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 of amplifying target loci of DNA molecules in a nucleic acid sample, the method comprising:
(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM; wherein the average length of the DNA molecules is less than 200 base pairs; and wherein a range of melting temperatures of the at least 1000 non-identical primers is less than 10° C.;
(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons; wherein a length of an annealing step of the PCR conditions is greater than 10 minutes; wherein the length of the target amplicons is less than 100 nucleotides; wherein at least 1,000 different target human loci are amplified, and wherein at least 50% of the amplified products comprise target loci.
2. The method of claim 1 , wherein a range of melting temperatures of the at least 1000 non-identical primers is less than 5° C.
3. The method of claim 1 , further comprising performing universal amplification on DNA molecules in the sample prior to step (a).
4. The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.
5. The method of claim 1 , wherein at least 90% of the target loci are amplified.
6. The method of claim 1 , wherein less than 20% of the amplified products are primer dimers.
7. The method of claim 1 , wherein the at least 1,000 non-identical primers comprise a 5′ region that is not specific for a target human locus followed by a region that is specific for a target human locus, an internal region that is not specific for the target human locus and forms a loop structure, and a 3′ region that is specific for the target humanlocus.
8. The method of claim 1 , wherein the sample comprises DNA from a pregnant mother and from her fetus, and wherein the method further comprises amplifying the amplified human loci and determining the presence or absence of a fetal chromosome abnormality from the sequencing data.
9. The method of claim 1 , wherein the sample comprises DNA from a single cell.
10. A method of amplifying target loci of DNA molecules in a nucleic acid sample, the method comprising:
(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM; wherein the average length of the DNA molecules is less than 200 base pairs;
(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons; wherein a length of an annealing step of the PCR conditions is greater than 10 minutes; wherein the length of the target amplicons is less than 100 nucleotides; wherein at least 1,000 different target human loci are amplified, wherein at least 50% of the amplified products comprise target loci; and wherein the sample comprises or is derived from maternal blood.
11. The method of claim 10 , wherein the method further comprises sequencing the amplified human loci and making a genetic diagnosis, wherein the genetic diagnosis comprises detecting a gene mutation or detecting a ploidy state of the target human loci.
12. The method of claim 1 , wherein the length of the annealing step of the reaction conditions is between 10 and 60 minutes.
13. The method of claim 1 , wherein the concentration of primers in the reaction volume are less than 10 nM.
14. The method of claim 1 , wherein the concentration of primers in the reaction volume are less than 5 nM.
15. The method of claim 1 , wherein the library comprises at least 7,500 non-immobilized, non-identical primers that hybridize to at least 7,500 non-identical target human loci, and wherein the 7,500 non-identical target human loci are amplified.
16. The method of claim 1 , The library of claim 1 , wherein a range of guanine-cytosine (GC) content among the different test primers in the library is less than 30%.
17. The method of claim 14 , wherein a range of melting temperatures among the different primers in the library is less than 20° C.
18. The method of claim 15 , wherein a range of the length among the different target amplicons is less than 50 nucleotides.
19. A method of amplifying target loci of DNA molecules in a nucleic acid sample, the method comprising:
(a) contacting the sample and a library of at least 1,000 non-immobilized, non-identical primers that hybridize to at least 1,000 non-identical target loci to produce a reaction mixture in which the concentration of each primer is less than 20 nM; wherein the average length of the DNA molecules is less than 200 base pairs;
(b) subjecting the reaction mixture to PCR conditions to produce amplified products comprising target amplicons; wherein a length of an annealing step of the PCR conditions is greater than 10 minutes; wherein the length of the target amplicons is less than 100 nucleotides; wherein at least 1,000 different target human loci are amplified, wherein at least 50% of the amplified products comprise target loci; and wherein the target human loci comprise single nucleotidepolymorphisms.