thus, the total number of complementary primers and sense primers ispreferably more than 5. In preferred embodiments at least 4 sense primers and 4 complementary primers are used; at least 7 sense primers and 7 complementary primers are used; and at least 10 sense primers and 10 complementary primers are used. Thenumber of complementary primers need not equal the number of sense primers. For example, a 2 sense primer and 3 complementary primer combination may be used.
When certain organic solvents such as DMSO are added to the amplification reaction and elevated temperature is used, substantial amplification as well as stringency is observed. If elevated temperature is used to increase strand displacement, aheat-stable nucleic acid polymerase such as a thermostable DNA polymerase may be employed. The temperature selected depends on the temperature range within which the enzyme is active.
The primers, nucleic acid polymerase, and nucleotides must be used in sufficient quantity to support the degree of amplification desired. Nucleotide analogs can be substituted or added provided the base pairing, DNA polymerase, andstrand-displacing functions are not adversely affected to the point that the amplification does not proceed to the desired extent.
"Strand displacement" refers to the dissociation of a nucleic acid strand from its nucleic acid template in a 5' to 3' direction occurring due to template-directed nucleic acid synthesis by a nucleic acid polymerase. The newly synthesized anddisplaced nucleic acids have substantially the same nucleotide sequence which is complementary to the template nucleic acid sequence.
Preferably, a strand separation agent is used to enhance the strand displacement. Strand separation agents include enzymes such as helicases, the enzyme RecA which has helicase activity, and single-stranded nucleic acid binding protein. The useof helicases for separating strands of nucleic acid is described in Cold Spring Harbor Symposia on Quantitative Biology, Vol. XLIII, "DNA: Replication and Recombination" (New York: Cold Spring Harbor Laboratory, 1978) and the use of RecA is reviewed byRadding, Ann. Rev. Genetics, 16: 405 (1982). Strand separation is discussed by Kornberg, A, DNA Replication W. H. Freeman & Co., San Francisco, Calif., 1980. Zarling et al., U.S. Pat. No. 5,223,414 (June 1993) discusses the use of RecA in nucleicacid hybridization and amplification.
Amplification, as discussed above, refers to increasing the number of target sequences. The degree of amplification can be increased by using conditions or agents which increase rates or extent of priming, or primer elongation. For example, astand separation agent may increase the strand displacement rate of a DNA polymerase.
A wide variety of methods are available to detect a target sequence. For example, the nucleotide substrates or the primers can include a detectable label which is incorporated into newly synthesized DNA The resulting labeled amplificationproduct is then separated from the unused labeled nucleotides or primers and the label is detected in the separated product fraction.
Substances which serve as useful detectable labels are well known in the art and include radioactive isotopes (e.g., .sup.32P, .sup.3H, .sup.125I, and .sup.14C), fluorescent compounds, chemiluminescent compounds, chromophores, as well as ligandssuch as biotin and haptens which, while not directly detectable, can be readily detected by reaction with labeled forms of their specific binding partners, e.g., avidin and antibodies, respectively.
Another approach is to detect the amplification product by hybridization with a detectable nucleic acid probe and measuring the resulting hybrids in any conventional manner. In particular, the product can be assayed by hybridizing achemiluminescent acridinium ester-labeled nucleic acid probe to the target sequence, selectively hydrolyzing the acridinium ester present on unhybridized probe, and measuring the chemiluminescence produced from the remaining acridinium ester in aluminometer. (See, e.g., Arnold et al., supra, PCT US88/02746, and Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) (both references hereby incorporated by reference herein).
EXAMPLES
The present invention will now be illustrated, but is not intended to be limited, by the following examples.
Example 1
Amplification of an RNA Target
This example illustrates the amplification of a ribosomal RNA(rRNA) target sequence by producing a DNA template, and amplifying the DNA template using a primer array and a 5'-exo-minus DNA polymerase. Production of the DNA template from rRNA,and amplification of the template, was carried out in the same reaction vessel containing the necessary reagents for production and amplification of the DNA template.
