Nucleic acid enrichment and detection
Disclosed is a hybridisation capture method based on the pyrophosphorolysis reaction. According to the present invention, there is provided a method for increasing the ratio of a first nucleic acid sequence to second nucleic acid sequence in a sample.
1 . A method comprising:
a) hybridizing a probe A with a nucleic acid sample to produce:
(i) first duplexes that comprise probe A and a first nucleic acid sequence in the sample, and
(ii) second duplexes that comprise probe A and a second nucleic acid sequence in the sample;
wherein said first duplexes have a greater degree of complementarity than said second duplexes;
b) enzymatically treating the sample to partially digest probe A in the first duplexes and optionally the second duplexes, wherein probe A is digested to a greater extent in the first duplexes than in the second duplexes by virtue of the greater degree of complementarity of the first duplexes than the second duplexes, and probe A in the first duplexes is becomes shorter than probe A in the second duplexes; and
c) enriching for the first or second nucleic acid sequence relative to the other nucleic acid sequence by the differential digestion in step (b), wherein:
the first nucleic acids become released from probe A during digestion; or
the method further comprises, after step (b), exposing the product of step (b) to conditions that denature the first nucleic acid sequences but not the target nucleic acids from a digested probe; and
the enrichment occurs through the physical separation of the nucleic acid sequences released from probe A from those that remain hybridized to probe A.
2 . The method of claim 1 , wherein the physical separation of the nucleic acid sequences is achieved through capture of probe A onto a solid support.
3 . The method of claim 2 , wherein the solid support is a bead.
4 . The method of claim 3 , wherein said bead is a magnetic or paramagnetic bead.
5 . The method of claim 2 , wherein said probe A comprises a capture moiety through which said probe is captured onto the solid support through chemical binding.
6 . The method of claim 5 , wherein said chemical binding is achieved through avidin-biotin interaction.
7 . The method of claim 1 , wherein the enzymatic digestion of probe A is achieved through pyrophosphorolysis.
8 . The method of claim 7 , wherein said sample and said probe are provided in a reaction mixture comprising a pyrophosphorolyzing enzyme and a source of pyrophosphate ion.
9 . The method of claim 1 , wherein the enzymatic digestion of probe A is achieved through exonucleolysis.
10 . The method of claim 9 , wherein said sample and said probe are provided in a reaction mixture comprising an exonuclease.
11 . The method of claim 1 , wherein probe A comprises a 5′ or 3′ tail region that is noncomplementary to either of said first nucleic acid sequence or said second nucleic acid sequence.
12 . The method of claim 11 , wherein probe A is captured onto a solid support, and wherein said solid support comprises a capture oligonucleotide which hybridizes to said tail region of probe A.
13 . The method of claim 12 , wherein said capture oligonucleotide comprises a capture moiety through which said capture oligonucleotide is bound to said solid support.