US 12,264,357 B2
Universal Sanger sequencing from next-gen sequencing amplicons
Heather Sanders, San Juan Capistrano, CA (US); Hai-Rong Li, San Juan Capistrano, CA (US); Feras Hantash, San Juan Capistrano, CA (US); and Frederic Waldman, San Juan Capistrano, CA (US)
Assigned to Quest Diagnostics Investments LLC, Secaucus, NJ (US)
Filed by Quest Diagnostics Investments LLC, Secaucus, NJ (US)
Filed on Aug. 9, 2021, as Appl. No. 17/397,280.
Application 17/397,280 is a continuation of application No. 16/171,675, filed on Oct. 26, 2018, granted, now 11,085,079.
Application 16/171,675 is a continuation of application No. 14/655,103, granted, now 10,138,519, previously published as PCT/US2013/078039, filed on Dec. 27, 2013.
Claims priority of provisional application 61/747,062, filed on Dec. 28, 2012.
Prior Publication US 2022/0033901 A1, Feb. 3, 2022
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6869 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6876 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6869 (2013.01); C12Q 1/6876 (2013.01); C12Q 2600/16 (2013.01)] 9 Claims
OG exemplary drawing
 
1. A method of generating an amplicon for next generation sequencing (NSG) for at least one region of interest comprising:
(a) adding to a sample of DNA:
(i) a first primer set specific for the region of interest consisting of two primers each comprising a 5′ region encoding an adapter and a 3′ region specific for the region of interest, wherein the first primer set binds with a Tm of about 60° C. immediately upstream and downstream of the region of interest; and
(ii) a second primer set consisting of two primers each of which comprises:
(A) a 5′ sequence selected from GTAAAACGACGGCCAGT (SEQ ID NO: 1) or AACAGCTATGACCATG (SEQ ID NO: 2);
(B) a spacer comprising at least 10 nucleotides; and
(C) a 3′ adapter that is substantially identical to the adaptor region,
wherein the adapter has a Tm of about 55° C. and the second primer set has a Tm of at least 70° C.; and
(b) performing a plurality of PCR cycles with a first annealing temperature and a second annealing temperature.