IP Library › Granted Patent US 12,662,704
Granted Patent B2
US 12,662,704 · App. 17/196,722 · Granted Jun 23, 2026

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 12,662,704
App. No.
17/196,722
Filed
Mar 9, 2021
Granted
Jun 23, 2026
Kind
B2
Art Unit
1681
USPC
506/2
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 (15)

1 . A method comprising:

(a) performing multiplex targeted pre-amplification of at least 10 target loci from cell-free DNA isolated from a biological sample in a single reaction volume using at least 10 different target-specific primers to obtain amplified DNA, wherein the biological sample is a blood, plasma, serum, or urine sample;

(b) tagging the amplified DNA with a plurality of different molecular barcodes and a sequencing adapter to obtain molecularly barcoded amplified DNA; and

(c) performing high-throughput sequencing of the molecularly barcoded amplified DNA to sequence the at least 10 amplified target loci to generate sequence reads with a depth of at least 200 reads per target locus, wherein the target loci comprises a cancer-associated mutation, and wherein the target loci are single nucleotide variant (SNV) loci.

2 . The method of claim 1 , wherein at least 20 target loci are pre-amplified in a single reaction volume using at least 20 different target-specific primers to obtain amplified DNA.

3 . The method of claim 1 , wherein at least 50 target loci are pre-amplified in a single reaction volume using at least 50 different target-specific primers to obtain amplified DNA.

4 . The method of claim 1 , wherein at least 100 target loci are pre-amplified in a single reaction volume using at least 100 different target-specific primers to obtain amplified DNA.

5 . The method of claim 1 , wherein the amplified DNA is tagged with up to 1024 molecular barcodes.

6 . The method of claim 1 , wherein the amplified DNA is tagged with between 1024 to 65536 molecular barcodes.

7 . The method of claim 1 , wherein the biological sample is not obtained from a pregnant woman.

8 . The method of claim 1 , wherein 10 to 200 target loci are pre-amplified in a single reaction volume.

9 . The method of claim 1 , further comprising tagging the amplified DNA with a sample-specific barcode.

10 . The method of claim 1 , wherein the amplified DNA is tagged by ligation.

11 . The method of claim 1 , further comprising identifying the SNV loci with a limit of detection of less than 0.01%.

12 . The method of claim 1 , wherein the method does not comprise PCR amplification in a microfluidic device.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 17, 2021
From: BABIARZ, JOSHUA; CONSTANTIN, TUDOR POMPILIU; EUBANK, LANE A.; GEMELOS, GEORGE; HILL, MATTHEW; KIRKIZLAR, HUSEYIN ESER; RABINOWITZ, MATTHEW; SAKARYA, ONUR; SIGURJONSSON, STYRMIR; ZIMMERMANN, BERNHARD
To: NATERA, INC.
Reel/Frame 055620/0185 →
Continuity (40)
Continuation 16934407 · Jul 21, 2020
Continuation 16743724 · Jan 15, 2020
Continuation 16399268 · Apr 30, 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 13683604 · Nov 21, 2012
Continuation In Part 13683604 · Nov 21, 2012
Continuation 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 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 20210198743A1 · Jul 1, 2021
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