The primer array used in this example comprised thirteen primers. The primers were prepared by standard phosphoramidite chemistry. Seven of the primers were complementary primers (with respect to the rRNA target). These primers had thefollowing nucleic acid sequences (written 5' to 3'):
TABLE-US-00001 SEQ. ID. NO. 1: CGCGTCTAGT CCTACTCAGG TGTTC SEQ. ID. NO. 2: CCCCAGATTC AGACTAGG SEQ. ID. NO. 3: CGGTCATTCA CTAGTATTTA GCCTT SEQ. ID. NO. 4: TTTCTTCATA GTACTGGTTC ACT SEQ. ID. NO. 5: ATTTCACTCC CTTAACAAGG GTT SEQ. ID. NO. 6: TTGTAAGCTA CAGGTTTCAG GT SEQ. ID. NO. 7: ACGGGCAATT GGTCTGCGAC C
Six of the primers were sense primers (with respect to the rRNA target). These primers had the following nucleic acid sequences (written 5' to 3'):
TABLE-US-00002 SEQ. ID. NO. 8: AGCAAAGACC CGGAGGTCCG AAT SEQ. ID. NO. 9: GAATACATAG GTATGCAAAG CGA SEQ. ID. NO. 10: CCTGCCGAAC TGAAACATCT TAGT SEQ. ID. NO. 11: CAGAGGAAAA GAAATCGAAG AGAT SEQ. ID. NO. 12: TGTAGGATTG AGGATAAAGG AT SEQ. ID. NO. 13: GGACTCCTAG TTGAACACAT CTGGAAAGA.
Target 23S rRNA was obtained from Chlamydia trachomatis (See, Glisin et al., Biochemistry 13:2633 (1974)), and diluted in buffer (50 mM Tris-HCl (PH 8.3), 37.5 mM KCl, 1.5 mM MgCl.sub.2, 10 mM DTT). Variable amounts of rRNA target were combinedwith primers (7.times.10.sup.-32 mole) in 20 .mu.l of buffer (7% DMSO, 50 mM Tris-HCl (pH 8.3) 75 mM KCl, 3 mM MgCl.sub.2, and 2 mM DTT) and incubated at 60.degree. C. for 30 minutes to pre-hybridize the primers. The pre-hybridized primers were thentreated with 0.2 mM ATP, 0.2 mM TTP, 0.2 mM GTP, 0.2 mM CTP, 200 units of Maloney Murine Leukemia Virus reverse transcriptase (MMLV-RT), and 2 units of Bst DNA polymerase I. Amplification was carried out at 37.degree. C. for 60 minutes, then at60.degree. C. for 120 minutes.
The amplification products were then denatured and assayed using acridinium ester labeled probe according to Nelson et al., supra. Denaturation was carried out by incubating at 95.degree. C. for 5 minutes. Probe labeling with acridinium esterwas carried out as described by Nelson et al., supra. The acridinium ester labeled probe was synthesized to have the nucleic acid sequence corresponding to SEQ. ID. NO. 27. The acridinium ester labeled probe (2.times.10.sup.-32 moles) was incubatedwith amplified product at 60.degree. C. for 15 minutes in 100 .mu.l of hybridization buffer (100 mM lithium succinate (PH 5.2), 8% lithium dodecyl sulfate, 1.5 mM EDTA, and 1.5 mM EGTA). The acridinium ester present on unhybridized probe was thenhydrolyzed by adding a reagent solution (0.15 M sodium tetraborate, pH 7.6, and 5% (v/v) Triton X-100) and incubating at 60.degree. C. for 10 minutes. The remaining chemiluminescence was measured in a luminometer. The results shown in Table 1demonstrate a titration of the target nucleic acid in the amplification method of the present invention.
TABLE-US-00003 TABLE 1 Chlamydia rRNA (Moles) RLU (mean) 4 .times. 10.sup.-18 307893 4 .times. 10.sup.-19 54771 4 .times. 10.sup.-20 3644 4 .times. 10.sup.-21 2354 0 2307
Example 2
Amplification of a Clinical Specimen
This example illustrates the amplification of an rRNA target sequence present in a clinical sample (cervical swab sample). The oligonucleotides, amplification procedure, and detection procedure used in this example were as described in Example1. This example differs from Example 1 in the type of sample used, and sample preparation.
