Methods for simultaneous amplification of target loci

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.

What is claimed is: 
     
         1 . A method for preparing a biological sample containing DNA from a first individual and a second individual, comprising:
 isolating cell-free DNA from the biological sample that comprises the DNA from the first individual and DNA from the second individual; and   amplifying the isolated cell-free DNA or DNA derived therefrom at a plurality of polymorphic loci, wherein the plurality of polymorphic loci comprises between 100 and 1,000 polymorphic loci, wherein the amplifying comprises amplifying the between 100 and 1,000 polymorphic loci from the isolated cell-free DNA or DNA derived therefrom using between 100 and 1,000 distinct primers configure to target the between 100 and 1,000 polymorphic loci.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises enriching the DNA in the sample at a plurality of polymorphic loci prior to the amplifying step. 
     
     
         3 . The method of  claim 2 , wherein the enrichment comprises:
 obtaining a plurality of target-specific primers designed to hybridize to regions of DNA upstream and downstream of the polymorphic loci;   hybridizing the target-specific primers to the DNA; and   amplifying the DNA using the polymerase chain reaction to form amplicons.   
     
     
         4 . The method of  claim 2 , further comprising preferentially enriching the DNA at a plurality of loci to minimize an average degree of allelic bias. 
     
     
         5 . The method of  claim 1 , wherein the polymorphic loci are SNP loci. 
     
     
         6 . The method of  claim 5 , wherein the polymorphic loci comprise SNP loci on chromosome 1, chromosome 2, and/or chromosome 3. 
     
     
         7 . The method of  claim 1 , wherein the plurality of polymorphic loci comprises at least 200 polymorphic loci, wherein the amplifying comprises amplifying the at least 200 polymorphic loci from the cell-free DNA isolated from the biological sample. 
     
     
         8 . The method of  claim 1 , wherein the biological sample is a blood sample or part thereof, and wherein the cell-free DNA comprises DNA from a fetus. 
     
     
         9 . The method of  claim 1 , wherein the biological sample is a blood sample or part thereof, and wherein the cell-free DNA comprises DNA from a transplant. 
     
     
         10 . The method of  claim 1 , wherein the distinct primers are circularizing probes. 
     
     
         11 . The method of  claim 1 , wherein the distinct primers are configured to target the polymorphic loci via hybridization. 
     
     
         12 . The method of  claim 1 , wherein the distinct primers comprise PCR primers. 
     
     
         13 . The method of  claim 1 , wherein the total amount of cfDNA in the sample is between 10 pg and 100 pg. 
     
     
         14 . The method of  claim 13 , wherein less than or equal to 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, or 0.005% of the cfDNA molecules that have a first locus have a mutation in the first locus. 
     
     
         15 . The method of  claim 13 , wherein amplifying the isolated cell-free DNA comprises amplifying from 72%, 69%, 66%, 63%, 59%, or 56% of available cfDNA template fragment molecules. 
     
     
         16 . The method of  claim 1 , wherein method further comprises performing high-throughput sequencing on the amplified DNA. 
     
     
         17 . A method for preparing a biological sample containing DNA from a first individual and a second individual, comprising:
 isolating cell-free DNA from the biological sample that comprises the DNA from the first individual and DNA from the second individual; and   enriching the isolated cell-free DNA or DNA derived therefrom at a plurality of SNP loci to obtain enriched DNA, wherein the plurality of SNP loci comprises between 100 and 1,000 SNP loci; and   amplifying the enriched DNA at the plurality of SNP loci, wherein the amplifying comprises amplifying the between 100 and 1,000 SNP loci from the enriched DNA using between 100 and 1,000 distinct primers configure to target the between 100 and 1,000 SNP loci.   
     
     
         18 . The method of  claim 17 , wherein the biological sample is a blood sample or part thereof, and wherein the cell-free DNA comprises DNA from a fetus. 
     
     
         19 . The method of  claim 17 , wherein the biological sample is a blood sample or part thereof, and wherein the cell-free DNA comprises DNA from a transplant. 
     
     
         20 . The method of  claim 17 , wherein method further comprises performing high-throughput sequencing on the amplified DNA.