Methods for simultaneous amplification of target loci
First Claim
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1. A method of amplifying target loci in a nucleic acid sample, the method comprising:
- (a) contacting a nucleic acid sample comprising target loci with a library of test primers comprising at least 1,000 different primer pairs to produce a reaction mixture in one reaction volume;
wherein each primer pair includes a forward test primer and a reverse test primer that hybridize to the same target locus, and wherein the primers do not include molecular inversion probes (MIPs);
(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons;
wherein the concentration of each test primer is less than 10 nM;
wherein the length of the annealing step of the reaction conditions is greater than 10 minutes;
wherein at least 1,000 different target loci are simultaneously amplified; and
wherein (i) less than 20% of the amplified products are test primer dimers, (ii) at least 80% of the amplified products are target amplicons, and (iii) at least 80% of the target loci are amplified; and
(c) sequencing the amplified products.
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Abstract
The invention provides methods for simultaneously amplifying multiple nucleic acid regions of interest in one reaction volume as well as methods for selecting a library of primers for use in such amplification methods. The invention also provides library of primers with desirable characteristics, such as minimal formation of amplified primer dimers or other non-target amplicons.
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Citations
19 Claims
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1. A method of amplifying target loci in a nucleic acid sample, the method comprising:
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(a) contacting a nucleic acid sample comprising target loci with a library of test primers comprising at least 1,000 different primer pairs to produce a reaction mixture in one reaction volume;
wherein each primer pair includes a forward test primer and a reverse test primer that hybridize to the same target locus, and wherein the primers do not include molecular inversion probes (MIPs);(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons;
wherein the concentration of each test primer is less than 10 nM;
wherein the length of the annealing step of the reaction conditions is greater than 10 minutes;
wherein at least 1,000 different target loci are simultaneously amplified; and
wherein (i) less than 20% of the amplified products are test primer dimers, (ii) at least 80% of the amplified products are target amplicons, and (iii) at least 80% of the target loci are amplified; and(c) sequencing the amplified products. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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Specification