PRIMER MIDDLE SEQUENCE INTERFERENCE PCR TECHNOLOGY

• US 20160237472A1
• Filed: 11/28/2013
• Published: 08/18/2016
• 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: YUE, Suwen, LIAO, Tongbing, JIANG, Bisheng, QU, Yue, JIANG, Yukang, JIANG, Hong
• Granted Patent: US 10,036,062 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• 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

1-14. -14. (canceled)

15. 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 amplifying the target sequence using:

(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 specifically bind and amplify the target sequence while primer dimer formation and non-specific amplification therefrom are inhibited;

(b) a first primer, a second primer, and an oligonucleotide which is complementary to an Intermediate Domain (ID) sequence of 5-11 consecutive bases within the first primer or the second primer, wherein the oligonucleotide (As Oligo) does not affect the primers specifically binding and amplifying the target sequence while inhibiting primer dimer formation and non-specific amplification therefrom; and

/or (c) a first primer and a second primer, wherein the first primer or second primer comprises an Intermediate Domain (ID) sequence of 5-7 consecutive bases and an anti-sense sequence complementary to the ID sequence, wherein the anti-sense sequence is capable of intra-molecular binding to the ID sequence such that the primer is folded, and wherein both the first and second primers specifically bind and amplify the target sequence while primer dimer formation and non-specific amplification therefrom are inhibited.

16. The method of claim 15, wherein in (a), the ID sequence is located toward the 3′

end of the primer sequences.

17. The method of claim 16, wherein the 3′

end base of the ID sequence is the fourth or fifth base from the 3′

end of the primer sequences.

18. The method of claim 15, wherein in (a), the 3′

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

19. The method of claim 18, wherein the first one or two bases at the 3′

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

20. The method of claim 18, wherein the primers end with C or A at the 3′

ends.

21. The method of claim 15, wherein in (a), the method further comprises using a single-stranded DNA binding protein (SSB) in the amplification of the target sequence.

22. The method of claim 15, wherein in (a), the identical ID sequence is of 6-8 consecutive bases within the primers.

23. The method of claim 15, wherein in (a), the identical ID sequence comprises a mutation compared to the target sequence or complementary sequence thereof, wherein the mutated base is at the 5′

end or 3′

end of the ID sequence or a middle base within the ID sequence.

24. The method of claim 15, wherein in (a), the identical ID sequence comprises a modified base.

25. The method of claim 24, wherein the modified base is an RNA base, a 2′

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

26. The method of claim 15, wherein in (a), the second and third bases from the 3′

ends of the primers are not CG/GC.

27. The method of claim 15, wherein in (b), the 3′

end OH group of the oligonucleotide is blocked.

28. The method of claim 15, wherein in (b), the oligonucleotide (As Oligo) is 6-10 bases and 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 (lock nucleic acid, LNA), and PNA (peptide nucleic acid), Morpholino, N3′

→

N5′

Phosphoramidate, wherein the oligonucleotide comprises 1-8 modified bases.

29. The method of claim 15, wherein in (b), the oligonucleotide (As Oligo) is immobilized on a solid support selected from a micro-sphere and a micro-well, which can be used in multiple real-time fluorescence PCR reactions in micro-array PCR.

30. The method of claim 15, wherein in (c), 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.

31. The method of claim 30, 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′

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

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

32. The method of claim 31, wherein the fluorescence reporter comprises 6-FAM-dT or Cy3-dT, and the fluorescence quencher comprises dabcyl or 5′

dG quenching base.

33. The method of claim 15, wherein the PCR reaction comprises dUTP instead of dTTP, a mineral oil sealing condition, and a uracil-DNA glycosylase (UDG) to degrade leaked products that cause colloidal aerosol cross-contamination,

wherein the release of a primer into the PCR reaction is postponed by:

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

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

34. The method of claim 33, wherein a primer of the PCR reaction is reversibly immobilized on a solid support and is released into the PCR reaction to initiate amplification.

35. The method of claim 15, wherein the method comprises amplifying the target sequence using (a) and (b), (a) and (c), (b) and (c), or all of (a)-(c).