| 摘要 |
<p>Method of obtaining a library of tags able to define a specific state of a biological sample, comprising the following successive steps: (1) extracting in a single-step mRNA from a small amount of a biological sample using oligo(dT)25 covalently bound to paramagnetic beads, (2) generating a double strand cDNA library, from said mRNA, (3) cleaving the obtained cDNAs using Sau3A I, (4) separating the cleaved cDNAs in two aliquots, (5) ligating the cDNA contained in each of said two aliquots via said Sau3A I restriction site to a linker consisting of one double-strand cDNA molecule having one of the following formulas: GATCGTCCC-X1 or GATCGTCCC-X2, wherein X1 and X2, which comprise 30-37 nucleotides and are different, include a 20-25 bp PCR priming site 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 DNA polymerase 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 bp and having a Tm of 55°-65°C, (10) isolating the ditags having between 20 and 28 bp from the amplification products obtained in step (9) by digesting said amplification products with Sau3A I and separating the digested products, (11) ligating the ditags obtained in step (10) to form concatemers, purifiying said concatemers and separating the concatemers having more than 300 bp, (12) cloning and sequencing said concatemers and (13) analysing the different obtained tags.</p> |
