Patent ID: 8440401

Claim:
A method of generating a single-strand region on a polynucleotide molecule comprising: (a) providing in a reaction mixture, a polynucleotide molecule having a single modified nucleotide on one strand of a duplex at a specific location; and (b) enzymatically cleaving the polynucleotide molecule at the modified nucleotide, by adding two or more enzymes to a reaction mixture wherein at least one of the enzymes is a DNA glycosylase and at least one of the enzymes is a cleaving enzyme; and wherein the DNA glycosylase and the cleaving enzyme having an activity ratio of at least 2:1 in the reaction mixture, the activity ratio being the ratio of a first rate of forming an abasic site by excision of the modified nucleotide from the polynucleotide by DNA glycosylase to a second rate of cleaving at the abasic site in the polynucleotide by the cleaving enzyme, the first and second rates determined for a single predetermined molar amount of the modified nucleotide in the polynucleotide.