IP Library › Granted Patent US 12,305,229
Granted Patent B2
US 12,305,229 · App. 17/214,403 · Granted May 20, 2025

Methods for simultaneous amplification of target loci

Inventors: Bernhard Zimmermann (Manteca, CA); Matthew Hill (Belmont, CA); Philippe Lacroute (Sunnyvale, CA); Michael Dodd (San Francisco, CA); Alexander Wong (Mountain View, CA)
Assignee: Natera, Inc.
C12Q1/686C12Q1/68C12Q1/6811C12Q1/6848C12Q1/6874C12Q1/6883C12Q2600/156
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Quick Facts
Patent No.
US 12,305,229
App. No.
17/214,403
Granted
May 20, 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 (26)

1. A method of amplifying target loci in a nucleic acid sample, the method comprising:

tagging a nucleic acid sample comprising cell-free nucleic acids including target loci, wherein the tagging includes attaching at least one of a plurality of molecular barcodes to a plurality of the cell-free nucleic acids to create tagged nucleic acids;

contacting one or more of the tagged nucleic acids with a plurality of primers;

hybridizing the plurality of primers to a plurality of the target loci to produce a reaction mixture;

subjecting the reaction mixture to primer extension reaction conditions and producing amplified products comprising target amplicons;

wherein at least two primers of the plurality of primers that are hybridized to target loci during the hybridizing step each have a melting temperature lower than an annealing temperature for the primer extension reaction conditions, wherein the length of the annealing step of the reaction conditions is greater than 3 minutes, and wherein at least two of the plurality of the target loci are included in the target amplicons; and

performing high-through sequencing on the amplified products.

2. The method of claim 1 , wherein melting temperature is determined according to Primer3 program.

3. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than a melting temperature of at least two of the primers.

4. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the highest melting temperature of the primers.

5. The method of claim 1 , wherein the annealing temperature is at least 8° C. greater than the highest melting temperature of the primers.

6. The method of claim 1 , wherein the annealing temperature is at least 3° C. greater than the average melting temperature of the primers.

7. The method of claim 1 , wherein the annealing temperature is at least 8° C. greater than the average melting temperature of the primers.

8. The method of claim 1 , wherein the range of melting temperatures of the primers is less than 5° C.

9. The method of claim 1 , wherein the ΔG values for each possible combination of two primers in the library are all equal to or greater than −5 kcal/mol.

10. The method of claim 1 , wherein 10 to 20,000 target loci are amplified with 10 to 20,000 primers.

11. The method of claim 1 , wherein 50 to 2,000 target loci are amplified with 50 to 2,000 primers.

12. The method of claim 1 , wherein 100 to 1,000 target loci are amplified with 100 to 1,000 primers.

13. The method of claim 1 , wherein at least 90% of the amplified products are target amplicons.

14. The method of claim 1 , wherein at least 90% of the target loci are amplified.

15. The method of claim 1 , wherein less than 20% of the amplified products are primer dimers.

16. The method of claim 1 , wherein the concentration of each primer in the library is less than 20 nM.

17. The method of claim 1 , wherein the primers have 2, 1, or 0 guanines or cytosines in the last 5 bases at the 3′ end of the primers.

18. The method of claim 1 , wherein the length of the annealing step of the reaction conditions is 5-60 minutes.

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

20. The method of claim 1 , wherein the nucleic acid sample comprises cell-free DNA, and wherein the cell-free DNA comprises tumor DNA.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Mar 26, 2021
From: ZIMMERMANN, BERNHARD; HILL, MATTHEW; DODD, MICHAEL; WONG, ALEXANDER
To: GENE SECURITY NETWORK, INC.
Reel/Frame 055738/0648 →
PROPRIETARY INFORMATION AND INVENTIONS AGREEMENT Recorded Mar 26, 2021
From: LACROUTE, PHILIPPE
To: GENE SECURITY NETWORK, INC.
Reel/Frame 055743/0696 →
CHANGE OF NAME Recorded Mar 26, 2021
From: GENE SECURITY NETWORK INC.
To: NATERA, INC.
Reel/Frame 055745/0962 →
Continuity (9)
Continuation 16752339 · Jan 24, 2020
Continuation 16412301 · May 14, 2019
Continuation 15336630 · Oct 27, 2016
Continuation 14538982 · Nov 24, 2014
Provisional Application 62066514 · Oct 21, 2014
Provisional Application 61994791 · May 16, 2014
Provisional Application 61987407 · May 1, 2014
Provisional Application 61982245 · Apr 21, 2014
Related Publication 20210222230A1 · Jul 22, 2021
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