Strand displacement methods employing competitor oligonucleotides for isolating one strand of a double-stranded nucleic acid
AssigneeApplera Corporation
InventorsClaudia Chiesa, Gary P. Schroth, Michael Egholm
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Abstract
The present invention provides methods and kits for isolating one strand of a double-stranded target nucleic acid. The method capitalizes on the differences in the kinetics and thermodynamic stabilities between conventional DNA/DNA, DNA/RNA and RNA/RNA duplexes and heteroduplexes in which one strand of the heteroduplexe is a nucleobase polymer having a net positively charged or net neutral backbone, such as a PNA.
Claims25
A method of isolating one strand of a double-stranded target nucleic acid, comprising:
- (i) contacting a double-stranded target nucleic acid comprising a first strand and a second strand with a competitor oligo capable of hybridizing to the first strand under conditions in which the first strand dissociates from the second strand and hybridizes with the competitor oligo to form a first-strand:competitor oligo heteroduplex;
- and (ii) isolating the dissociated second strand.
A method of isolating one strand of a double-stranded target nucleic acid, comprising:
- (i) contacting a double-stranded target nucleic acid comprising a first strand and a second strand with a competitor oligo capable of hybridizing to the first strand under conditions in which the first strand dissociates from the second strand and hybridizes with the competitor oligo to form a first-strand:competitor oligo heteroduplex;
- (ii) isolating the heteroduplex, and (iii) dissociating the heteroduplex and isolating the first strand.
A method of isolating one strand of a double-stranded target nucleic acid, comprising:
- (i) dissociating the double-stranded target nucleic acid into a first strand and a second strand;
- (ii) contacting the dissociated target nucleic acid with a competitor oligo capable of hybridizing to the first strand under conditions which favor first-strand:competitor oligo heteroduplex formation and disfavor reannealing of the first and second strands;
- and (iii) isolating the dissociated second strand.
A method of isolating one strand of a double-stranded target nucleic acid, comprising:
- (i) dissociating the double-stranded target nucleic acid into a first strand and a second strand;
- (ii) contacting the dissociated target nucleic acid with a competitor oligo capable of hybridizing to the first strand under conditions which favor first-strand:competitor oligo heteroduplex formation and disfavor reannealing of the first and second strands;
- (iii) isolating the heteroduplex, and (iv) dissociating the heteroduplex and isolating the first strand.
The method of 13, in which the capture moiety is a solid support.
A method of isolating one strand of a double-stranded target nucleic acid, comprising the steps of:
- (i) dissociating the double-stranded target nucleic acid into a first strand and a second strand;
- (ii) contacting the dissociated target nucleic acid with a competitor oligo capable of hybridizing to only the first strand under conditions which kinetically favor competitor oligo first-strand hybrid formation and kinetically disfavor reannealing of the first and second strands, said competitor oligo being conjugated with a moiety that facilitates capture of competitor oligo:first-strand hybrids;
- (iii) capturing the competitor oligo:first strand hybrid, and (iv) dissociating the heteroduplex and isolating the first strand.
Dependencies are inferred from the claim text.