Reagents and methods of PCR

• US 9,758,813 B2
• Filed: 05/18/2015
• Issued: 09/12/2017
• Est. Priority Date: 03/12/2009
• Status: Active Grant

• Current Assignee: Brandeis University
• Original Assignee: Brandeis University
• Inventors: Wangh, Lawrence J., Rice, John E., Rice, Nicholas, Jia, Yanwei
• Primary Examiner(s): Wilder, Cynthia B
• Application Number:
  US14/715,299
• Publication Number:
  US 20150322503A1
• Time in Patent Office: 848 Days
• Field of Search: 435 912
• 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′ exonuclease 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. A reaction mixture for a primer-dependent DNA amplification reaction including primer extension by a DNA polymerase for amplifying at least one DNA target sequence, said reaction mixture comprising:

(a) at least one primer pair;

(b) a DNA polymerase;

(c) dNTPs; and

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

(i) comprising two separate strands, each of the strands 6-50 nucleotides long;

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

C.; and

(iii) that has a terminal region on each end of its two separate 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,

wherein said at least one double-stranded oligonucleotide additive is present in the reaction mixture at a concentration sufficient to suppress mispriming of an amplification reaction performed using the reaction mixture.

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

3. The amplification reaction mixture of claim 2 wherein the 2-4 modifying groups is three modifying groups.

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

5. The amplification reaction mixture of claim 2 wherein the 2-4 modifying groups is four modifying groups.

6. 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.

7. The amplification reaction mixture of claim 1 wherein the modifying groups are Dabcyl.

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

9. The amplification reaction mixture of claim 8 wherein the mixture of two additives consists of three strands.

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

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

12. 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.

13. The amplification reaction mixture according to claim 1 wherein the concentration of said at least one double-stranded oligonucleotide additive is not more than 1000 nM.

14. The amplification reaction mixture according to claim 1, further including a reverse transcriptase.

15. The amplification reaction mixture of claim 1 wherein the at least one double-stranded additive includes from one to four single-stranded overhangs.

16. The amplification reaction mixture of claim 15 wherein the at least one double-stranded additive comprises at least one strand that, when not hybridized, forms a stem-loop structure.

17. 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.

18. A modified double-stranded oligonucleotide comprising two separate strands, each of the strands 6-50 nucleotides long, that is at least fifty percent double-stranded at 32°

C., that has a terminal region on each end of its two separate strands, and that includes 2-4 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, said modified oligonucleotide being capable of inhibiting the 5′

exonuclease domains of DNA polymerases.

19. The modified oligonucleotide of claim 18 wherein the double-stranded oligonucleotide is DNA.

20. The modified oligonucleotide of claim 18 wherein said modifying groups are attached to terminal nucleotides.