IP Library › Granted Patent US 12,410,476
Granted Patent B2
US 12,410,476 · App. 17/868,238 · Granted Sep 9, 2025

Methods for simultaneous amplification of target loci

Inventors: Matthew Rabinowitz (San Francisco, CA); Matthew Micah Hill (Belmont, CA); Bernhard Zimmermann (Manteca, CA); Johan Baner (San Francisco, CA); George Gemelos (Portland, OR); Milena Banjevic (Los Altos Hills, CA); Allison Ryan (Belmont, CA); Styrmir Sigurjonsson (San Jose, CA); Zachary Demko (San Francisco, CA)
Assignee: Natera, Inc.
C12Q1/6883C12Q1/6809C12Q1/6811C12Q1/6844C12Q1/6848C12Q1/6851C12Q1/6855C12Q1/6869C12Q1/6874C12Q2600/156
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Quick Facts
Patent No.
US 12,410,476
App. No.
17/868,238
Granted
Sep 9, 2025
Kind
B2
Abstract

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.

Claims (35)

1. A method for preparing a non-naturally occurring composition of amplified DNA derived from a biological sample useful for measurement of an amount of DNA of a first individual in a biological sample of a second individual, comprising:

(a) extracting cell-free DNA from the biological sample, wherein the biological sample comprises DNA of the first individual and DNA of the second individual;

(b) preparing a non-naturally occurring composition of amplified DNA by performing targeted PCR amplification on the cell-free DNA extracted in (a) or DNA derived therefrom to amplify about 200 to about 20,000 SNP target loci on one or more chromosomes expected to be disomic using about 200 to about 20,000 target-specific primers or primer pairs in the same reaction; and

(c) analyzing the non-naturally occurring composition of amplified DNA obtained in (b) by performing high-throughput sequencing to measure an amount of one or more alleles at a plurality of amplified target loci.

2. The method of claim 1 , wherein the biological sample is a blood, serum, plasma, or urine sample.

3. The method of claim 1 , wherein the cell-free DNA extracted in (a) comprises DNA of a fetus.

4. The method of claim 1 , wherein the cell-free DNA extracted in (a) comprises DNA of a transplant.

5. The method of claim 1 , wherein the extracting step comprises size selection to enrich for shorter cell-free DNA.

6. The method of claim 1 , wherein the target-specific primers or primer pairs are each designed to amplify less than about 100 bp of DNA.

7. The method of claim 1 , wherein the target-specific primers or primer pairs are each designed to amplify about 65-80 bp of DNA.

8. The method of claim 1 , wherein the targeted PCR amplification amplifies about 200 to about 1,000 SNP target loci on one or more chromosomes expected to be disomic.

9. The method of claim 1 , wherein the targeted PCR amplification amplifies about 200 to about 2,000 SNP target loci on one or more chromosomes expected to be disomic.

10. The method of claim 1 , wherein the targeted PCR amplification amplifies about 500 to about 5,000 SNP target loci on one or more chromosomes expected to be disomic.

11. The method of claim 1 , wherein the targeted PCR amplification amplifies about 1,000 to about 10,000 SNP target loci on one or more chromosomes expected to be disomic.

12. The method of claim 1 , wherein the non-naturally occurring composition of amplified DNA obtained in (b) comprises amplified SNP loci on chromosome 1.

13. The method of claim 1 , wherein the non-naturally occurring composition of amplified DNA obtained in (b) comprises amplified SNP loci on chromosome 2.

14. The method of claim 1 , wherein the non-naturally occurring composition of amplified DNA obtained in (b) comprises amplified SNP loci on chromosome 3.

15. The method of claim 1 , wherein step (c) comprises performing a barcoding PCR to add a sequencing tag and a sample-specific barcode to obtain a barcoded sequencing library, pooling a plurality of barcoded sequencing libraries and sequencing the pool of barcoded sequencing libraries together in one sequencing lane, and for each library measuring an amount of each allele at the amplified target loci.

16. The method of claim 1 , wherein the high-throughput sequencing is sequencing-by-synthesis.

17. The method of claim 1 , wherein the method further comprises measuring the amount of DNA of the first individual in the biological sample of the second individual using the amount of one or more alleles at the amplified target loci.

