Patent ID: 6727068
Filing Date: 2004-04-27
Classification: C12N,C12Q

Abstract:
A method for constructing a nucleic acid library comprising:a) obtaining a population of end-labeled double-stranded cDNA molecules; b) dividing said population into a first portion and a second portion; c) digesting said first portion with at least one sequence-specific endonuclease wherein said first portion is divided into one pool per endonuclease; d) digesting said second portion with at least one (Y) endonuclease having a degenerate recognition sequence and fractionating the digested cDNA into YXz pools where Y is the number of degenerate endonucleases, X is the the extent of degeneracy and z is the number of bases; e) isolating nucleic acid fragments from said endonuclease digestions of said first and second portions using said end-labels; f) digesting the fragments of said first portion with said at least one endonuclease of d) sequence and fractionating the digested cDNA into YXz pools where Y is the number of degenerate endonucleases, X is the the extent of degeneracy and z is the number of bases; g) digesting the fragments of said second portion with said at least one endonuclease of c) wherein the second portion pools are divided into one pool per endonuclease; h) removing labeled nucleic acid fragments from said first and second portions while retaining unlabeled fragments; i) adding a population of adapters to each of the pools, said population containing adapters specific for the endonucleases used, wherein each adapter comprises a first region specific to a particular endonuclease used and a second region containing a primer binding site; j) hybridizing and ligating said adapters to said unlabeled fragments; k) amplifying the fragments using the adapters; and l) separating the amplified fragments by size.