Generation and amplification of nucleic acids from ribonucleic acids

• US 6,300,069 B1
• Filed: 05/03/1999
• Issued: 10/09/2001
• Est. Priority Date: 05/03/1999
• Status: Expired due to Term

• Current Assignee: Qiagen GmbH (Qiagen NV)
• Original Assignee: Qiagen GmbH (Qiagen NV)
• Inventors: Missel, Andreas, Korfhage, Christian, Kang, Jie, Löffert, Dirk
• Primary Examiner(s): Sisson, Bradley L.
• Application Number:
  US09/304,452
• Time in Patent Office: 890 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/91.3, 435/183, 536/23.1, 536/24.3, 536/24.37, 536/25.3
• US Class Current: 435/6.12
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer

Abstract

Novel compositions and methods useful for the generation of nucleic acids from a ribonucleic acid template and further nucleic acid replication are disclosed. It is shown that the generation and amplification of nucleic acids by methods that utilize two or more different polymerases, such as reverse transcriptase-polymerase chain reaction (RT-PCR), are dramatically more sensitive and efficient in the presence of a homopolymeric nucleic acid. Homopolymeric nucleic acids have been found to reduce or negate the inhibitory effect reverse transcriptases have on DNA polymerase activity. It is demonstrated that this inhibition-relieving effect of homopolymeric nucleic acids is general in nature; independent of the chemical species of homopolymer used, or the chemical composition of the polymerization reaction mixture.

Claims

1. In a method for generating a nucleic acid from an RNA template comprising the steps:

a) adding said RNA template to a reaction mixture, said reaction mixture comprising at least one reverse transcriptase and at least one nucleic acid polymerase, or derivatives thereof; and

b) incubating said reaction mixture under conditions sufficient to allow polymerization of a nucleic acid molecule complementary to a portion of said RNA template,

wherein the improvement comprises a reaction mixture further comprising a homopolymeric oligonucleotide at least 50 bases in length.

2. The method of claim 1, wherein said reverse transcriptase is selected from the group consisting of:

AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C. hydrogenoformans DNA Polymerase, SUPERSCRIPT I (a variant reverse transcriptase isolated from Murine Leukemia Virus), and SUPERSCRIPT II (a variant reverse transcriptase isolated from Murine Leukemia Virus).

3. The method of claim 1, wherein said reaction mixture further comprises a nucleic acid primer.

4. The method of claim 1, wherein said DNA molecule complementary to a portion of said RNA template is amplified by a process of DNA replication.

5. The method of claim 3, wherein said primer is complementary to a portion of said RNA template.

6. The method of claim 4, wherein said process of DNA replication comprises a polymerase chain reaction (PCR).

7. The method of claim 4, wherein the combined reactions of DNA generation and amplification occurs in a coupled reaction mixture.

8. The method of claim 7, wherein the reaction proceeds between about 23°

C. to about 100°

C.

9. The method of claim 8, wherein reverse transcription occurs between about 37°

C. to about 70°

C. and said PCR amplification occurs between about 60°

C. to about 95°

C.

10. The method of claim 9, wherein said PCR amplification comprises at least 25 cycles of denaturation, annealization, and polymerization.

11. The method of claim 10, wherein said reverse transcription occurs between about 37°

C. to about 60°

C., denaturation occurs at about 94°

C., annealization occurs at about 60°

C., and polymerization occurs at about 72°

C.

12. In a method for generating a nucleic acid from an RNA template comprising the steps:

a) adding said RNA template to a reaction mixture, said reaction mixture comprising at least one reverse transcriptase, at least one nucleic acid polymerase, and one or more ribonucleotide triphosphate or derivatives thereof; and

b) incubating said reaction mixture under conditions sufficient to allow polymerization of a nucleic acid molecule complementary to a portion of said RNA template,

wherein the improvement comprises a reaction mixture further comprising a homopolymeric oligonucleotide at least 50 bases in length.

13. The method of claim 12 wherein said nucleic acid is DNA, said polymerase is DNA polymerase, and said ribonucleic acid triphosphate derivative is selected from the group consisting of;

dATP, dCTP, dGTP, dTTP, dITP, dUTP, α

-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and digoxigenin-dUTP.

14. The method of claim 12, wherein said homopolymeric oligonucleotide is a homopolymeric ribonucleic acid.

15. The method of claim 12, wherein said homopolymeric oligonucleotide is a homopolymeric deoxyribonucleic acid.

16. The method of claim 13, wherein said DNA polymerase is a thermostable polymerase.

17. The method of claim 14, wherein said homopolymeric ribonucleic acid is selected from the group consisting of;

homopolymeric poly(A), homopolymeric poly(U), homopolymeric poly(C), and homopolymeric poly(G).

