METHODS FOR SIMULTANEOUS AMPLIFICATION OF 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.
45 Citations
46 Claims
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1-23. -23. (canceled)
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24. A method for amplifying and sequencing DNA, comprising:
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ligating adaptors to cell-free DNA isolated from a biological sample, wherein the adaptors each comprises a universal priming site; 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, wherein at least one of the primers comprises a sample index and a sequencing tag; performing high-throughput sequencing to sequence the amplified DNA comprising the target loci. - View Dependent Claims (25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
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46. A method for amplifying and sequencing DNA, comprising:
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ligating adaptors to cell-free DNA isolated from a biological sample, wherein the adaptors each comprises a universal priming site, 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, wherein at least one of the primers comprises a sample index and a sequencing tag; performing high-throughput sequencing to sequence the amplified DNA comprising the target loci, wherein the target loci are SNP loci, and wherein amplified DNAs of multiple samples are pooled and sequenced in a single sequencing lane.
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Specification