Cervical swab samples were collected and suspended in a buffered solution containing an anionic detergent (60 mM phosphate buffer (pH 6.8), 6% lithium dodecyl sulfate, 2 mM EDTA, and 2 mM EGTA). The clinical specimen was spiked with 10.sup.-18moles of 23S Chamydia trachomatis rRNA, hybridized with the primers, amplified, and detected, as described in Example 1. A successful amplification of target nucleic acid in the clinical sample was indicated by the clinical sample with Chlamydia rRNAhaving an RLU over 50 fold greater than a clinical sample without Chlamydia rRNA.
Example 3
Specificity of Amplification
This example illustrates the amplification of a specific target sequence using probes directed to a target sequence and a non-target sequence (i.e., a region of the transcript outside the amplicon). The target nucleic acid used in this examplewas derived from gene 10 of T7 bacteriophage. In particular, an XmaI and BanI digest of pGEMX1 containing part of gene 10 of T7 bacteriophage was sub-cloned into pUC19. Expression of the insert with T7 RNA polymerase yielded an RNA product 863 bases inlength and this transcript was used to produce a DNA template which was then amplified.
The primer array, used to amplify the target sequence, was made up of four complementary primers (with respect to the RNA transcript) having the following nucleic acid sequences (written 5' to 3'):
TABLE-US-00004 SEQ. ID. NO. 14 CTATAGGGAG ACCACAACGG TTT SEQ. ID. NO. 15 CTAGAAATAA TTTTGTTTAA CTT SEQ. ID. NO. 16 AGAAGGAGAT ATACATATGG CT SEQ. ID. NO. 17 CATGACTGGT GGACAGCAAA TG
and four primers having the same sense as the RNA transcript, having the following nucleic acid sequences (written 5' to 3'):
TABLE-US-00005 SEQ. ID. NO.18 GAGCGAACGC AGTCAGGA SEQ. ID. NO.19 GAGAAGTGGT CACGGAGGTA CGAGCGAACG CAGT SEQ. ID. NO.20 CCAGATGCAA CGTACCATGT SEQ. ID. NO.21 GGGAACTGAG CGGATTTACC GC
Hybridization, amplification, and detection were carried out as described in Example 1. Acridinium ester-labeled probes, SEQ. ID. NO: 22 and SEQ. ID. NO. 27 were used to detect the presence of the target sequence. A non-target acridiniumester labeled probe (SEQ. ID. NO. 23) was used as a control to determine non-specific amplification occurring outside the primer region (e.g., 3' of the complementary primers and 5' the sense primers). The results shown in Table 2 demonstrate that theamplification is specific.
TABLE-US-00006 TABLE 2 Probe Sample RLU (mean) SEQ. ID. 22 + (4 .times. 10.sup.-19 mole) 240474 SEQ. ID. 27 SEQ. ID. 23 (4 .times. 10.sup.-19 mole) 3091 SEQ. ID. 23 no target 2720
Example 4
Enhanced Strand Displacement
This example demonstrates the ability of a non-complementary region, attached to a primer, to enhance strand displacement and amplification. Strand displacement was measured using two different combinations of complementary primers. Bothcombinations contained a 5'-.sup.32p labeled primer (SEQ. ID. NO. 19) and an additional primer. The additional primer in one combination corresponded to SEQ. ID. NO. 24. The additional primer in the other combination corresponded to SEQ. ID. NO.25. The SEQ. ID. NO. 25 primer was similar to the SEQ. ID. NO. 24 primer, but had five fewer complementary nucleotides and contained a 5' non-complementary region. Amplification was carried out on an oligodeoxynucleotide template corresponding to aportion of the T7 bacteriophage gene 10. The template, corresponding to SEQ. ID. NO: 26, was synthesized as described in Example 1.
