Patent ID: 6225062
Filing Date: 2001-05-01
Classification: C12Q

Abstract:
A method of determining the sequence of nucleotides in a target ribonucleic acid under isothermal conditions comprising the steps of:(a) combining in a single reaction vesselan RNA sample containing a target ribonucleic acid,first and second polynucleotide primers, wherein the first primer specifically hybridizes with a target sequence near the 3' end of the target ribonucleic acid, and the second primer specifically hybridizes to the 3' end of an antisense copy of the target ribonucleic acid, and wherein at least the first or second primer has a detectable label, and wherein at least one of the first or second primer has an RNA polymerase transcription initiation signal at its 5' end which signal does not specifically hybridize to the target ribonucleic acid,ribonucleotide triphosphates for RNA synthesis,deoxyribonucleotide triphosphates for DNA synthesis,at least one type of chain terminating nucleotide triphosphate,and enzymes with the activity of reverse transcriptase, RNase H, RNA Polymerase and a low discrimination DNA Polymerase having reduced discrimination between dNTP and ddNTP substrates;(b) incubating the combined reactants under isothermal conditions for a length of time sufficient to generate chain-termninated reaction products, and(c) detecting chain termninated reaction products after electrophoretic separation, said chain terminated reaction products reflecting the sequence of nucleotides in the target ribonucleic acid.