Patent ID: 6403339
Filing Date: 2002-06-11
Classification: C12Q

Abstract:
A method for detecting a nucleotide sequence by means of polymerase chain reaction, wherein the nucleotide sequence is a constituent of a double-stranded nucleic acid molecule which is in solution and which is comprised of a strand and a counter-strand, the method which comprises the following steps:adding a first primer to the solution, wherein the first primer is complementary to a first segment of the strand located at a 3â€²-terminus of a central segment, and adding a second primer, therein the second primer is complementary to a 3â€²-terminus of the central segment of the counter-strand; contacting the solution with a third primer whose 5â€²-terminal end is bound to a solid phase, wherein the third primer is complementary to the central segment of the strand comprising the nucleotide sequence; closing a reaction vessel containing the solution; repeatedly heating and cooling the solution with the following steps: heating to 92Â° C.-100Â° C.; cooling to 40Â° C.-60Â° C.; heating to 72Â° C.-75Â° C; anddetecting the nucleic acid molecule during or after the heating and cooling step in a closed reaction vessel by forming or removing an interaction allowing a nonradiative energy transfer or a direct energy transfer between a first and a second fluorophoric molecule if the nucleic sequence is present, wherein the first fluorophoric molecule is bound to the solid phase or to the third primer, and the second fluorophoric molecule is bound to the second primer.