Multiplex amplification of polynucleotides

• US 10,689,695 B2
• Filed: 10/17/2017
• Issued: 06/23/2020
• Est. Priority Date: 12/04/2002
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Andersen, Mark, Ruff, David
• Primary Examiner(s): Thomas, David C
• Application Number:
  US15/785,693
• Publication Number:
  US 20180135104A1
• Time in Patent Office: 980 Days
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6851 Quantitative amplification
• 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 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2561/113 Real time assay

Abstract

The present invention provides methods, reagents and kits for carrying out a variety of assays suitable for analyzing polynucleotides or samples that include an amplification step performed in a multiplex fashion. Also provided are methods for analyzing and improving the efficiency of amplification and for carrying out gene expression analysis.

Claims

1. A kit for carrying out a two-step amplifications reaction comprising:

(a) a first container or reaction vessel comprising a plurality of amplification primer sets suitable for carrying out a multiplex amplification reaction to produce a plurality of different amplicons and one or more single-stranded oligonucleotide probes complementary to all or a part of one or more of the plurality of different amplicons; and

(b) a second container or reaction vessel comprising a single set of amplification primers suitable for carrying out a singleplex amplification reaction to produce a single amplicon, and a single-stranded oligonucleotide probe complementary to all or a part of the single amplicon.

2. The kit of claim 1, wherein the single set of amplification primers in the second container or reaction vessel is the same as or nested to one of the plurality of amplification primer sets in the first container or reaction vessel.

3. The kit of claim 1, wherein the plurality of amplification primer sets comprises at least 100 different amplification primer sets.

4. The kit of claim 1, wherein the kit further comprises a third container or reaction vessel comprising a DNA polymerase.

5. The kit of claim 1, wherein the kit further comprises a third container or reaction vessel comprising a reverse transcriptase.

6. The kit of claim 1, wherein at least 60% of the plurality of amplification primer sets in the first container or reaction vessel are at approximately an equimolar concentration relative to one another.

7. The kit of claim 6, wherein all of the plurality of amplification primer sets in the first container or reaction vessel are at approximately an equimolar concentration relative to one another.

8. The kit of claim 1, wherein the concentration of each of the plurality of amplification primer sets is at a concentration less than 100 nM for each primer.

9. The kit of claim 1, wherein the concentration of each of the plurality of amplification primer sets is at a concentration of about 30 to 45 nM for each primer.

10. The kit of claim 1, wherein the one or more single-stranded oligonucleotide probes in the first container or reaction vessel is a TaqMan probe.

11. The kit of claim 1, wherein the single-stranded oligonucleotide probe in the second container or reaction vessel is detectably labeled.

12. The kit of claim 1, wherein the single-stranded oligonucleotide probe in the second container or reaction vessel is a TaqMan probe.

13. The kit of claim 1, wherein at least one step of the two-step amplification reaction is a reverse transcription polymerase chain reaction (RT-PCR).

14. The kit of claim 1, wherein the first container or reaction vessel further comprises a DNA polymerase and/or a reverse transcriptase.

15. The kit of claim 1, wherein the second container or reaction vessel further comprises a DNA polymerase and/or a reverse transcriptase.

16. The kit of claim 4, wherein the DNA polymerase is at a concentration of about 4-6 U/20 μ

L reaction volume.

17. The kit of claim 8, wherein the concentration of the single set of amplification primers in the second container or reaction vessel is at a concentration of about 300-900 nM for each primer.

18. The kit of claim 2, wherein the single set of amplification primers in the second container or reaction vessel and one of the plurality of amplification primer sets in the first container or reaction vessel are the same.

19. The kit of claim 1, wherein the plurality of amplification primer sets in the first container or reaction vessel and the single set of amplification primers in the second container or reaction vessel all have a Tm that is within 10 degrees of one another.

20. The kit of claim 1, wherein the one or more single-stranded oligonucleotide probes in the first container or reaction vessel is as the same as the single-stranded oligonucleotide probe in the second container or reaction vessel.