The primers (4.times.10.sup.-32 mole) were prehybridized with template (4.times.10.sup.-13 mole) in 20 .mu.l of buffer (50 mM Tris-HCl (pH 8.3), 37.5 mM KCl, 1.5 mM MgCl.sub.2, 10 mM DTT), by incubating at 70.degree. C. for 5 minutes, and thereaction was then allowed to cool to room temperature (about 20-25.degree. C.) over the course of about 2 hours. Amplification was carried out by adding 0.1 mM ATP, 0.1 mM TTP, 0.1 mM GTP, 0.1 mM CTP, 4 units Bst DNA polymerase I and incubating at50.degree. C. Time points were taken at 5, 15, and 30 minutes by transferring aliquots of the reaction to a formamide/EDTA solution (50% formamide 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA). The samples were loaded and run on a denaturing 15% polyacrylamidegel. Bands corresponding to the elongation product and primer were excised from the gel and analyzed by Cerenkov counting in a scintillation counter.
The results shown in Table 3 demonstrate that strand displacement is more efficient using primers with a 5' non-complementary region. The "% product" refers to the amount of elongation product divided by the sum of the amount of elongationproduct and primer. The higher percentage of product observed with the primer having a 5' non-complementary region indicates increased amplification and strand displacement.
TABLE-US-00007 TABLE 3 Primer (SEQ. ID. NO).sup.1 24 24 24 25 25 25 Time 5 min 15 min 30 min 5 min 15 min 30 min % Product 4.3 5.8 5.9 18 25 35 .sup.1In addition to the primers listed in Table 4, each reaction mixture contained a radiolabeledprimer having the nucleotide sequence of SEQ. ID. NO. 19.
The present embodiments of this invention are to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes whichcome within the meaning and range of equivalency of the claims therefore are intended to be embraced therein.
Other embodiments are within the following claims.
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2725nucleic acidsinglelinear TAGT CCTACTCAGG TGTTG 25ic acidsinglelinear 2CCCCAGATTC AGACTAGG leic acidsinglelinear 3CGGTCATTGA CTAGTATTTAGCCTT 2523nucleic acidsinglelinear 4TTTCTTCATA GTACTGGTTC ACT 2323nucleic acidsinglelinear 5ATTTCACTCC CTTAACAAGG GTT 2322nucleic acidsinglelinear 6TTGTAAGCTA CAGGTTTCAG GT 222c acidsinglelinear 7ACGGGCAATT GGTCTGCGAC C 2eicacidsinglelinear 8AGCAAAGACC CGGAGGTCCG AAT 2323nucleic acidsinglelinear 9GAATACATAG GTATGCAAAG CGA 2324nucleic acidsinglelinear CGAAC TGAAACATCT TAGT 2424nucleic acidsinglelinear GAAAA GAAATCGAAG AGAT 2422nucleic acidsinglelinearGATTG AGGATAAAGG AT 2229nucleic acidsinglelinear CCTAG TTGAACACAT CTGGAAAGA 2923nucleic acidsinglelinear GGGAG ACCACAACGG TTT 2323nucleic acidsinglelinear AATAA TTTTGTTTAA CTT 2322nucleic acidsinglelinear GAGATATACATATGG CT 2222nucleic acidsinglelinear CTGGT GGACAGCAAA TG 22ic acidsinglelinear AACGC AGTCAGGA leic acidsinglelinear GTGGT CACGGAGGTA CGAGCGAACG CAGT 342c acidsinglelinear 2GGAA CGTACCATGT 2eicacidsinglelinear 2TGAG CGGATTTACC GC 2227nucleic acidsinglelinear 22GAACAACGCC AGTTTGATCT CCAGCAG 2726nucleic acidsinglelinear 23CAACCTCAAA GGCCCATAAC GTTGCG 2629nucleic acidsinglelinear 24GTCAGGACTT CACCGCCAAA TACCTTCAA 295cacidsinglelinear 25GAAATTAATA CGACTCACTA TAGGGAGACT TCACCGCCAA ATACCTTCAA 5leic acidsinglelinear 26CATGACTGGT GGACAGCAAA TGGGTACTAA CCAAGGTAAA GGTGTAGTTG 5GAGA TAAACTGGCG TTGTTCTTGA AGGTATTTGG CGGTGAAGTC CTGCGT TCGCTCGTAC CTCCGTGACCACTTCTC cleic acidsinglelinear 27CCCGTAGACG AAAGGAGAGA AAGACCG 27
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