Patent ID: 6461814
Filing Date: 2002-10-08
Classification: C12N

Abstract:
A method of obtaining a set of tags from a source of interest comprising the steps of:(a) providing a double-stranded cDNA, having a poly A or a poly T tail, (b) cleaving said double-stranded cDNA with a punctuating restriction endonuclease providing a cDNA fragment, (c) ligating to said cDNA fragment a 5â€² adapter comprising a recognition sequence for a type IIs restriction endonuclease which allows DNA cleavage at a site in the cDNA fragment distant from the recognition sequence for a type IIs restriction endonuclease providing a first cDNA construct, (d) cleaving said first cDNA construct with said type IIs restriction endonuclease providing a second cDNA construct, (e) ligating to said second cDNA construct a 3â€² adapter, thereby producing a recognition sequence for a punctuating endonuclease located 3â€² adjacent to said second cDNA construct, providing a third cDNA construct, (f) digesting said third cDNA construct with said punctuating restriction endonuclease providing a set of tags comprising cDNA sequences, and (g) determining the nucleotide sequences of said set of tags.