IMPROVED URACIL-EXCISION BASED MOLECULAR CLONING

摘要

The invention provides an improvement to a uracil-specific excision reagent (USER ) based cloning technique comprising generating a single-stranded extension on a polynucleotide molecule, the single-stranded extension having a desired length and sequence composition, comprising: (a) introducing a cassette into a polynucleotide molecule at a predetermined location; (b) cleaving the polynucleotide molecule with a nicking endonuclease specific for a nicking site in the cassette and with a restriction endonuclease specific for a restriction site in the cassette; and (c) dissociating the cleaved polynucleotide molecule between the nicking site and the restriction site to generate the single-strand extension with the desired length and sequence composition; wherein said improvement to said USER based cloning technique comprises using a proof-reading polymerase to generate said polynucleotide molecule. Also provided is a deoxyuridine-excision based method of fusing duplex nucleic acid sequence to form a fused duplex product referred to herein as 'USER fusion'. The invention further provides related materials and methods.