Patent ID: 6395887
Filing Date: 2002-05-28
Classification: C12N,C12Q

Abstract:
A method for selectively amplifying in a nucleic acid sample DNA fragments having sequences corresponding to 3â€² ends of mRNAs, comprising the steps of:(a) contacting the mRNAs with oligonucleotide primers comprising a 5â€² sequence incapable of hybridizing to a polyA tail of the mRNAs, and a 3â€² sequence that hybridizes to a portion of the polyA tail of the mRNAs and n non-polyA nucleotides immediately upstream of the polyA tail, wherein n is at least one; (b) reverse transcribing the mRNA to produce a first strand cDNA complementary to the mRNA that includes the oligonucleotide primer; (c) synthesizing a second DNA strand complementary to the first strand cDNA to form a duplex; (d) cleaving the duplex with at least one sequence-specific cleaving agent to provide a number of duplex cleavage fragments; (e) ligating an adapter to the cleavage fragments, the adapter consisting of two partially hybridized nucleic acid strands, wherein portions of the two strands are non-complementary to each other and portions of the two strands are complementary to each other; and (f) amplifying the ligated cleaved fragments of step (e) using a set of primers, in which for each set the first primer comprises the 5â€² sequence incapable of hybridizing to a polyA tail of the mRNAs, and the 3â€² sequence that hybridizes to a portion of the polyA tail of the mRNAs and at least n+1 non-polyA nucleotides immediately upstream of the polyA tail, and a second primer whose sequence comprises at least a portion of the sequence of one strand of the adapter in the non-complementary portion, thereby selectively amplifying a DNA fragment comprising sequence complementary to an 3â€² region of an mRNA.