US 11,807,897 B2
Methods of preparing nucleic acids for sequencing
Anthony John Iafrate, Newton, MA (US); Long Phi Le, Boston, MA (US); Zongli Zheng, Boston, MA (US); Jason Myers, Golden, CO (US); and Joshua Stahl, Boulder, CO (US)
Assigned to The General Hospital Corporation, Boston, MA (US); and ARCHERDX, LLC, San Francisco, CA (US)
Filed by The General Hospital Corporation, Boston, MA (US); and ARCHERDX, LLC, San Francisco, CA (US)
Filed on Oct. 2, 2019, as Appl. No. 16/590,878.
Application 16/590,878 is a continuation of application No. 14/605,363, filed on Jan. 26, 2015, granted, now 10,450,597.
Claims priority of provisional application 61/931,959, filed on Jan. 27, 2014.
Prior Publication US 2020/0270671 A1, Aug. 27, 2020
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12Q 1/6809 (2018.01)
CPC C12Q 1/6806 (2013.01) [C12Q 1/6809 (2013.01)] 4 Claims
 
1. A method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising:
(a) contacting a target nucleic acid molecule comprising the known target nucleotide sequence with an initial target-specific primer under hybridization conditions;
(b) performing a template-dependent extension reaction that is primed by a hybridized initial target-specific primer and that uses the target nucleic acid molecule as a template;
(c) contacting an extension product of step (b) with a plurality of tailed random primers under hybridization conditions;
(d) performing a template-dependent extension reaction that is primed by a hybridized tailed random primer and that uses the extension product of step (b) as a template;
(e) amplifying a portion of an extension product of step (d) with a first tail primer and a first target-specific primer; and
(f) sequencing an amplification product from step (e) using a first or second sequencing primer;
wherein each of the plurality of tailed random primers comprises (i) a common sequence and (ii) a 3′-end comprising about 3 to about 15 random nucleotides;
wherein the initial target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid;
wherein the first target-specific primer comprises a 3′ portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence and is nested with respect to the initial target-specific primer; and
wherein the first tail primer comprises a nucleic acid sequence identical to a portion of the common sequence.