US 11,725,230 B2
Selective degradation of wild-type DNA and enrichment of mutant alleles using nuclease
Gerassimos Makrigiorgos, Chestnut Hill, MA (US); and Chen Song, Boston, MA (US)
Assigned to Dana-Farber Cancer Institute, Inc., Boston, MA (US)
Appl. No. 15/739,301
Filed by Dana-Farber Cancer Institute, Inc., Boston, MA (US)
PCT Filed Jun. 24, 2016, PCT No. PCT/US2016/039167
§ 371(c)(1), (2) Date Dec. 22, 2017,
PCT Pub. No. WO2016/210224, PCT Pub. Date Dec. 29, 2016.
Claims priority of provisional application 62/183,854, filed on Jun. 24, 2015.
Prior Publication US 2018/0187242 A1, Jul. 5, 2018
Int. Cl. C12Q 1/68 (2018.01); C12Q 1/6806 (2018.01); C12P 19/34 (2006.01); C12N 15/10 (2006.01); C12N 9/22 (2006.01); C12Q 1/6883 (2018.01); C12N 9/00 (2006.01); C07H 21/04 (2006.01)
CPC C12Q 1/6806 (2013.01) [C12N 9/22 (2013.01); C12N 15/10 (2013.01); C12N 15/1093 (2013.01); C12P 19/34 (2013.01); C12Q 1/68 (2013.01); C12Q 1/6883 (2013.01)] 20 Claims
 
1. A method for preparing a target mutant nucleic acid for subsequent enrichment relative to a wild type nucleic acid comprising
subjecting a nucleic acid sample comprising a double-stranded wild type nucleic acid and a double-stranded target nucleic acid suspected of containing a mutation to a condition that destabilizes the double stranded wild type and target mutant nucleic acids;
contacting the destabilized double stranded wild type and target mutant nucleic acids with a pair of oligonucleotide probes, one of which is complementary to the wild type nucleic acid top strand and the other is complementary to the wild type nucleic acid bottom strand, to permit hybridization of the probes to their corresponding sequences on the wild type and target mutant nucleic acids thereby forming complementary wild-type-probe duplexes on top and bottom strands, and partially complementary target mutant-probe duplexes, wherein at least one of the probes overlaps the suspected mutation; and
exposing the complementary wild-type-probe duplexes and the partially complementary target mutant-probe duplexes to a double strand-specific nuclease (DSN),
wherein the DSN preferentially cleaves the complementary wild type-probe duplexes relative to the partially complementary target mutant-probe duplexes.