Methods for multiplex PCR amplification of target loci in a nucleic acid sample
Methods for non-invasive prenatal paternity testing are disclosed herein. The method uses genetic measurements made on plasma taken from a pregnant mother, along with genetic measurements of the alleged father, and genetic measurements of the mother, to determine whether or not the alleged father is the biological father of the fetus. This is accomplished by way of an informatics based method that can compare the genetic fingerprint of the fetal DNA found in maternal plasma to the genetic fingerprint of the alleged father.
1. A method of amplifying target loci in a nucleic acid sample, the method comprising:
(a) performing multiplex PCR on a nucleic acid sample comprising target loci to simultaneously amplify at least 1,000 distinct target loci using either (i) at least 1,000 different primer pairs or (ii) at least 1,000 target-specific primers and a universal or tag-specific primer, wherein the multiplex PCR is performed in a single reaction volume to produce amplified products comprising target amplicons and
(b) sequencing the amplified products using high-throughput sequencing;
wherein the concentration of each primer in the primer pairs or each target-specific primer is less than 20 nM; and wherein the length of an annealing step of the multiplex PCR amplification is greater than 10 minutes.
2. The method of claim 1 , comprising obtaining primers for use in step (a) by empirically or in silico identifying one or more primers that form a primer dimer with another primer in a library of potential primers with the greatest frequency, eliminating the one or more identified primers from the library of potential primers, and using the primers remaining in the library in step (a).
3. The method of claim 1 , comprising performing universal amplification on nucleic acids in the sample prior to step (a), wherein the universal amplification optionally comprises universal PCR, whole genome amplification, ligation-mediated PCR, degenerate oligonucleotide primer PCR, or multiple displacement amplification.
4. The method of claim 1 , wherein the multiplex PCR comprises fully nested, semi-nested, or hemi-nested PCR; or wherein each primer pair comprises a forward and a reverse primer in which the 3′ ends of the forward and reverse primers are designed to hybridize to a region of DNA separated from a polymorphic site in a target locus by a small number of bases, wherein the small number is from 1 to 20 bases.
5. The method of claim 4 , wherein each primer pair comprises an inner forward primer in which the 3′ end of the inner forward primer is designed to hybridize to a region of DNA separated from a polymorphic site in a target locus by a small number of bases, wherein the small number is from 2 to 60 bases.
6. The method of claim 1 , wherein each primer pair comprises a forward and a reverse primer in which the 3′ end of the forward and reverse primer is designed to hybridize to a region of DNA separated from a polymorphic site in a target locus by a small number of bases, wherein the small number is from 2 to 20 bases.
7. The method of claim 1 , comprising simultaneously amplifying at least 5,000 distinct target loci.
8. The method of claim 1 , wherein at least 90% of the amplified products map to the target loci.
9. The method of claim 1 , wherein the multiplex PCR amplification comprises 20 cycles of PCR, and the average degree of allelic bias between the sample and the target amplicons is no more than a factor of 1.2.
10. The method of claim 1 , wherein the nucleic acid sample is isolated from a blood, plasma, or serum sample.
11. The method of claim 10 , wherein the nucleic acid sample comprises DNA from a fetus.
12. The method of claim 10 , wherein the nucleic acid sample comprises DNA from a transplant.
13. The method of claim 1 , wherein the target loci are present in the human genome.
14. The method of claim 1 , wherein the target loci comprise human single nucleotide polymorphisms.
15. The method of claim 1 , wherein the length of the target amplicons is less than 100 nucleotides.
16. The method of claim 1 , wherein the nucleic acid sample comprises DNA molecules with an average length of less than 200 base pairs, and wherein the target amplicons are less than 100 nucleotides in length.