Method for producing a polynucleotide for use in single primer amplification and phosphorothioate-containing oligonucleotides as primers in nucleic acid amplification.

摘要

A method is disclosed for extending an extender probe to produce a single stranded polydeoxynucleotide that is free of unreacted extender probe and has two segments that are non-contiguous and complementary with each other. The method comprises the steps of (1) providing in combination (a) a polynucleotide having two non-contiguous, non-complementary nucleotide sequences S1 and S2 wherein S2 is 5' of S1 and is at least ten deoxynucleotides long, (b) an extender probe comprised of two deoxynucleotide sequences, wherein the sequence at the 3'-end of the extender probe (EP1) is hybridizable with S1 and the other of the deoxynucleotide sequences (EP2) is substantially identical to S2 and (c) means for modifying the 3'-end of extender probe that does not hybridize with the polynucleotide and (2) extending the extender probe along the polynucleotide wherein extender probe not hybridized to the polynucleotide becomes modified at its 3'-end. A method is also disclosed for forming a polynucleotide sequence complementary to a single stranded target polynucleotide sequence ("target sequence"). In the method a polynucleotide primer having a 3'-terminus comprised of a nucleotide monophosphate, which has at least one phosphate-sulfur bond, is hybridized to the 3'-end of the target sequence. The polynucleotide primer is extended along the target sequence and the extended polynucleotide primer is then dissociated from the target sequence. The method finds particular application in the area of nucleic acid amplification.