Primer middle sequence interference PCR technology

• US 10,036,062 B2
• Filed: 11/28/2013
• Issued: 07/31/2018
• Est. Priority Date: 11/30/2012
• Status: Active Grant

• Current Assignee: Beijing Tag-Array Molecular Test Co., Ltd.
• Original Assignee: Beijing Tag-Array Molecular Test Co., Ltd.
• Inventors: Jiang, Hong, Yue, Suwen, Liao, Tongbing, Jiang, Bisheng, Qu, Yue, Jiang, Yukang
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/648,678
• Publication Number:
  US 20160237472A1
• Time in Patent Office: 1,706 Days
• Field of Search: 435 912
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2549/126 using oligonucleotides as clamps

Abstract

Provided is a primer middle sequence interference PCR method, and the method uses one segment of a non-complemented or same-sequence base of the middle sequence of primers to perform antisense interference inside and outside the primer molecules, so as to competitively destroy the polymerization among the primers to selectively inhibit amplification of the primer dimer (PD).

Claims

1. A method for inhibiting primer dimer formation and non-specific amplification of a target sequence therefrom in a polymerase chain reaction (PCR), the method comprising preparing a PCR reaction system for amplifying the target sequence, said PCR reaction system comprising:

(a) a first primer and a second primer which, when aligned in the 5′

to 3 direction, share an identical Intermediate Domain (ID) sequence of 5-9 consecutive bases, wherein both primers comprise a 3′

region configured to specifically bind and amplify the target sequence;

(b) a 5′

antisense sequence of 5-7 consecutive bases, which antisense sequence is complementary to said Intermediate Domain (ID) sequence, wherein the antisense sequence is configured to competitively bind said intermediate Domain to disturb polymerization between the primers, is configured to bind to the ID sequence intra-molecularly such that the first and/or second primer is folded, and does not affect the primers specifically binding and amplifying the target sequence while inhibiting primer dimer formation and non-specific amplification therefrom.

2. The method of claim 1, wherein in the 3′

to 5′

direction, location of the ID sequence on the primer sequences starts from the fourth or fifth base from the 3′

terminal OH end of the primer sequences.

3. The method of claim 1, wherein in (a), the 3′

end regions of the primers do not contain two or more bases that are complementary between the primers.

4. The method of claim 3, wherein the first one or two bases at the 3′

end regions of the primers do not contain any base that is complementary between the primers.

5. The method of claim 3, wherein the primers'"'"' last base is C or A at the 3′

terminal OH ends.

6. The method of claim 1, wherein in (a), the method further comprises using a single-stranded DNA binding protein (SSB) in the amplification of the target sequence.

7. The method of claim 1, wherein in (a), said first primer and said second primer which, when aligned in the 5′

to 3′

direction, share an identical Intermediate Domain (ID) sequence of 6-8 consecutive bases.

8. The method of claim 7, wherein in (a), the identical ID sequence comprises a mutation compared to the target sequence or complementary sequence thereof.

9. The method of claim 7, wherein in (a), the identical ID sequence comprises a modified base.

10. The method of claim 9, wherein the modified base is an RNA base, a 2′

-F RNA base, 5F-dU, 5Br-dU, 8-OH-dG, or 8-OH-dA.

11. The method of claim 1, wherein in (a), the second and third bases from the 3′

terminal OH ends of the primers are not CG/GC.

12. The method of claim 1, wherein in (b), the antisense sequence comprises 2′

-O-Methyl (OMe) RNA, 2′

-O-methoxy-Ethyl (MOE) RNA, 2′

-Amino-RNA, 2′

-Fluoro-RNA, 2′

-O, 4′

-C-methylene bridge RNA, and PNA (peptide nucleic acid), Morpholino, or N3′

→

N5′

Phosphoramidate.

13. The method of claim 1, wherein the PCR reaction system further comprises dUTP instead of dTTP, a mineral oil sealing condition, and an uracil-DNA glycosylase (UDG) to degrade leaked products that cause colloidal aerosol cross-contamination,

wherein said preparing a PCR reaction system comprises:

dissolving the primer in 20% (w/v) Dextran pre-added to the bottom of a container for the PCR;

adding other PCR reagents and the mineral oil sealing in order and allowing the PCR reaction to settle; and

denaturing the PCR reaction system by heat to release the primer into the PCR reaction to initiate amplification,

wherein the residual above the mineral oil sealing without the primer cannot be amplified and only contains leaked products that can be degraded by UDG.

14. The method of claim 13, wherein a primer of the PCR reaction is reversibly immobilized on a solid support and is released into the PCR reaction to initiate amplification.

15. The method of claim 1, wherein the first primer comprises the ID sequence and the anti-sense sequence complementary to the ID sequence while the second primer does not, or vice versa.

16. The method of claim 15, wherein the primer comprising the ID sequence and the anti-sense sequence is fluorescently labeled, and wherein a base from the third base from the 3′

terminal OH end of the primer to a base in the ID sequence is labeled with a fluorescence reporter, while the 5′

terminal COOH end of the primer is labeled with a fluorescence quencher, or vice versa.

17. The method of claim 16, wherein the fluorescence reporter comprises 6-FAM-dT or Cy3-dT, and the fluorescence quencher comprises dabcyl or 5′

dG quenching base.