Precircle probe nucleic acid amplification methods
The invention is directed to novel methods of multiplexing nucleic acid reactions, including amplification, detection and genotyping. The invention relies on the use of precircle probes that are circularized in the presence of the corresponding target nucleic acids, cleaved, and then amplified.
1. A method for detecting a target sequence comprising a first and second target domain in a sample, said method comprising:
a) hybridizing said target sequence to a precircle probe to form a first hybridization complex, said precircle probe comprising:
i) a first targeting domain;
ii) a second targeting domain;
iii) at least a first universal priming site; and
iv) a cleavage site;
wherein said first and second targeting domains hybridize to said first and second target domains;
b) contacting said first hybridization complex with a ligase to form a closed circular probe;
c) cleaving said closed circular probe at said cleavage site to form a cleaved probe;
d) amplifying said cleaved probe to form a plurality of amplicons; and
e) detecting said amplicons to detect the presence of said target sequence in said sample, wherein said sample is a purified sample.