18. A method for preparing a non-naturally occurring composition of amplified DNA derived from a biological sample useful for measurement of an amount of DNA of a first individual in a biological sample of a second individual, comprising:

(a) preparing a non-naturally occurring composition of amplified DNA by performing targeted PCR amplification to amplify about 200 to about 20,000 SNP target loci on one or more chromosomes expected to be disomic using about 200 to about 20,000 target-specific primers or primer pairs in a single reaction, wherein the reaction mixture comprises cell-free DNA extracted from the biological sample or DNA derived therefrom, wherein the biological sample is a blood, serum, plasma, or urine sample comprising DNA of the first individual and DNA of the second individual; and

(b) analyzing the non-naturally occurring composition of amplified DNA obtained in (a) by performing high-throughput sequencing to measure a quantity of each allele at a plurality of amplified target loci that comprise an allele present in the first individual but not the second individual.

19. The method of claim 18 , wherein the cell-free DNA extracted from the biological sample comprises DNA of a fetus.

20. The method of claim 18 , wherein the cell-free DNA extracted from the biological sample comprises DNA of a transplant.

21. The method of claim 18 , wherein the extracting step comprises size selection to enrich for shorter cell-free DNA.

22. The method of claim 18 , wherein the target-specific primers or primer pairs are each designed to amplify less than about 100 bp of DNA.

23. The method of claim 18 , wherein the target-specific primers or primer pairs are each designed to amplify about 65-80 bp of DNA.

24. The method of claim 18 , wherein the targeted PCR amplification amplifies about 200 to about 1,000 SNP target loci on one or more chromosomes expected to be disomic.

25. The method of claim 18 , wherein the targeted PCR amplification amplifies about 200 to about 2,000 SNP target loci on one or more chromosomes expected to be disomic.

26. The method of claim 18 , wherein the targeted PCR amplification amplifies about 500 to about 5,000 SNP target loci on one or more chromosomes expected to be disomic.

27. The method of claim 18 , wherein the targeted PCR amplification amplifies about 1,000 to about 10,000 SNP target loci on one or more chromosomes expected to be disomic.

28. The method of claim 18 , wherein step (b) comprises performing a barcoding PCR to add a sequencing tag and a sample-specific barcode to obtain a barcoded sequencing library, pooling a plurality of barcoded sequencing libraries and sequencing the pool of barcoded sequencing libraries together in one sequencing lane, and for each library measuring a quantity of each allele at the amplified target loci.

29. The method of claim 18 , wherein the high-throughput sequencing is sequencing-by-synthesis.

30. The method of claim 18 , wherein the method further comprises measuring the amount of DNA of the first individual in the biological sample of the second individual using the quantity of each allele at the plurality of amplified target loci.

Continuity (41)
Continuation 16817117 · Mar 12, 2020
Continuation 16353636 · Mar 14, 2019
Continuation 16140298 · Sep 24, 2018
Continuation 14918544 · Oct 20, 2015
Continuation In Part 14877925 · Oct 7, 2015
Continuation In Part 14692703 · Apr 21, 2015
Continuation In Part 14538982 · Nov 24, 2014
Continuation In Part 14225356 · Mar 25, 2014
Continuation In Part 13780022 · Feb 28, 2013
Continuation In Part 13683604 · Nov 21, 2012
Continuation In Part 13683604 · Nov 21, 2012
Continuation In Part PCTUS2012058578 · Oct 3, 2012
Continuation In Part PCTUS2012058578 · Oct 3, 2012
Continuation In Part 13335043 · Dec 22, 2011
Continuation In Part 13300235 · Nov 18, 2011
Continuation In Part 13300235 · Nov 18, 2011
Continuation In Part 13300235 · Nov 18, 2011
Continuation In Part 13300235 · Nov 18, 2011
Continuation In Part 13110685 · May 18, 2011
Continuation In Part 13110685 · May 18, 2011
Continuation In Part 13110685 · May 18, 2011
Continuation In Part 13110685 · May 18, 2011
Provisional Application 62148173 · Apr 15, 2015
Provisional Application 62147377 · Apr 14, 2015
Provisional Application 62146188 · Apr 10, 2015
Provisional Application 62066514 · Oct 21, 2014
Provisional Application 61994791 · May 16, 2014
Provisional Application 61987407 · May 1, 2014
Provisional Application 61982245 · Apr 21, 2014
Provisional Application 61683331 · Aug 15, 2012
Provisional Application 61675020 · Jul 24, 2012
Provisional Application 61634431 · Feb 29, 2012
Provisional Application 61542508 · Oct 3, 2011
Provisional Application 61571248 · Jun 23, 2011
Provisional Application 61516996 · Apr 12, 2011
Provisional Application 61448547 · Mar 2, 2011
Provisional Application 61462972 · Feb 9, 2011
Provisional Application 61426208 · Dec 22, 2010
Provisional Application 61398159 · Jun 21, 2010
Provisional Application 61395850 · May 18, 2010
Related Publication 20230053752A1 · Feb 23, 2023
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