Methods for high level multiplexed polymerase chain reactions and homogeneous mass extension reactions

• US 9,896,724 B2
• Filed: 05/22/2015
• Issued: 02/20/2018
• Est. Priority Date: 07/31/2003
• Status: Active Grant

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Inventors: Beaulieu, Martin, van den Boom, Dirk Johannes
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US14/720,179
• Publication Number:
  US 20160102347A1
• Time in Patent Office: 1,005 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• 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 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Provided herein are optimized methods for performing multiplexed detection of a plurality of sequence variations. Also provided are methods for performing multiplexed amplification of target nucleic acid.

Claims

1. A multiplex method of genotyping a plurality of polymorphic loci, comprising:

(a) simultaneously amplifying a plurality of nucleic acid-target regions with amplification primer pairs under amplification conditions whereby at least 60% of 7 or more nucleic acid target regions attempted are amplified by 7 or more amplification primer pairs to produce an amplified mixture of nucleic acid-target regions containing polymorphic loci, wherein the amplification conditions comprise a ratio of the concentration of MgCl₂ to the concentration of each one of the dNTPs of less than or equal to 7;

1, whereby at least 60% of the genotypes for said 7 or more nucleic acid target regions attempted are determined;

(b) after (a), contacting the amplified mixture of nucleic acid-target regions with genotyping primers in the presence of at least one chain terminating reagent under primer mass extension reaction conditions whereby the primers are extended up to, or through, the respective polymorphic loci, wherein there is one genotyping primer for each polymorphic locus;

(c) determining the masses of the extended genotyping primers; and

(d) based on the masses of the extended genotyping primers determined in (c), determining at least 60% of the genotypes for said 7 or more nucleic acid target regions attempted, thereby genotyping a plurality of polymorphic loci.

2. The method of claim 1, wherein in (c), the masses of the extended genotyping primers are determined by mass spectrometry.

3. The method of claim 2, wherein the mass spectrometry is Matrix Assisted Laser Desorption Ionization (MALDI) mass spectrometry.

4. The method of claim 1, wherein the free Mg²⁺ concentration is between 1.0-2.0 mM.

5. The method of claim 4, wherein the concentration of each dNTP is between about 400-700 μ

M.

6. The method of claim 1, wherein the amplification reaction conditions comprise water, genomic DNA, a buffer, dNTPs, the primer pairs, MgCl₂, and a polymerase.

7. The method of claim 1, wherein the ratio of the concentration of MgCl₂ to the concentration of each one of the dNTPs is 7:

1.

8. The method of claim 6, wherein the polymerase is Taq polymerase.

9. The method of claim 6, wherein the amplification reaction conditions comprise about 100 nM primer pairs, and between about 2.6 up to about 4.8 mM MgCl₂.

10. The method of claim 1, wherein a sequence tag is attached to one or more primer pairs.

11. The method of claim 1, wherein only a single primer pair is utilized to amplify each particular nucleic acid-target region.

12. The method of claim 1, wherein a sequence tag is attached to a primer in a primer pair.

13. The method of claim 1, wherein the extension reaction conditions comprise at least 55 thermocycles.

14. The method of claim 13, wherein the extension conditions comprise at least 75 thermocycles.

15. The method of claim 14, wherein the extension conditions comprise between 75 to 100 thermocycles.

16. The method of claim 1, wherein the amplification conditions comprise about 45 thermocycles.

17. The method of claim 1, wherein the concentration of each genotyping primer is selected to even out the mass intensities of the extended genotyping primers.

18. The method of claim 1, wherein the masses of the extended genotyping primers are distinct in an allele-specific manner.

19. The method of claim 1, wherein the separation between the masses of each of the extended genotyping primers is at least 20 Daltons.

20. The method of claim 1, further comprising inactivating one or more amplification reactants.

21. The method of claim 20, wherein the one or more amplification reactants includes a nucleotide.

22. The method of claim 21, wherein the nucleotide is inactivated by a phosphatase.

23. The method of claim 20, wherein the one or more amplification reactants includes a primer.

24. The method of claim 23, wherein the amplification reactant that is a primer is inactivated by a nuclease.

25. The method of claim 24, wherein the nuclease is an exonuclease.

26. The method of claim 1, wherein for each amplification primer pair, one primer is in lower concentration than the other primer.

27. The method of claim 26, wherein the lower concentration primer for a particular nucleic acid-target is in the same orientation as the genotyping primer for that nucleic acid-target region.

28. The method of claim 1, wherein at least 90% of the attempted genotypes are determined.

29. The method of claim 28, wherein at least 95%, 96%, 97%, 98% or 99% of the attempted genotypes are determined.

30. The method of claim 1, wherein the ratio of the concentration of MgCl₂ to the concentration of each one of the dNTPs is less than or equal to 6:

1.

31. The method of claim 1, wherein the ratio of the concentration of MgCl₂ to the concentration of each one of the dNTPs is less than or equal to 5:

1.