METHODS FOR SIMULTANEOUS AMPLIFICATION OF TARGET LOCI
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.
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.