US 11,884,970 B2
Denaturation-enhanced DNA mutation testing for limited biological specimens
Gerassimos Makrigiorgos, Chestnut Hill, MA (US); and Cloud P. Paweletz, Boston, MA (US)
Assigned to DANA-FARBER CANCER INSTITUTE, INC., Boston, MA (US)
Appl. No. 17/057,936
Filed by Dana-Farber Cancer Institute, Inc., Boston, MA (US)
PCT Filed May 24, 2019, PCT No. PCT/US2019/033876
§ 371(c)(1), (2) Date Nov. 23, 2020,
PCT Pub. No. WO2019/226970, PCT Pub. Date Nov. 28, 2019.
Claims priority of provisional application 62/737,033, filed on Sep. 26, 2018.
Claims priority of provisional application 62/676,082, filed on May 24, 2018.
Prior Publication US 2021/0214778 A1, Jul. 15, 2021
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6848 (2018.01); C12Q 1/6806 (2018.01)
CPC C12Q 1/6848 (2013.01) [C12Q 1/6806 (2013.01); C12Q 2600/156 (2013.01)] 21 Claims
OG exemplary drawing
 
1. A method for increasing the number of compartments in which a target sequence is identified by an assay, comprising:
(a) obtaining a sample comprising double-stranded DNA comprising a target sequence, wherein the sample comprises double-stranded DNA comprising complementary strands of unequal length;
(b) producing blunt ends on the double-stranded DNA;
(c) denaturing the double-stranded DNA in a sample to form single-stranded DNA;
(d) partitioning the sample of formed single-stranded DNA into a multitude of compartments, wherein a majority of the compartments contain either one or no single-stranded DNAs comprising the target sequence; and
(e) assaying to identify compartments containing target sequences;
wherein the producing blunt ends of step (b) and denaturing of step (c) increases the number of compartments in which the target sequence is identified by the assaying of step (e) compared to a method that does not comprise the producing blunt ends of step (b) and denaturing of step (c).