Patent ID: 6544741
Filing Date: 2003-04-08
Classification: C12N

Abstract:
A method for constructing a normalized cDNA library comprising the steps of:(a) identifying and synthesizing oligonucleotides specifically complementary to the 3â€² region of target mRNA; (b) annealing the oligonucleotides of step (a) to a composition comprising non-target RNA and target RNA under conditions sufficient to produce RNA-DNA heteroduplexes specifically between the oligonucleotides and the target RNA; (c) cleaving the heteroduplexes with an RNAse H; (d) adding oligo(dT) or oligo(dU) primers or a variant thereof that have sequences for a first restriction endonuclease recognition site; (e) synthesizing by reverse transcription first-strand cDNA; (f) synthesizing second-strand cDNA, producing double-stranded cDNA molecules; (g) ligating to both ends of the resulting double-stranded cDNA molecules adapters having cohesive ends of a second restriction endonuclease site which is different from the first restriction endonuclease site; (h) removing double-stranded cDNA fragments less than about 150 bp long; (i) digesting the remaining cDNA molecules with the first restriction endonuclease; (j) digesting the resulting cDNA with the second restriction endonuclease; (k) ligating the doubly digested isolated cDNA into a vector predigested with the first and second restriction endonucleases so as to permit cloning of cDNA complementary to the non-target mRNA.