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 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 reaction 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; (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 within a nucleic acid-target region; (c) determining the masses of the extended genotyping primers by mass spectrometry; 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 the mass spectrometry is selected from among Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF), Electrospray Ionization (ESI), Ultraviolet-Matrix-Assisted Laser Desorption/Ionization (UV-MALDI), Infrared-Matrix-Assisted Laser Desorption/Ionization (IR-MALDI), Orthogonal-Time-of-Flight (0-TOF), Axial-Time-of-Flight (A-TOF), Ion Cyclotron Resonance (ICR), Fourier Transform Linear/Reflectron (RETOF), Quadrupole mass spectrometry, Quadrupole ion trap mass spectrometry, and combinations thereof.
3. The method of claim 2 , wherein the mass spectrometry is Matrix Assisted Laser Desorption/Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry.
4. The method of claim 1 , wherein the amplification reaction conditions comprise water, genomic DNA, a buffer, dNTPs, the primer pairs, MgCl 2 , and a polymerase.
5. The method of claim 4 , wherein the concentration of each dNTP is between about 400 μM to about 700 μM.
6. The method of claim 4 , wherein the concentration of each dNTP is about 200 μM.
7. The method of claim 4 , wherein the amplification reaction conditions comprise a free Mg 2+ concentration of between 1.0 mM to 2.0 mM, wherein the free Mg 2+ concentration is equal to the total MgCl 2 concentration less the total combined concentration of the dNTPs.
8. The method of claim 7 , wherein the free Mg 2+ concentration is between 1.1-1.9 mM, between 1.2-1.8 mM, between 1.3-1.7 mM, or between 1.4-1.6 mM.
9. The method of claim 7 , wherein the free Mg 2+ concentration is about 1.5 mM.
10. The method of claim 4 , wherein the ratio of the concentration of MgCl 2 to the concentration of each of the dNTPs is ≤10:1, ≤9:1, ≤8:1, ≤7:1, ≤6:1 or ≤5:1.
11. The method of claim 10 , wherein the ratio of the concentration of MgCl 2 to the concentration of each of the dNTPs is ≤7:1.
12. The method of claim 1 , wherein the amplification reaction conditions comprise about 100 nM primer pairs, and between about 2.6 mM up to about 4.8 mM MgCl 2 .
13. The method of claim 12 , wherein the amplification reaction conditions comprise about 500 μM of each dNTP and about 3.5 mM MgCl 2 .
14. The method of claim 1 , wherein only a single primer pair is utilized to amplify each nucleic acid-target region.
15. The method of claim 1 , wherein between 7 nucleic acid target regions to about 50 or more nucleic acid target regions are attempted to be amplified.
16. The method of claim 1 , wherein the extension reaction conditions comprise at least 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more thermocycles.
17. The method of claim 1 wherein, in (d), at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, up to 100% of the attempted genotypes are determined.
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 , wherein the chain terminating reagent comprises a dideoxynucleotide.
21. The method of claim 1 , wherein more than 7 nucleic acid target regions are attempted to be amplified.