Patent ID: 6110677
Filing Date: 2000-08-29
Classification: C12Q

Abstract:
A method for modifying an oligonucleotide, said method comprising:(a) combining said oligonucleotide with a polynucleotide and a 5'-nuclease, said oligonucleotide having a 3' portion capable of reversibly hybridizing to said polynucleotide and a 5'-portion which does not hybridize to the polynucleotide,(b) incubating said oligonucleotide, said polynucleotide and said nuclease under isothermal conditions, whereby a duplex formed by hybridization of the 3' portion of the oligonucleotide to the polynucleotide is in equilibrium with unhybridized oligonucleotide and unhybridized polynucleotide, said isothermal conditions being at or near the melting temperature of said complex, and(c) while maintaining said isothermal conditions, cleaving said oligonucleotide with said nuclease when said 3'-portion is hybridized to said polynucleotide to provide:(i) a first fragment including said 5'-portion and no more than one nucleotide from the 5'-end of said 3'-portion, and(ii) a second fragment that is 3' of said first fragment with reference to the intact oligonucleotide, thereby modifying said oligonucleotide, wherein said first fragment and said second fragment are continuously produced under said isothermal conditions.