Patent ID: 6806049
Filing Date: 2004-10-19
Classification: C12Q

Abstract:
A method for analyzing expression frequencies of genes, which comprises the following steps:(a) forming a vector primer to which each cDNA is ligated, by annealing the vector primer with each mRNA derived from a cell of which expression frequencies of genes is to be analyzed, and synthesizing the cDNA, said vector primer comprising a linear plasmid vector having a single-stranded poly(T) sequence at one 3â€² end of one strand of the linear plasmid vector, said linear plasmid vector comprising a recognition sequence for a first restriction enzyme, a recognition sequence for a type IIS restriction enzyme, and a recognition sequence for a second restriction enzyme in order from the poly(T) sequence wherein (1) the first restriction enzyme and the second restriction enzyme each digest the vector primer at one position, (2) the cleavage site of the type IIS restriction enzyme is positioned beyond the recognition sequence of the second restriction enzyme, and (3) the vector primer digested with the first restriction enzyme and the type IIS restriction enzyme can be cyclized, (b) digesting the vector primer to which the cDNA is ligated, with the second restriction enzyme and a third restriction enzyme that does not digest the vector primer and forms a digested end of the same shape as a digested end obtained with the second restriction enzyme, to excise an upstream region of the cDNA, and cyclizing the vector primer, (c) digesting the cyclized vector primer with the first restriction enzyme and the type IIS restriction enzyme to excise a downstream region of the cDNA so that a tag consisting of a part of the cDNA is left, and cyclizing the vector primer again, (d) performing polymerase chain reaction (PCR) by using the vector primer as a template and primers to amplify the tag, wherein said primers are oligonucleotides having nucleotide sequences corresponding to known nucleotide regions on each side of the tag that are maintained in the vector primer following digestion in step (c), (e) ligating the amplification products to form a concatemer of the tags, wherein the tags are separated by known nucleotide sequences introduced by the primers for tag amplification so that no ditags are present in the concatemer, and (f) determining the nucleotide sequence of the concatemer and investigating types and frequencies of tags occurring in the nucleotide sequence.