18. The method of claim 15, wherein said homopolymeric deoxyribonucleic acid is selected from the group consisting of;

homopolymeric poly(dA), homopolymeric poly(dI), homopolymeric poly(dC), homopolymeric poly(dG), and homopolymeric poly(dT).

19. The method of claim 16, wherein said DNA polymerase is selected from the group consisting of;

Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENTM (a variant DNA polymerase isolated from Thermococcus litoralis), and DEEPVENT®

(a variant DNA polymerase isolated from Pyrococcus sp.).

20. In a composition suitable for reverse transcription of RNA, comprising at least one member of the group consisting of:

a reverse transcriptase, a DNA polymerase, one or more oligonucleotide primers, any one or more nucleotides or derivatives thereof, a buffering agent, a salt, and additives useful in RT-PCR, wherein the improvement comprises a homopolymeric oligonucleotide at least 50 bases in length.

21. The composition of claim 20, wherein said homopolymeric oligonucleotide is a homopolymeric ribonucleic acid.

22. The composition of claim 20, wherein said homopolymeric oligonucleotide is a homopolymeric deoxyribonucleic acid.

23. The composition of claim 20, wherein the composition contains at least 10 ng of homopolymer.

24. The composition of claim 20 further comprising a reverse transcriptase.

25. The composition of claim 20 further comprising a DNA polymerase.

26. The composition of claim 20 further comprising one or more oligonucleotide primers.

27. The composition of claim 20 further comprising a buffering agent.

28. The composition of claim 20 further comprising a salt solution.

29. The composition of claim 20 further comprising an additive useful in RT-PCR.

30. The composition of claim 20 further comprising one or more nucleotides.

31. The composition of claim 20, wherein said homopolymeric oligonucleotide is at least 100 bases in length.

32. The composition of claim 21, wherein said homopolymeric ribonucleic acid is selected from the group consisting of, homopolymeric poly(A), homopolymeric poly(U), homopolymeric poly(C), and homopolymeric poly(G).

33. The composition of claim 22, wherein said homopolymeric deoxyribonucleic acid is selected from the group consisting of;

homopolymeric poly(dA), homopolymeric poly(dI), homopolymeric poly(dC), homopolymeric poly(dG), and homopolymeric poly(dT).

34. The composition of claim 23, wherein the composition contains between 10 to 2000 ng of homopolymer.

35. The composition of claim 24, wherein said reverse transcriptase is selected from the group consisting of:

AMV-RT, M-MLV-RT, HIV-RT, EIAV-RT, RAV2-RT, C. hydrogenoformans DNA Polymerase, SUPERSCRIPT I (a variant reverse transcriptase isolated from Murine Leukemia Virus), and SUPERSCRIPT II (a variant reverse transcriptase isolated from Murine Leukemia Virus).

36. The composition of claim 25, wherein said DNA polymerase is selected from the group consisting of;

Taq, Tbr, Tfl, Tru, Tth, Tli, Tac, Tne, Tma, Tih, Tfi, Pfu, Pwo, Kod, Bst, Sac, Sso, Poc, Pab, Mth, Pho, ES4, VENT®

(a variant DNA polymerase isolated from Thermococcus litoralis), and DEEPVENT®

(a variant DNA polymerase isolated from Pyrococcus sp.).

37. The composition of claim 26, wherein said primer is selected from the group consisting of;

random primers, homopolymers, and primers specific to a target RNA template.

38. The composition of claim 27, wherein said buffering agent is selected from the group consisting of;

TRIS, TRICINE, BIS-TRICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS.

39. The composition of claim 28, wherein said salt solution is selected from the group consisting of;

potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, ammonium acetate, magnesium chloride, magnesium acetate, magnesium sulfate, manganese chloride, manganese acetate, manganese sulfate, sodium chloride, sodium acetate, lithium chloride, and lithium acetate.

40. The composition of claim 29, wherein said additive useful in RT-PCR is selected from the group consisting of:

DMSO, glycerol, formamide, betain, tetramethylammonium chloride, PEG, non-ionic surfacant, ectoine, polyols, E. coli SSB protein, Phage T4 gene 32 protein, and BSA.

41. The composition of claim 30, wherein said nucleotide is a deoxyphosphonucleotide or derivative thereof.

42. The composition of claim 41, wherein said deoxyphosphonucleotide is selected from the group consisting of;

DATP, dCTP, dGTP, dTTP, dITP, dUTP, (α

-thio-dNTPs, biotin-dUTP, fluorescein-dUTP, and digoxigenin-dUTP.

43. The composition of claim 34, wherein the composition contains between 100 to 1000 ng of homopolymer.

44. The composition of claim 43, wherein the composition contains between 200 to 600 ng of homopolymer.

45. The composition of claim 20, wherein said homopolymeric oligonucleotide is less than 5000 bases in length.