Use of RNA/DNA chimeric primers for improved nucleic acid amplification reactions

• US 8,460,874 B2
• Filed: 07/03/2008
• Issued: 06/11/2013
• Est. Priority Date: 07/03/2007
• Status: Active Grant

• Current Assignee: Infiniplex Limited
• Original Assignee: GenAphora Ltd.
• Inventors: Peleg, Ofer
• Primary Examiner(s): HORLICK, KENNETH R
• Application Number:
  US12/667,261
• Publication Number:
  US 20100291635A1
• Time in Patent Office: 1,804 Days
• Field of Search: 435/6.12, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• 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 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

Methods are provided for amplification of a nucleic acid sequence. The method use RNA/DNA chimeric oligonucleotides as primers. The primers have RNA residues scattered along their length and no two ribonucleotides in the prime are adjacent to one another. The methods are useful for reducing non-specific amplification products, such as primer dimers. The invention also provides kits comprising RNA/DNA chimeric oligonucleotide primers for practicing the amplification methods.

Claims

1. A method of reducing or eliminating formation of artifacts and non-specific amplification products in a DNA-dependent DNA polymerase amplification reaction, the method comprising:

conducting a nucleic acid amplification reaction using at least one RNA/DNA chimeric oligonucleotide as a forward or as a reverse primer or as a probe, and using a DNA-dependent DNA polymerase;

wherein the chimeric oligonucleotide comprises at least one ribonucleotide located within 10 nucleotides adjacent to the 3′

end of the chimeric oligonucleotide;

wherein the at least one ribonucleotide impedes DNA synthesis from primer-primer dimers or primer-probe dimers; and

wherein no two ribonucleotides in the chimeric oligonucleotide are adjacent to one another.

2. The method of claim 1 wherein the forward primer and the reverse primer are both RNA/DNA chimeric oligonucleotides.

3. The method of claim 1 wherein all the primers and probes used in the amplification reaction have an identical base as the 3′

terminal base.

4. The method of claim 3 wherein the identical 3′

terminal base of the chimeric oligonucleotides is selected from A or T.

5. The method of claim 1 wherein said at least one ribonucleotide is a base that is adjacent to a complementary base to said 3′

end of at least one chimeric oligonucleotide.

6. The method of claim 1 wherein said at least one ribonucleotide in each chimeric oligonucleotide is located within one to 5 nucleotides upstream to at least one of the nucleotides that is complementary to its own 3′

end or the 3′

end of another primer or probe in the reaction mixture.

7. The method of claim 6 wherein said at least one ribonucleotide in each chimeric oligonucleotide is adjacent to at least one of the nucleotides that is complementary to its own 3′

end or the 3′

end of another primer or probe in the reaction mixture.

8. The method of claim 1 wherein the DNA-dependent DNA polymerase amplification reaction is selected from the group consisting of:

exponential rolling circle amplification (ERCA), rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), self-sustained sequence replication (3SR), amplification with Qβ

replicase, and cycle sequencing.

9. The method of claim 8, wherein the DNA-dependent DNA polymerase amplification reaction is a real-time quantitative PCR (qPCR).

10. A kit for carrying out a DNA-dependent DNA polymerase amplification reaction comprising:

(i) at least one RNA/DNA chimeric oligonucleotide as a forward or as a reverse primer or as a probe;

wherein the chimeric oligonucleotide comprises at least one ribonucleotide located within 10 nucleotides adjacent to the 3′

end of the chimeric oligonucleotide;

wherein the at least one ribonucleotide impedes DNA synthesis from primer-primer dimers or primer-probe dimers; and

wherein no two ribonucleotides in the chimeric oligonucleotide are adjacent to one another;

(ii) a DNA-dependent DNA polymerase;

(iii) the necessary reagents and buffers to carry out the amplification reaction.

11. The kit of claim 10 wherein the forward primer and the reverse primer are both RNA/DNA chimeric oligonucleotides.

12. The kit of claim 10 wherein all the primers and probes used in the kit have an identical base as the 3′

terminal base.

13. The kit of claim 12 wherein the identical 3′

terminal base of the chimeric oligonucleotides is selected from A or T.

14. The kit of claim 10 wherein said at least one ribonucleotide is a base that is adjacent to a complementary base to said 3′

end of at least one chimeric oligonucleotide.

15. The kit of claim 10 wherein said at least one ribonucleotide in each chimeric oligonucleotide is located within one to 5 nucleotides upstream to at least one of the nucleotides that is complementary to its own 3′

end or the 3′

end of another primer or probe in the kit.

16. The kit of claim 15 wherein said at least one ribonucleotide in each chimeric oligonucleotide is adjacent to at least one of the nucleotides that is complementary to its own 3′

end or the 3′

end of another primer or probe in the kit.

17. The kit of claim 10 wherein the DNA-dependent DNA polymerase amplification reaction is selected from the group consisting of:

exponential rolling circle amplification (ERCA), rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), polymerase chain reaction (PCR), real-time quantitative PCR (qPCR), self-sustained sequence replication (3SR), amplification with Qβ

replicase, and cycle sequencing.

18. The kit of claim 17 wherein the DNA-dependent DNA polymerase amplification reaction is a real-time quantitative PCR (qPCR).

19. The kit according to claim 10 further comprising the means to detect the products of the amplification reaction.