Reagents and methods for PCR

• US 9,034,605 B2
• Filed: 03/11/2010
• Issued: 05/19/2015
• Est. Priority Date: 03/12/2009
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Wangh, Lawrence J., Rice, John, Rice, Nicholas, Jia, Yanwei
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US13/256,038
• Publication Number:
  US 20120088275A1
• Time in Patent Office: 1,895 Days
• Field of Search: 435/91.2, 435/91.21
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2545/114 involving a quantitation step

Abstract

Modified double-stranded oligonucleotides that have terminal regions on each of their strands, that have a hybrid length of 6-50 nucleotides long, that have a melting temperature Tm of at least 32° C., and that include 2-4 modifying groups, each covalently attached to a different terminal region, preferably to a terminal nucleotide, said modifying groups being polycyclic substituents that do not have bulky portions that are non-planar, said modified oligonucleotide being capable of binding to the 5′ ex-nuclease domains of DNA polymerases and, when included in a PCR or other primer-dependent DNA amplification reaction at a concentration, generally not more than 2000 nM, that is effective for at least one of the functions of suppressing mispriming, increasing polymerase selectivity against 3′ terminal mismatches, increasing polymerase selectivity against AT-rich 3′ ends, reducing scatter among replicates, suppressing polymerase 5′ exonuclease activity, and inhibiting polymerase activity; as well as amplification reaction mixtures containing such modified double-stranded oligonucleotides, and amplification reactions, amplification assays and kits that include such modified double-stranded oligonucleotides.

Claims

1. An amplification reaction mixture comprising:

(a) at least one primer pair,

(b) a DNA polymerase,

(c) dNTP'"'"'s, and

(d) at least one double-stranded oligonucleotide additive;

(i) comprising two separate nucleic acid strands, each of the strands 6-50 nucleotides in length,

(ii) that is at least fifty percent double-stranded at 32°

C.,

(iii) having blunt ends or overhangs of 1-8 nucleotides,

(iv) that has terminal regions on each of its strands and includes 1-4 modifying groups, each covalently attached to a different terminal region, said modifying groups being polycyclic moieties that do not have bulky portions that are non-planar, and

(v) wherein said at least one double-stranded oligonucleotide additive is included in said reaction mixture at a concentration of 25-2000 nM.

2. The amplification reaction mixture of claim 1 wherein the at least one modifying groups is 2-4 modifying groups.

3. The amplification reaction mixture of claim 1 wherein the additive includes a first strand that is a primer or probe for a target sequence and a reverse complement strand that is partially complementary to the first strand.

4. The amplification reaction mixture of claim 1 wherein the modifying groups are covalently linked to terminal nucleotides of said at least one double-stranded oligonucleotide additive.

5. The amplification reaction mixture of claim 1 comprising at least one modifying group selected from the group consisting of Dabcyl, digoxigenin (DIG), coumarin (CMN), and QSY 21 (QSY).

6. The amplification reaction mixture of claim 2 wherein the at least one additive is a mixture of two additives.

7. The amplification reaction mixture of claim 6 wherein the mixture consists of three strands.

8. The amplification reaction mixture of claim 1, wherein the at least one double-stranded oligonucleotide additive consists of natural nucleotides.

9. The amplification reaction mixture of claim 1 wherein the at least one double-stranded oligonucleotide additive is DNA.

10. The amplification reaction mixture according to claim 1 wherein the at least one additive is not a primer or probe for said at least one target sequence.

11. A method for amplifying at least one DNA target sequence comprising contacting said at least one DNA target sequence with an amplification reaction mixture according to claim 1 and subjecting the reaction mixture to a primer-dependent DNA amplification reaction having a primer annealing temperature and a primer extension temperature.

12. The method of claim 11 wherein contacting the at least one DNA target sequence with the reaction mixture consists of adding the at least one DNA target sequence in single-stranded form to the reaction mixture.

13. The method of claim 11 that includes reverse transcribing RNA to obtain the at least one DNA target sequence.

14. The method of claim 11 wherein the at least one modifier is 2-4 modifiers.

15. The method of claim 14 wherein the mixture includes a first additive having a double-stranded oligonucleotide that has a Tm that is in the range of the primer annealing temperature to not more than 5°

C. below the primer annealing temperature and a second additive having a double-stranded oligonucleotide that has a Tm that is higher than the primer annealing temperature.

16. The method of claim 15 wherein each additive includes 3-4 modifying groups.

17. The method according to claim 11, wherein the primer-dependent amplification reaction is a polymerase chain reaction (PCR) amplification reaction.

18. The method according to claim 11, wherein the primer-dependent amplification is a LATE-PCR amplification reaction.

19. A kit of reagents for amplifying at least one DNA target sequence, said kit comprising reagents for the amplification reaction mixture of claim 1.

20. The kit according claim 19 further including reverse transcriptase.

21. The amplification reaction mixture of claim 5 wherein the 1-4 modifying groups are selected from the group consisting of Dabcyl, digoxigenin (DIG), coumarin (CMN), and QSY 21 (QSY).