Multiplex amplification of polynucleotides

• US 9,822,405 B2
• Filed: 06/26/2015
• Issued: 11/21/2017
• 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:
  US14/752,396
• Publication Number:
  US 20150292002A1
• Time in Patent Office: 879 Days
• Field of Search: None
• 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 method of generating a plurality of target sequences of interest, comprising the step of:

amplifying one or more target polynucleotides in the presence of a plurality of amplification primers suitable for amplifying a plurality of target sequences of interest and in the presence of a plurality of labeled oligonucleotide probes,

wherein each of the plurality of labeled oligonucleotide probes is complementary to a region of an amplified target sequence of interest and has the same label suitable for monitoring amplification as a function of time.

2. The method of claim 1 in which the product of the amplification is further subjected to at least one assay selected from the group consisting of single polynucleotide polymorphism analysis, genotyping analysis, gene expression analysis, fingerprinting analysis, analysis of gene mutations for genetic diagnoses, analysis of rare expressed genes in cells, nucleic acid sequencing, nucleic acid mini-sequencing and gene expression analysis.

3. The method of claim 1 in which the product of the amplification is further subjected to at least one assay selected from the group consisting of chromatography, electrophoresis, and staining with a dye or hybridization probe.

4. The method of claim 1 in which the product of the amplification is divided into a plurality of aliquots.

5. The method of claim 2 in which the product of the amplification is divided into a plurality of aliquots and wherein said at least one assay is performed on at least one of said aliquots.

6. The method of claim 5 wherein the number of aliquots is equal to the number of primer pairs used in said amplifying.

7. The method of claim 1 in which the amplification is carried out in the presence of uracil N-glycosylase.

8. The method of claim 1 wherein the one or more target polynucleotides is a plurality of target polynucleotides.

9. The method of claim 1 wherein the one or more target polynucleotides comprise a cDNA library.

10. The method of claim 1 wherein the amplifying is achieved with a thermostable DNA polymerase.

11. The method of claim 1 wherein the plurality of labeled oligonucleotides probes is selected from the group consisting of 5 ′

-exonuclease probes, stem-loop beacon probes, and stemless beacon probes.

12. The method of claim 1 wherein the label is a fluorophore.

13. The method of claim 1 wherein the amplifying is by a polymerase chain reaction carried out for a number of cycles such that the amplification remains in the linear range.

14. The method of claim 1 wherein the amplifying comprises 10 to 20 polymerase chain reaction cycles.

15. The method of claim 1 wherein the amplifying comprises 2 to 14 polymerase chain reaction cycles.

16. The method of claim 1 wherein at least 50% of the amplification primers are present at concentrations in the range from 30 nM to 100 nM for each primer.

17. The method of claim 1 wherein at least 50% of the amplification primers are present at approximately equimolar concentration from 30 nM to 100 nM for each primer.