Patent ID: 7498131
Filing Date: 2009-03-03
Classification: C12Q

Abstract:
1. A method for determining the presence or absence of at least two target sequences in a nucleic acid sample, wherein the method comprises the steps of: (a) providing to a nucleic acid sample at least one circularizable probe for each target sequence to be detected in the sample, wherein (b) allowing the first and the second target specific sections of the circularizable probe to anneal to the first and second parts of target sequences so that the 5′ end of the first and the 3′ end of the second target specific sections of the circularizable probe are annealed adjacently on the target sequence; (c) providing a means for connecting the first and second target specific sections that are adjacently annealed to the target sequence and permitting the first and the second target specific sections to be connected, resulting in a connected circular probe that corresponds to a target sequence in the sample; (d) adding to said annealed circular probe a primer pair comprising (e) amplifying the mixture, thereby producing a population of corresponding amplified connected probes or amplicons each of which is a linear monomeric representation of the connected circularizable probes; (f) determining the presence or absence of a target sequence in the sample by detecting the presence or absence of the corresponding amplified linked probe or amplicon, with the proviso that, if the blocking region comprises a recognition site for a restriction endonuclease, the connected circularizable probe is subjected to restriction enzyme cleavage prior to the amplification step, and the presence of said amplicon is indicative of the presence of, and the absence of said amplicon is indicative of the absence of, said target sequence in the sample.