US 12,305,229 B2
Methods for simultaneous amplification of target loci
Bernhard Zimmermann, Manteca, CA (US); Matthew Hill, Belmont, CA (US); Philippe Lacroute, Sunnyvale, CA (US); Michael Dodd, San Francisco, CA (US); and Alexander Wong, Mountain View, CA (US)
Assigned to Natera, Inc., San Carlos, CA (US)
Filed by Natera, Inc., San Carlos, CA (US)
Filed on Mar. 26, 2021, as Appl. No. 17/214,403.
Application 17/214,403 is a continuation of application No. 16/752,339, filed on Jan. 24, 2020, granted, now 11,390,916.
Application 16/752,339 is a continuation of application No. 16/412,301, filed on May 14, 2019, granted, now 10,597,709, issued on Mar. 23, 2020.
Application 16/412,301 is a continuation of application No. 15/336,630, filed on Oct. 27, 2016, granted, now 10,351,906, issued on Jul. 16, 2019.
Application 15/336,630 is a continuation of application No. 14/538,982, filed on Nov. 24, 2014, granted, now 9,677,118, issued on Jun. 13, 2017.
Claims priority of provisional application 62/066,514, filed on Oct. 21, 2014.
Claims priority of provisional application 61/994,791, filed on May 16, 2014.
Claims priority of provisional application 61/987,407, filed on May 1, 2014.
Claims priority of provisional application 61/982,245, filed on Apr. 21, 2014.
Prior Publication US 2021/0222230 A1, Jul. 22, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6811 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/686 (2018.01); C12Q 1/6874 (2018.01); C12Q 1/6883 (2018.01)
CPC C12Q 1/686 (2013.01) [C12Q 1/68 (2013.01); C12Q 1/6811 (2013.01); C12Q 1/6848 (2013.01); C12Q 1/6874 (2013.01); C12Q 1/6883 (2013.01); C12Q 2600/156 (2013.01)] 20 Claims
 
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.