Method and kit for direct isothermal sequencing of nucleic acids

Abstract

Direct determination of the sequence of an RNA sample is performed under isothermal conditions. An RNA sample containing a target nucleic acid is combined in a single reaction vessel with a reaction mixture containing two polynucleotide primers, a first primer that specifically hybridizes with a target sequence near the 3′ end of the target nucleic acid, and a second primer that specifically hybridizes to the 3′ end of an antisense copy of the target nucleic acid. At least one of the primers is labeled with a detectable label, and 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 RNA target. The reaction mixture also contains ribonucleotide triphosphates for RNA synthesis, deoxyribonucleotide triphosphates for DNA synthesis, at least one type of dideoxynucleotide triphosphate chain-terminator, and enzymes with the activity of reverse transcriptase, RNAse H, RNA Polymerase and a low discrimination DNA Polymerase such as Thermo Sequenase™. The combined reactants are incubated under isothermal conditions for a length of time sufficient to generate chain-terminated reaction products, and the chain-termninated reaction products are then detected after electrophoretic separation.