Methods for non-invasive prenatal paternity testing
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:
performing multiplex polymerase chain reaction (PCR) on a nucleic acid sample comprising 1,000 target loci in a single reaction volume, wherein the single reaction volume comprises (i) 1,000 different target loci-specific PCR primer pairs or (ii) 1,000 different target loci-specific PCR primers and a universal or tag-specific PCR primer, wherein the concentration of each PCR primer in the primer pairs or each target-specific PCR primer is 20 nM or less and the length of an annealing step of the multiplex PCR is 10 minutes or more, thereby producing amplified products comprising greater than 50% target amplicons that map to target loci.
2. The method of claim 1 , comprising performing universal amplification on nucleic acids in the sample prior to performing the multiplex PCR, wherein the universal amplification optionally comprises universal PCR, whole genome amplification, ligation-mediated PCR, degenerate oligonucleotide primer PCR, or multiple displacement amplification.
3. 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′ 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 selected from the group consisting of 2, 3, 4, 5, 6, 7 to 10, 11 to 15, or 16 to 20 bases.
4. The method of claim 1 , comprising simultaneously amplifying at least 5,000 distinct target loci.
5. The method of claim 1 , comprising simultaneously amplifying at least 10,000 distinct target loci.
6. The method of claim 1 , wherein at least 90% of the amplified products map to the target loci.
7. The method of claim 1 , wherein the concentration of each primer in the primer pairs or each target-specific primer is 10 nM or less.
8. The method of claim 1 , wherein the target loci are present in the human genome, or wherein the target loci comprise human single nucleotide polymorphisms.
9. The method of claim 1 , wherein the nucleic acid sample comprises DNA from a fetus.
10. The method of claim 1 , wherein the nucleic acid sample comprises maternal DNA from the pregnant mother of a fetus and fetal DNA, wherein the target loci comprise a human single nucleotide polymorphism (SNP), and wherein the method comprises measuring the amount of one or more alleles at the target loci comprising a human SNP.
11. The method of claim 1 , wherein the nucleic acid sample comprises DNA from a tumor.
12. The method of claim 1 , wherein the nucleic acid sample comprises DNA from an individual, and wherein the DNA comprises DNA from a cancer and DNA not from a cancer.
13. The method of claim 1 , wherein the nucleic acid sample comprises DNA from a transplant.
14. The method of claim 1 , wherein the length of the target amplicons is less than 100 nucleotides.
15. The method of claim 1 , wherein the nucleic acid sample comprises DNA from a single cell.
16. The method of claim 1 , wherein the annealing step comprises annealing at a temperature of 60° C. to 65° C.
17. A method of amplifying target loci in a nucleic acid sample, the method comprising:
performing multiplex PCR on a nucleic acid sample comprising DNA molecules having an average length of less than 200 base pairs and comprising 1,000 target loci in a single reaction volume, wherein the single reaction volume comprises (i) 1,000 different target loci-specific PCR primer pairs or (ii) 1,000 different target loci-specific PCR primers and a universal or tag-specific PCR primer, wherein the concentration of each PCR primer in the primer pairs or each target-specific PCR primer is 20 nM or less and the length of an annealing step of the multiplex PCR is 10 minutes or more, thereby producing amplified products comprising greater than 50% target amplicons that are less than 100 nucleotides in length and map to target loci.
18. The method of claim 17 , wherein each primer pair comprises a forward primer in which the 3′ end of the forward primer is designed to hybridize to a region of DNA separated from a polymorphic site in a target locus by 2 to 20 bases.
19. The method of claim 17 , wherein each primer pair comprises a forward primer in which the 3′ end of the forward primer is designed to hybridize to a region of DNA separated from a polymorphic site in a target locus by 2 to 10 bases.
20. The method of claim 17 , wherein the amplified products comprise greater than 90% target amplicons.
21. The method of claim 17 , wherein the primer pairs are selected by eliminating one or more primers that form primer dimers.