Construction And Identification Of YunNan GeJiu Lung Cancer Cell YTMLC CDNA Library

Objective In order to isolate and screen tissue-specificgenes of human lung cancer , a directional cDNA library from human lung cancer YTMLC90 was contracted by SMART (switching mechanism at 5' end of RNAtranscript) technique. Methods The total RNA was separated from cultured human lung cancer cell and the frist-strand cDNA was synthesized through reverse transcription by a modified oligo (dT) primer (contained SfiI B site) while the SMART oligonucleotide (contained Sfi I A site) was utilized as a template so that the the frist-strand cDNA could be extended over the 5' end of mRNA. The double-strand cDNA was amplified by LD-PCR (long-distance PCR) with the above two primers and then digested by Sfi I( I A & I B) restriction enzyme. After cDNA size fractionation through CHROMA SPIN column, the double-strand cDNA was ligated into the Sfi I digedted λ TripIE×2 vector and then the recombinant DNA was packaged invitro. Results The unamplified human lung cancer cell cDNA library consists of 1.01×106 pfu/ml independent clones in which the percentage of recombinant clones is about 93. 2%. The titer of the amplified cDNAlibrary is5.24×109pfu/ml and the exogenous inserts of the recombinants is 750-3000bp. Consults The human lung cancer cell cDNA library has an excellent qualityand lays solid foundation for screening and cloning new tissue-specific genes of human lung cancer.