Compositions, methods and kits for nucleic acid synthesis and amplification

• US 9,005,895 B2
• Filed: 06/20/2011
• Issued: 04/14/2015
• Est. Priority Date: 06/21/2010
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Woo, Cora L., Berkman, Jennifer, Yim, Priscilla W.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/164,177
• Publication Number:
  US 20120003645A1
• Time in Patent Office: 1,394 Days
• Field of Search: None
• 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 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2549/125 using sterilising/blocking agents, e.g. albumin

Abstract

The present invention is directed to compositions, methods and kits useful for the synthesis of nucleic acid molecules. More specifically, compositions, methods and kits are provided for the amplification of nucleic acid molecules in a one-step RT-PCR procedure comprising one or more agents used to increase tolerance to PCR inhibitors.

Claims

1. A method of performing RT-PCR of a nucleic acid sample comprising:

mixing a composition comprising;

at least one active reverse transcriptase;

at least one active DNA polymerase; and

a combination of PCR inhibitor blocking agents consisting essentially of 0.2% w/v-0.8% w/v fish gelatin and 500 ng/μ

l to 6000 ng/μ

l BSA configured to increase tolerance to one or more PCR inhibitors when present in the nucleic acid sample, with;

a) a nucleic acid sample;

b) one or more labeled probes;

c) one or more primers; and

performing a RT-PCR on the nucleic acid sample.

2. The method of claim 1, wherein the labeled probe is a TaqMan®

probe.

3. The method of claim 1, wherein the one or more PCR inhibitors is selected from the group consisting of hematin, humic acid and heparin.

4. The method of claim 1, wherein the RT-PCR is performed in a single tube or reaction.

5. The method of claim 1, further comprising the step of removing the composition from storage at freezing temperatures prior to mixing with the nucleic acid sample, the one or more labeled probes and the one or more primers, wherein the composition does not require thawing.

6. The method of claim 1, wherein the increased tolerance is indicated by a decrease in C_t.

7. The method of claim 6, wherein the C_t is decreased by at least one C_t.

8. The method of claim 1, wherein the tolerance is increased by at least 10% when compared to methods using compositions without the combination of PCR inhibitor blocking agents.

9. The method of claim 1, wherein the concentration of fish gelatin is 0.8% w/v to 4% w/v.

10. The method of claim 1, wherein the concentration of fish gelatin is 0.5% w/v.

11. A method for amplifying a nucleic acid molecule, the method comprising:

mixing a nucleic acid template with a composition comprising one or more reverse transcriptases, one or more DNA polymerases, and a combination of PCR inhibitor blocking agents consisting essentially of 0.2% w/v-0.8% w/v fish gelatin and 500 ng/μ

l to 6000 ng/μ

l BSA, to form a reaction mixture, wherein each of the PCR inhibitor blocking agents is present at a concentration sufficient to reduce PCR inhibition by the PCR inhibitors when present; and

incubating the reaction mixture under conditions sufficient to amplify a nucleic acid molecule complementary to all or a portion of the nucleic acid template.

12. The method of claim 11, wherein the nucleic acid template is RNA.

13. The method of claim 12, wherein the combination of PCR inhibitor blocking agents increases tolerance to one or more PCR inhibitors and wherein the increase in tolerance is indicated by a decrease in C_t.

14. The method of claim 13, wherein the decrease in C_t is by at least 1 C_t.

15. The method of claim 11, wherein the nucleic acid template is DNA.

16. The method of claim 11, wherein at least one of the PCR inhibitors is selected from the group consisting of hematin, humic acid or heparin.

17. The method of claim 11, wherein the concentration of fish gelatin is 0.4% w/v to 0.8% w/v.

18. The method of claim 17, wherein the concentration of fish gelatin is 0.4% w/v.

19. A method for nucleic acid synthesis, the method comprising:

mixing one or more first nucleic acid molecules with one or more polymerases, and a combination of PCR inhibitor blocking agents consisting essentially of 0.2% w/v-0.8% w/v fish gelatin and 500 ng/μ

l to 6000 ng/μ

l BSA; and

incubating the mixture under conditions sufficient to make one or more second nucleic acid molecules complementary to all or a portion of the one or more first nucleic acid molecules.

20. The method of claim 19, wherein the one or more first nucleic acid molecules is RNA.

21. The method of claim 19, wherein the one or more first or second nucleic acid molecules is DNA.

22. The method of claim 19, wherein the step of mixing the one or more first nucleic acid molecules with one or more polymerases, and the combination of PCR inhibitor blocking agents further comprises one or more reverse transcriptases in the mixture.

23. The method of claim 19, wherein the concentration of fish gelatin is 0.4% w/v to 4% w/v.

24. The method of claim 23, wherein the concentration of fish gelatin is 0.5% w/v.