Methods for amplification of cell-free DNA using ligated adaptors and universal and inner target-specific primers for multiplexed nested PCR
Matthew Rabinowitz, San Francisco, CA (US); Matthew Hill, Belmont, CA (US); Bernhard Zimmermann, Manteca, CA (US); Johan Baner, San Francisco, CA (US); George Gemelos, Portland, OR (US); Milena Banjevic, Los Altos Hills, CA (US); Allison Ryan, Belmont, CA (US); Styrmir Sigurjonsson, San Jose, CA (US); and Zachary Demko, San Francisco, CA (US)
Assigned to Natera, Inc., San Carlos, CA (US)
Filed by Natera, Inc., San Carlos, CA (US)
Filed on Feb. 20, 2020, as Appl. No. 16/796,748.
Application 16/796,748 is a continuation of application No. 16/399,957, filed on Apr. 30, 2019, granted, now 10,590,482.
Application 16/399,957 is a continuation of application No. 16/012,667, filed on Jun. 19, 2018, granted, now 11,408,031.
Application 16/012,667 is a continuation of application No. 13/335,043, filed on Dec. 22, 2011, granted, now 10,113,196, issued on Oct. 30, 2018.
Application 13/335,043 is a continuation in part of application No. 13/300,235, filed on Nov. 18, 2011, granted, now 10,017,812, issued on Jul. 10, 2018.
Application 13/300,235 is a continuation in part of application No. 13/110,685, filed on May 18, 2011, granted, now 8,825,412, issued on Sep. 2, 2014.
Claims priority of provisional application 61/542,508, filed on Oct. 3, 2011.
Claims priority of provisional application 61/571,248, filed on Jun. 23, 2011.
Claims priority of provisional application 61/516,996, filed on Apr. 12, 2011.
Claims priority of provisional application 61/448,547, filed on Mar. 2, 2011.
Claims priority of provisional application 61/462,972, filed on Feb. 9, 2011.
Claims priority of provisional application 61/426,208, filed on Dec. 22, 2010.
Claims priority of provisional application 61/398,159, filed on Jun. 21, 2010.
Claims priority of provisional application 61/395,850, filed on May 18, 2010.
Prior Publication US 2020/0190570 A1, Jun. 18, 2020
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 sequence;
performing a first PCR to simultaneously amplify at least 10 target loci using a first 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 a second 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.