MICROASSAY FOR SERIAL ANALYSIS OF GENE EXPRESSION AND APPLICATIONS THEREOF

摘要

Method of obtaining a library of tags able to define a specific state of abiological sample, comprising the following successive steps: (1) extractingin a single-step mRNA from a small amount of a biological sample usingoligo(dT)25 covalently bound to paramagnetic beads, (2) generating a doublestrand cDNA library, from said mRNA, (3) cleaving the obtained cDNAs usingSau3A I, (4) separating the cleaved cDNAs in two aliquots, (5) ligating thecDNA contained in each of said two aliquots via said Sau3A I restriction siteto a linker consisting of one double-strand cDNA molecule having one of thefollowing formulas: GATCGTCCC-X1 or GATCGTCCC-X2, wherein X1 and X2, whichcomprise 30-37 nucleotides and are different, include a 20-25 bp PCR primingsite with a Tm of 55~C-65~C, (6) digesting the products obtained in step (5)with the tagging enzyme BsmF I, (7) blunt-ending said BsmF I tags with a DNApolymerase and mixing the tags ligated with the different linkers, (8)ligating the tags obtained in step (7) to form ditags with a DNA ligase, (9)amplifying the ditags obtained in step (8) with primers comprising 20-25 bpand having a Tm of 55~-65~C, (10) isolating the ditags having between 20 and28 bp from the amplification products obtained in step (9) by digesting saidamplification products with Sau3A I and separating the digested products, (11)ligating the ditags obtained in step (10) to form concatemers, purifiying saidconcatemers and separating the concatemers having more than 300 bp, (12)cloning and sequencing said concatemers and (13) analysing the differentobtained tags.