Construction of a Skin cDNA Library of Cashmere Goat and Cloning of KAP6-2 Full-length cDNA

In order to understand the molecular mechanism of the characteristic of \ncashmere,we constructed a cDNA library using the SMART cDNA library \nconstruction kit (Clontech).Total RNA was isolated from the goat (Capra \nhircus ) skin tissue with hair follicle angen.Oligotex (QIAGEN) \nwas used to isolate mRNA from total RNA.The "anchor first-strand cDNA" \nsynthesized by reverse transcription with the SMART technique.The \nLD-PCR was performed using a modified oligo (dT) primer and an anchor \nprimer as the primer set,and anchor first-strand cDNA as the template \nto enrich the cDNA population for full-length sequences.After digestion \nwith Sfi I and size fractionation,SMART cDNA was ligated into the Sfi \nI-digested pBluescript II SK (with Sfi I A and B site).The ligation \nmixture was transformed into E.Coli 5α.The cDNA library contained \n1.8x105 independent clones.Randomly select cDNA clones and sequence \nwith the 5 prime end,a full-length KAP6-2 cDNA was found by compared \nwith those in the NCBI database (nr) using the Blast-N programs,it \nshowed 75.5% identity in amino acids with mouse KAP6-2 and the \naccession number in GenBank is AY316158.