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Abstract

The present invention relates to a method for selectively extending an oligonucleotide primer along a specific target polynucleotide sequence in a mixture of polynucleotides. Selective extension is achieved by controlling the concentration of the oligonucleotide primer by forming the oligonucleotide in situ by degrading the 3'-end of a modified oligonucleotide. The method comprises providing in combination the mixture of polynucleotides and the modified oligonucleotide having a 3'-end that is not extendable along any polynucleotide and extending the oligonucleotide primer selectively along the specific target polynucleotide sequence by controlling the degradation of the 3'-end of the modified oligonucleotide. In this way extension of the oligonucleotide primer along polynucleotides other than the specific target polynucleotide sequence is substantially reduced or avoided. In another aspect the invention is an improvement in a method for amplifying a target polynucleotide sequence. The method comprises combining the target polynucleotide sequence with reagents for amplifying the target polynucleotide sequence and subjecting the combination to conditions wherein the target polynucleotide sequence is amplified. The reagents comprise an oligonucleotide primer and a polymerase. The improvement comprises deriving the oligonucleotide primer from a modified oligonucleotide having a portion that hybridizes to the target polynucleotide sequence except for the 3'-end thereof, which has at least one nucleotide analog that is incapable of hybridizing to a polynucleotide. Kits for carrying out the above methods are also disclosed.

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First Claim