Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
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 for nested PCR amplification, comprising:
isolating cell-free DNA from a biological sample and ligating adaptors to the isolated cell-free DNA, wherein the adaptors each comprise a universal priming sequence;
performing a first PCR to simultaneously amplify at least 10 target loci using a first universal primer and at least 10 target-specific primers in a first reaction volume; and
performing a second, nested PCR to simultaneously amplify the at least 10 target loci using a second universal primer and at least 10 inner target-specific primers in a second reaction volume to obtain amplified DNA, wherein primer binding sites of the inner target-specific primers of the second PCR are internal to primer binding sites of the target-specific primers of the first PCR, wherein at least 80% of the amplified DNA maps to the target loci.
2. The method of claim 1 , wherein the biological sample is a blood, plasma, serum, or urine sample.
3. The method of claim 1 , wherein the adaptors each comprise a molecular barcode.
4. The method of claim 1 , wherein at least one of the primers comprises a sequencing tag.
5. The method of claim 1 , wherein the adaptors each comprise a molecular barcode, and wherein at least one of the primers comprises a sequencing tag.
6. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying between 100 and 5,000 target loci using the first universal primer and between 100 and 5,000 target-specific primers in the first reaction volume.
7. The method of claim 6 , wherein the second PCR comprises simultaneously amplifying between 100 and 5,000 target loci using the second universal primer and between 100 and 5,000 inner target-specific primers in the second reaction volume.
8. The method of claim 1 , wherein the first PCR comprises simultaneously amplifying between 100 and 1,000 target loci using the first universal primer and between 100 and 1,000 target-specific primers in the first reaction volume.
9. The method of claim 8 , wherein the second PCR comprises simultaneously amplifying between 100 and 1,000 target loci using the second universal primer and between 100 and 1,000 inner target-specific primers in the second reaction volume.
10. The method of claim 3 , wherein the isolated cell-free DNA are tagged with up to 1024 different molecular barcodes.
11. The method of claim 3 , wherein the isolated cell-free DNA are tagged with 1024-65536 different molecular barcodes.
12. The method of claim 1 , wherein the concentration of each target-specific primer of the first and/or second PCR is less than 20 nM.
13. The method of claim 1 , wherein the concentration of each target-specific primer of the first and/or second PCR is less than 10 nM.
14. The method of claim 1 , wherein the length of the annealing step of the first and/or second PCR is at least 3 minutes.
15. The method of claim 1 , wherein the length of the annealing step of the first and/or second PCR is at least 5 minutes.
16. The method of claim 1 , wherein at least 90% of the amplified DNA maps to the target loci.
17. The method of claim 1 , wherein the target loci are SNP loci.
18. The method of claim 1 , wherein the cell-free DNA comprises DNA from a fetus.
19. The method of claim 1 , wherein the cell-free DNA comprises DNA from a tumor.
20. The method of claim 1 , wherein the cell-free DNA comprises DNA from a transplant.
21. The method of claim 1 , wherein amplified DNAs of multiple samples are pooled and sequenced in a single sequencing lane.