Patent ID: 6670121
Filing Date: 2003-12-30
Classification: C12N,C12Q

Abstract:
A method for identifying and characterizing mRNA molecules, comprising the steps of:a) isolating and purifying poly-A mRNA from tissue samples; b) synthesizing double-stranded cDNA from the poly-A mRNA, wherein a first cDNA strand is synthesized using reverse transcriptase using an anchored oligo-T nucleotide having a 3â€² extension of two bases, wherein a first base is A, C or G and a second base is A, C, G or T, and having a 5â€² extension of 5-15 bases, wherein the 5â€² extension comprises a recognition site for a second restriction endonuclease with cleavage characteristics {fraction (16/14)} nucleotides downstream of the recognition sequence, and a second cDNA strand is synthesized using the first cDNA strand as a template to form the double-stranded cDNA; c) truncating the double-stranded cDNA with a first restriction endonuclease, thereby forming truncated cDNA fragments; d) hybridizing and ligating a first set of adaptor molecules to ends of the truncated cDNA fragments; e) purifying 3â€² ends of the truncated cDNA fragments comprising 3â€² poly-da nucleotides; f) removing 3â€² poly-dA nucleotides from the ends purified ends of the purified truncated cDNA fragments with the second restriction endonuclease; g) hybridizing and ligating a second set of adaptor molecules to the truncated cDNA with removed 3â€² poly-dA nucleotides; h) amplifying the truncated cDNA fragments with removed 3â€² poly-da nucleotides with a polymerase chain reaction, thereby forming amplification products, wherein 5â€² primers having two or three permutations at the 3â€² end and containing one or two artificial mismatches to a complementary adaptor sequence are used in the polymerase chain reaction; i) fractionating the amplification products according to their length; and, j) analyzing the amplification products.