Methods for high level multiplexed polymerase chain reactions and homogeneous mass extension reactions
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.
1. A method for performing multiplexed amplification of target nucleic acid, comprising:
(a) forming a mixture containing 7 or more pairs of primary and secondary primers and one or more target nucleic acids, wherein each primer pair amplifies a nucleic acid-target region of a target nucleic acid and a sequence tag is attached to one or both of the primary and secondary primers of one or more primer pairs; and
(b) amplifying a plurality of nucleic acid-target regions of (a) under amplification conditions, whereby at least 60% of the 7 or more nucleic acid-target regions attempted are amplified by the 7 or more amplification primer pairs to produce an amplified mixture of nucleic acid-target regions, wherein the amplification conditions comprise a multiplicity of thermocycles and amplification reactants comprising a polymerase, dNTPs and MgCl 2 , wherein the ratio of the concentration of MgCl 2 to the concentration of each one of the dNTPs is less than or equal to 10:1 and only a single primer pair is used to amplify each particular nucleic acid target-region.
2. The method of claim 1 , wherein the concentration of each dNTP is between about 400-700 μM.
3. The method of claim 1 , wherein the free Mg 2+ concentration is between 1.0-2.0 mM.
4. The method of claim 1 , wherein the polymerase is Taq polymerase.
5. The method of claim 1 , wherein the amplification reaction conditions comprise about 100 nM primer pairs, and between about 2.6 up to about 4.8 mM MgCl 2 .
6. The method of claim 1 , wherein a sequence tag is attached to both primers of one or more primer pairs.
7. The method of claim 1 , further comprising inactivating one or more amplification reactants, wherein the one or more amplification reactants includes a nucleotide and/or a primer.
8. The method of claim 7 , wherein the amplification reactant that is a nucleotide is inactivated by a phosphatase and/or the amplification reactant that is a primer is inactivated by a nuclease.
9. The method of claim 8 , wherein the nuclease is an exonuclease.
10. The method of claim 1 , wherein for each amplification primer pair, one primer is in lower concentration than the other primer.
11. The method of claim 1 , wherein the ratio of the concentration of MgCl 2 to the concentration of each one of the dNTPs is less than or equal to 9:1.
12. The method of claim 1 , wherein the multiplicity of thermocycles is about 45.
13. The method of claim 1 , wherein the amplification conditions excludes the addition of one or more of BSA, glycerol, DMSO and urea.
14. The method of claim 4 , wherein the Taq polymerase is at a concentration of 0.03 units/μl.
15. The method of claim 1 , whereby at least 90% of the 7 or more nucleic acid-target regions attempted are amplified by the 7 or more amplification primer pairs.
16. The method of claim 1 , wherein the free Mg 2+ concentration is between about 1.3 to about 1.7 mM.
17. The method of claim 1 , wherein the ratio of the concentration of MgCl 2 to the concentration of each one of the dNTPs is less than or equal to 7:1.
18. The method of claim 1 , wherein the ratio of the concentration of MgCl 2 to the concentration of each one of the dNTPs is less than or equal to 6:1.
19. The method of claim 1 , wherein the mixture of (a) contains 13 or more pairs of primary and secondary primers, whereby at least 60% of the 13 or more nucleic acid-target regions attempted are amplified by the 13 or more amplification primer pairs.
20. The method of claim 1 , wherein a sequence tag is attached to all primers.
21. The method of claim 1 , wherein the sequence tag is from 5 up to 20 nucleotides in length.
22. The method of claim 1 , wherein the sequence tag comprises the sequence of nucleotides of SEQ ID NO: 37.