Methods for simultaneous amplification of target loci
DC CAFCFirst Claim
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1. A method for amplifying and sequencing DNA, comprising:
- isolating cell-free DNA from a biological sample and tagging the isolated cell-free DNA, wherein each tagged DNA molecule comprises a molecular barcode;
performing a first PCR to simultaneously amplify at least 10 target loci using a universal primer and at least 10 target-specific primers in a single reaction volume;
performing a second, nested PCR to simultaneously amplify the at least 10 target loci using the universal primer and at least 10 inner target-specific primers in a single reaction volume;
performing high-throughput sequencing to sequence the amplified DNA comprising the target loci.
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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
21 Claims
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1. A method for amplifying and sequencing DNA, comprising:
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isolating cell-free DNA from a biological sample and tagging the isolated cell-free DNA, wherein each tagged DNA molecule comprises a molecular barcode; performing a first PCR to simultaneously amplify at least 10 target loci using a universal primer and at least 10 target-specific primers in a single reaction volume; performing a second, nested PCR to simultaneously amplify the at least 10 target loci using the universal primer and at least 10 inner target-specific primers in a single reaction volume; performing high-throughput sequencing to sequence the amplified DNA comprising the target loci. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20)
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21. A method for amplifying and sequencing DNA, comprising:
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isolating cell-free DNA from a biological sample, subjecting the isolated cell-free DNA to blunting ending and dA-tailing, and tagging the isolated cell-free DNA, wherein each tagged DNA molecule comprises a molecular barcode and a universal priming site, and wherein the cell-free DNA comprises DNA from mixed origin; performing a first PCR to simultaneously amplify at least 100 target loci using a universal primer and at least 100 target-specific primers in a single reaction volume; performing a second, one-sided nested PCR to simultaneously amplify the at least 100 target loci using the universal primer and at least 100 inner target-specific primers in a single reaction volume; performing high-throughput sequencing to sequence the amplified DNA comprising the target loci, wherein the target loci are SNP loci.
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Specification