Amplification of cell-free DNA using nested PCR

• US 10,590,482 B2
• Filed: 04/30/2019
• Issued: 03/17/2020
• Est. Priority Date: 05/18/2010
• Status: Active Grant

• Current Assignee: Natera Incorporated (Natera)
• Original Assignee: Natera Incorporated (Natera)
• Inventors: Ryan, Allison, Sigurjonsson, Styrmir, Banjevic, Milena, Gemelos, George, Hill, Matthew, Baner, Johan, Rabinowitz, Matthew, Demko, Zachary
• Primary Examiner(s): Vanni, G Steven
• Application Number:
  US16/399,957
• Publication Number:
  US 20190256907A1
• Time in Patent Office: 322 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: G16B 10/00 ICT specially adapted for evolutionary bioinformatics, e.g. phylogenetic tree construction or analysis

Abstract

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.

Claims

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 site;

performing a first PCR to simultaneously amplify at least 10 target loci using a 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 the 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 at least one of the primers comprises a sequencing tag.

4. The method of claim 1, wherein the PCR is one-sided nested PCR.

5. The method of claim 1, wherein the first PCR comprises simultaneously amplifying at least 50 target loci using the universal primer and at least 50 target-specific primers in the first reaction volume.

6. The method of claim 1, wherein the first PCR comprises simultaneously amplifying at least 100 target loci using the universal primer and at least 100 target-specific primers in the first reaction volume.

7. The method of claim 1, wherein the second PCR comprises simultaneously amplifying at least 50 target loci using the universal primer and at least 50 inner target-specific primers in the second reaction volume.

8. The method of claim 1, wherein the second PCR comprises simultaneously amplifying at least 100 target loci using the universal primer and at least 100 inner target-specific primers in the second reaction volume.

9. 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.

10. 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.

11. The method of claim 1, wherein the length of the annealing step of the first and/or second PCR is at least 3 minutes.

12. The method of claim 1, wherein the length of the annealing step of the first and/or second PCR is at least 5 minutes.

13. The method of claim 1, wherein at least 90% of the amplified DNA maps to the target loci.

14. The method of claim 1, wherein the target loci are SNP loci.

15. The method of claim 1, wherein the cell-free DNA comprises DNA from a fetus.

16. The method of claim 1, wherein the cell-free DNA comprises DNA from a tumor.

17. The method of claim 1, wherein the cell-free DNA comprises DNA from a transplant.

18. The method of claim 1, wherein the adaptors each comprise a molecular barcode.

19. The method of claim 18, wherein the isolated cell-free DNA are tagged with up to 1024 different molecular barcodes.

20. The method of claim 18, wherein the isolated cell-free DNA are tagged with 1024-65536 different molecular barcodes.