Patent ID: 6372436
Filing Date: 2002-04-16
Classification: C12N

Abstract:
A method for producing a cDNA library enriched for rare cDNAs and reduced in abundant cDNAs which comprises:(a) obtaining a pool of linear double-stranded cDNAs; (b) cloning a first portion of the pool of cDNAs into a first vector to create a first cDNA library; (c) cloning a second portion of the pool of cDNAs into a second vector to create a second cDNA library; (d) producing single-stranded linear cDNA inserts (target cDNA) from the first cDNA library; (e) producing single-stranded circles (target cDNA) from the second cDNA library; (f) producing a pool of abundant linear cDNAs (driver cDNA) from the first and the second DNA libraries by the following steps: (i) amplifying the cDNA inserts from the first and the second libraries by polymerase chain reaction using two pairs of appropriate primers which specifically hybridize with the first and second vectors, respectively; (ii) removing DNA sequences common to all of the amplified products from step (i); (iii) denaturing the amplified products from step (ii); (iv) partially reassociating the denatured products from step (iii) in a hybridization mixture under appropriate hybridization conditions so as to produce duplexes of abundant cDNAs, and (v) removing unreassociated cDNAs from step (iv), thereby producing the pools of abundant linear cDNAs from the first and the second cDNA libraries; (g) hybridizing the linear cDNA inserts from step (d) or the single-stranded circles from step (e) with an excess amount of the abundant cDNA pool produced from the second cDNA library or the first cDNA library, respectively, from step (v) under hybridization conditions to produce duplexes, and (h) isolating single-stranded linear cDNA inserts or single-stranded circles which remain after the hybridization of step (g), thereby producing cDNA or a cDNA library enriched for rare cDNAs and reduced in abundant cDNAs.