Nested PCR employing degradable primers

• US 20030228620A1
• Filed: 05/27/2003
• Published: 12/11/2003
• Est. Priority Date: 05/24/2002
• Status: Active Grant

• Current Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Invitrogen Corp. (Thermo Fisher Scientific Incorporated)
• Inventors: Du Breuil Lastrucci, Rusla M.
• Granted Patent: US 7,273,730 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2547/101 by confinement to a single tube/container
• CPC Class Codes: C12Q 2549/119 using nested primers

Abstract

The invention is directed to one-tube nested nucleic acid amplification methods, as well as compositions for performing these methods, which employ one or more outer primers containing one or more exo-sample nucleotides and inner primers. Nested amplification reactions are performed in these methods in the presence of an agent that degrades the exo-sample-nucleotide-containing primers in time course fashion during the PCR cycles.

Claims

1. A composition, comprising:

a template nucleic acid molecule comprising a target nucleic acid sequence;

at least one exo-sample-nucleotide-containing primer capable of annealing to the template at a specific nucleotide sequence in the template, at least one nucleotide polymerase enzyme and at least one agent capable of degrading an exo-sample nucleotide, with the proviso that the composition does not contain amplified template.

2. A composition according to claim 1, further comprising an additional primer capable of annealing to the template at a nucleotide sequence of the template such that extension of the exo-sample-nucleotide-containing primer is in the direction of the additional primer.

3. A method for amplifying a target nucleic acid sequence present in a template nucleic acid molecule, said method comprising:

(a) mixing the template with an amplification reaction mixture comprising;

(1) an outer primer pair each of which comprises a sequence with sufficient complementarity to a portion of the template to anneal to the template in an amplification reaction, wherein at least one member of the outer primer pair contains at least one exo-sample nucleotide;

(2) at least one inner primer that comprises a sequence with sufficient complementarity to a portion of the template to anneal to the template in an amplification reaction, wherein the portion of the template to which the inner primer anneals is between the portions of the template to which the outer primers anneal;

(3) an agent capable of degrading an exo-sample nucleotide; and

(b) conducting an amplification reaction.

4. The method of claim 3, wherein degrading an exo-sample nucleotide in an outer primer results in degradation of the outer primer.

5. The method of claim 3, wherein the agent is an enzyme.

6. The method of claim 5, wherein the enzyme is a glycosylase.

7. The method of claim 6, wherein the glycosylase is a DNA glycosylase.

8. The method of claim 6, wherein the glycosylase is selected from the group consisting of uracil-DNA glycosylase, UNGs, UNG1, TAG, alkA, MAG, MAG1, N-methyl-purine glycosylase, EndoIII, EndoVIII, EndoIX, NTG1, NTH, hydroxy-methyl-DNA glycosylase, formyluracil-DNA glycosylase;

2,6-dihydroxy-5N-formamiodopyrimidine DNA glycosylase, OGG1, OGG2, S3, PDG, and 5-methyl-cytosine-DNA glycosylase.

9. The method of claim 3, wherein the exo-sample nucleotide is a nucleotide selected from the group consisting of deoxyuridine, bromodeoxyuridine, 7-methylguanine, 5,6-dihdro-5,6-dihydroxydeoxythimidine, 3-methyldeoxadenosine, inosine, 5-bromo-deoxycitidine, 5-methyldeoxycitidine, 5-bromodeoxyuridine, O-6-methyl-deoxyguanosine, 5-iododeoxyuridine, 8-oxo-deoxyguanine, ethanoadenine, and 5,6-dihydrouracil.

10. The method of claim 3, wherein at least one of the outer primers is from about 6 nucleotides to about 1000 nucleotides in length.

11. The method of claim 3, wherein at least one of the inner primers is from about 6 nucleotides to about 1000 nucleotides in length.

12. The method of claim 3, wherein the target nucleic acid sequence is amplified by polymerase chain reaction.

13. The method of claim 3, wherein the target nucleic acid sequence is amplified by two or more polymerase chain reactions.

14. The method of claim 13, wherein the polymerase chain reaction is a reverse-transcriptase polymerase chain reaction or a rapid cycle capillary polymerase chain reaction.

15. A composition comprising:

(a) one or more exo-sample nucleotide degrading agents,

(b) one or more polymerases,

(c) one or more nucleoside triposphates, and

(d) one or more primers comprising at least one exo-sample nucleotide, with the proviso that the composition does not contain amplified nucleic acid.

16. The composition of claim 15, wherein said agent is a DNA glycosylase.

17. The composition of claim 16, wherein said DNA glycosylase is selected from the group consisting of uracil-DNA glycosylase, UNGs, UNG1, TAG, alkA, MAG, MAG1, N-methyl-purine glycosylase, EndoIII, EndoVIII, EndoIX, NTG1, NTH, hydroxy-methyl-DNA glycosylase, formyluracil-DNA glycosylase, 2,6-dihydroxy-5N-formamiodopyrimidine DNA glycosylase, OGG1, OGG2, S3, PDG, and 5-methyl-cytosine-DNA glycosylase.

18. The composition of any one of claims 16 or 17 wherein at least one glycosylase is thermostable.

19. The composition of claim 18, wherein said thermostable glycosylase retains at least 5% of the original glycosylase activity after heating to 95°

C. for 30 minutes.

20. The composition of claim 15, wherein said polymerase is a DNA polymerase.

21. The composition of claim 15, wherein said polymerase is a thermostable polymerase.

22. The composition of claim 15, wherein said polymerase is a thermostable DNA polymerase.

23. The composition of claim 22, wherein said thermostable DNA polymerase retains at least 5% of the original polymerase activity after heating to 95°

C. for 30 minutes.

24. A kit comprising instructions for performing nested polymerase chain reaction in which the relative concentration of one or more primers decreases with respect to one or more other primers during amplification of a target nucleic acid molecule.

25. The kit of claim 24 further comprising:

(a) one or more glycosylases,

(b) one or more buffers,

(c) one or more nucleoside triphosphates

(d) one or more containers,

(e) one or more polymerases,

(f) one or more containers containing water, and

(g) magnesium chloride or one or more solutions containing magnesium chloride.

26. The kit of claim 25 comprising one or more polymerases with one or more antibodies or one or more polymerases with one or more single stranded nucleic acid binding proteins.

27. The kit of claim 25 comprising one or more exo-sample-nucleotide-containing primers.

28. The kit of claim 25, wherein said glycosylase is thermostable.

29. The kit of claim 28, wherein said thermostable glycosylase retains at least 5% of the original glycosylase activity after heating to 95°

C. for 30 minutes.

30. The kit of claim 25 or claim 26, wherein said polymerase is a DNA polymerase.

31. The kit of claim 25 or claim 26, wherein said polymerase is a thermostable DNA polymerase.

32. The kit of claim 31, wherein said thermostable DNA polymerase retains at least 5% of the original polymerase activity after heating to 95°

C. for 30 minutes.