Methods for simultaneous amplification of target loci
DC CAFCFirst Claim
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1. A method for measuring an amount of DNA from a first individual in a biological sample of a second individual, comprising:
- pre-amplifying at least 50 polymorphic loci from cell-free DNA of mixed origin in a single reaction volume to obtain pre-amplified DNA, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual, wherein neither the first individual nor the second individual is a fetus;
dividing the pre-amplified DNA into multiple aliquots;
amplifying subpools of the polymorphic loci in parallel in individual reaction volumes to obtain amplified DNA, wherein each reaction volume comprises at least one aliquot of the pre-amplified DNA; and
pooling the amplified DNA into one pool;
sequencing the amplified DNA by high-throughput sequencing and measuring an amount of each allele at the polymorphic loci; and
determining the amount of the DNA from the first individual in the biological sample using the amount of each allele at the polymorphic 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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15 Claims
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1. A method for measuring an amount of DNA from a first individual in a biological sample of a second individual, comprising:
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pre-amplifying at least 50 polymorphic loci from cell-free DNA of mixed origin in a single reaction volume to obtain pre-amplified DNA, wherein the cell-free DNA is extracted from the biological sample and comprises DNA from the first individual and DNA from the second individual, wherein neither the first individual nor the second individual is a fetus; dividing the pre-amplified DNA into multiple aliquots;
amplifying subpools of the polymorphic loci in parallel in individual reaction volumes to obtain amplified DNA, wherein each reaction volume comprises at least one aliquot of the pre-amplified DNA; and
pooling the amplified DNA into one pool;sequencing the amplified DNA by high-throughput sequencing and measuring an amount of each allele at the polymorphic loci; and determining the amount of the DNA from the first individual in the biological sample using the amount of each allele at the polymorphic loci. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)
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