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 determining a transplant status, comprising
 (a) obtaining a nucleic acid sample from a blood, plasma, or serum sample of a transplant recipient, wherein the nucleic acid sample comprises a mixture of cell-free DNA from a transplant and cell-free DNA from the transplant recipient;   (b) amplifying at least 100 different target loci from the nucleic acid sample by contacting the nucleic acid sample with a library of at least 100 non-immobilized, non-identical primers that hybridize to the at least 100 different target loci to produce a reaction mixture, and subjecting the reaction mixture to PCR conditions to produce amplification products comprising target amplicons, wherein at least 50% of the amplification products each comprises at least one of the target loci;   (c) measuring an absolute or relative amount of transplant cell-free DNA from the amplification products, and determining a transplant status based on the measured amount of transplant cell-free DNA, wherein the transplant status is transplant rejection, tolerance, non-rejection based allograft injury, transplant function, transplant survival, chronic transplant injury, or tittering of pharmacological immunosuppression.   
     
     
         2 . The method of  claim 1 , wherein the length of the target amplicons is less than 100 nucleotides. 
     
     
         3 . The method of  claim 1 , wherein a range of melting temperatures among the different primers in the library is less than 10° C. 
     
     
         4 . The method of  claim 1 , wherein a range of melting temperatures among the different primers in the library is less than 5° C. 
     
     
         5 . The method of  claim 1 , further comprising performing universal amplification on DNA molecules in the nucleic acid sample prior to step (b). 
     
     
         6 . The method of  claim 1 , wherein at least 90% of the amplification products in step (b) are target amplicons. 
     
     
         7 . The method of  claim 1 , wherein at least 90% of the target loci are amplified in step (b). 
     
     
         8 . The method of  claim 1 , wherein less than 20% of the amplification products in step (b) are primer dimers. 
     
     
         9 . The method of  claim 1 , wherein the library of primers each comprises a 5′ region that is not specific for a target locus followed by a region that is specific for a target locus, an internal region that is not specific for the target locus and forms a loop structure, and a 3′ region that is specific for the target locus. 
     
     
         10 . The method of  claim 1 , wherein average length of DNA molecules in the nucleic acid sample is less than 200 base pairs. 
     
     
         11 . The method of  claim 1 , wherein an annealing step of the PCR conditions is between 10 and 60 minutes. 
     
     
         12 . The method of  claim 1 , wherein the concentration of each of the library of primers in the reaction mixture is less than 20 nM. 
     
     
         13 . The method of  claim 1 , wherein the concentration of each of the library of primers in the reaction mixture is less than 10 nM. 
     
     
         14 . The method of  claim 1 , wherein the library comprises at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 different target loci, and wherein the 1,000 non-identical target loci are amplified. 
     
     
         15 . The method of  claim 1 , wherein a range of guanine-cytosine (GC) content among the different primers in the library is less than 30%. 
     
     
         16 . The method of  claim 15 , wherein a range of melting temperatures among the different primers in the library is less than 20° C. 
     
     
         17 . The method of  claim 16 , wherein a range of lengths among the different target amplicons is less than 50 nucleotides. 
     
     
         18 . The method of  claim 1 , wherein the measuring comprises sequencing the amplification products by high-throughput sequencing. 
     
     
         19 . The method of  claim 1 , wherein the target loci comprise single nucleotide polymorphism (SNP) loci, wherein the cell-free DNA from the transplant and the cell-free DNA from the transplant recipient comprise different alleles at said SNP loci. 
     
     
         20 . The method of  claim 19 , wherein the transplant recipient is homozygous for a first allele, and the transplant is homozygous for a second allele or is heterozygous for the first allele and the second allele, at one or more of the SNP loci. 
     
     
         21 . A method for detecting tumor recurrence, comprising
 (a) identifying a plurality of tumor-specific mutations in a tumor tissue of a cancer patient;   (b) obtaining a nucleic acid sample from a blood, plasma, or serum sample of the cancer patient, wherein the nucleic acid sample comprises a mixture of cell-free DNA originated from tumor cells and cell-free DNA originated from normal cells;   (c) amplifying a plurality of different target loci from the nucleic acid sample by contacting the nucleic acid sample with a plurality of non-immobilized, non-identical primers that hybridize to the plurality of different target loci to produce a reaction mixture, and subjecting the reaction mixture to PCR conditions to produce amplification products comprising target amplicons, wherein at least 50% of the amplification products each comprises at least one of the target loci;   (d) measuring the presence of one or more of the tumor-specific mutations in the amplification products to detect tumor recurrence.   
     
     
         22 . The method of  claim 21 , wherein the length of the target amplicons is less than 100 nucleotides. 
     
     
         23 . The method of  claim 21 , wherein a range of melting temperatures among the plurality of primers is less than 10° C. 
     
     
         24 . The method of  claim 21 , wherein a range of melting temperatures among the plurality of primers is less than 5° C. 
     
     
         25 . The method of  claim 21 , further comprising performing universal amplification on DNA molecules in the nucleic acid sample prior to step (c). 
     
     
         26 . The method of  claim 21 , wherein at least 90% of the amplification products in step (c) are target amplicons. 
     
     
         27 . The method of  claim 21 , wherein at least 90% of the target loci are amplified in step (c). 
     
     
         28 . The method of  claim 21 , wherein less than 20% of the amplification products in step (c) are primer dimers. 
     
     
         29 . The method of  claim 21 , wherein the plurality of primers each comprises a 5′ region that is not specific for a target locus followed by a region that is specific for a target locus, an internal region that is not specific for the target locus and forms a loop structure, and a 3′ region that is specific for the target locus. 
     
     
         30 . The method of  claim 21 , wherein average length of DNA molecules in the nucleic acid sample is less than 200 base pairs. 
     
     
         31 . The method of  claim 21 , wherein an annealing step of the PCR conditions is between 10 and 60 minutes. 
     
     
         32 . The method of  claim 21 , wherein the concentration of each of the plurality of primers in the reaction mixture is less than 20 nM. 
     
     
         33 . The method of  claim 21 , wherein the concentration of each of the plurality of primers in the reaction mixture is less than 20 nM. 
     
     
         34 . The method of  claim 21 , wherein the plurality of primers comprises at least 25 non-immobilized, non-identical primers that hybridize to at least 25 different target loci, and wherein the 25 non-identical target loci are amplified. 
     
     
         35 . The method of  claim 21 , wherein a range of guanine-cytosine (GC) content among the plurality of primers is less than 30%. 
     
     
         36 . The method of  claim 35 , wherein a range of melting temperatures among the plurality of primers is less than 20° C. 
     
     
         37 . The method of  claim 36 , wherein a range of lengths among the different target amplicons is less than 50 nucleotides. 
     
     
         38 . The method of  claim 21 , wherein the measuring comprises sequencing the amplification products by high-throughput sequencing. 
     
     
         39 . The method of  claim 21 , wherein the target loci comprise single nucleotide polymorphism (SNP) loci, wherein the cell-free DNA originated from tumor cells and the cell-free DNA originated from normal cells comprise different alleles at said SNP loci. 
     
     
         40 . The method of  claim 39 , wherein the cell-free DNA originated from normal cells is homozygous for a first allele, and the cell-free DNA originated from tumor cells is homozygous for a second allele or is heterozygous for the first allele and the second allele, at one or more of the SNP loci.