| abstract |
The present invention provides a method for determination of the identity of at least one nucleotide in a RNA-molecule comprising the steps of: (i) providing the RNA-molecule, an oligonucleotide primer binding to a predetermined position of the RNA molecule, a reverse transcriptase, deoxynucleotides and other necessary reagents, in a reaction vessel; (ii) performing a primer extension reaction, whereby the oligonucleotide primer is extended on the RNA-molecule through incorporation of at least one deoxynucleotide by the action of a reverse transcriptase, resulting in the release of a PPi molecule only upon incorporation of a deoxynucleotide; and (iii) detecting the presence or absence of incorporation, thereby indicating the nucleotide identity of the RNA molecule in the relevant position. In a preferred embodiment, the sequencing of the invention is coupled to the Pyrosequencing™ reaction. A variant of the method employs incorporation of modified nucleotides, with an optionally cleavable linker arm to which is attached a label. |