| 摘要 |
<p>A method for detecting nucleotide variations of a target nucleotide sequence from a sample is provided to detect at least two of the nucleotide variations simultaneously and accurately without error through a simple amplification and a hybridization without a restriction enzyme treatment or a sequence analyzing process. A method for detecting at least two nucleotide variations of a target nucleotide sequence from a sample including the target nucleotide sequence comprises the steps of: (a) hybridizing the target nucleotide sequence with a primer selected from the group consisting of: (i) a first nucleotide variation specific primer 1(NVS-P1) which is specifically hybridized into a variation-occurring region of a target nucleotide sequence having a nucleotide corresponding to a first nucleotide variation, includes a nucleotide complementary to the nucleotide corresponding to the first nucleotide variation and has a detectable mark at a 5'-terminal; (ii) a target specific primer 1(TSP1) which is hybridized into a specific region of a target nucleotide sequence which is located at upstream of the variation-occurring region where the NVS-P1 primer is hybridized; (iii) a second nucleotide variation specific primer 2(NVS-P2) which is specifically hybridized into a variation-occurring region of a target nucleotide sequence having a nucleotide corresponding to a second nucleotide variation at the same site where the first nucleotide variation occurs or an adjacent site thereof, includes a nucleotide complementary to the nucleotide corresponding to the second nucleotide variation and has a detectable mark at a 5'-terminal; and (iv) a target specific primer 2(TSP2) which is located at downstream of the variation-occurring region where the NVS-P2 primer is hybridized; (b) amplifying the target nucleotide sequence through at least two cycles of a primer annealing, a primer elongation and a denaturation; (c) hybridizing an amplification product obtained from the step(b) into a first probe and a second probe, wherein the first probe is specifically hybridized into a product amplified by the primer set(i) and (ii) and the second probe is specifically hybridized into a product amplified by the primer set(iii) and (iv); and (d) detecting a hybridization signal obtained from the step(c), wherein the detectable mark is a chemical mark, an enzyme mark, a radioactivity mark, a fluorescence mark, a luminescent mark, a chemiluminescent mark, an FRET(fluorescence resonance energy transfer) mark or a metal mark.</p> |
