BioSample

The poly (A)+ RNA was dephosphorylated with bacterial alkaline phosphatase (BAP) and then decapped with tabacco acid pyrophosphatase (TAP). The decapped intact mRNA was ligated with DNA-RNA linker including EcoRI site by treatment of T4 RNA ligase and the first strand cDNA was synthesized from oligo dT-selected mRNA by priming with dT-tailed vector. The dT-tailed vector was adjusted to have about 60nt. The cDNA vector was circularized with E. coli DNA ligase after digestion of EcoRI which site is also included in vector. An RNA strand converted to a DNA strand by Okayama-Berg method. The obtained cDNA vectors were used for transformation of competent cells E. coli Top10F' by electroporation method. The cDNA libraries constructed by this method are full-length enriched cDNA library. After analyzing and sequencing about 2,000 - 3,000 colonies in original cDNA library, the abundant cDNAs were selected and amplified by PCR reaction using vector region primer including T7 promotor as 5' primer and N(dT)14 as 3' primer. The PCR products were used as template for synthesis of biotinylated single stranded RNA by in vitro transcription reaction. The synthesized RNA probes were hybridized with antisense single stranded cDNAs prepared from original liberary and incubated with avidin-gel. After removing DNA-RNA hybrids by centrifuge, the subtracted cDNA libraries were constructed by transformaion of the remaining DNA into competent cells E. coli Top10F' with electroporation method.