Methods for high level multiplexed polymerase chain reactions and homogeneous mass extension reactions
First Claim
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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.
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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.
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Citations
31 Claims
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1. A multiplex method of genotyping a plurality of polymorphic loci, comprising:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)
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Specification