Construction And Identification Of Yunnan Gejiu Lung Cancer Cell GLC-82 CDNA Library

Objective To construct a full-length cDNA library from humanglandular lung cancer cell GLC. Methods The total RNA was separated from cultured human glandular lung cancer cell GLC and the frist-strand cDNA was synthesized with the help of Moloney murine leukemia virus (MMLV) reverse transcriptase (RT). A modified oligo(dT) primer(containing Sfi Ⅰ B site) primers the first-strand synthesis reaction ,and the SMART IV Oligo(containing Sfi Ⅰ A site) serves as a short ,extended template at the 5'end of the mRNA. When the RT reaches the 5 'end, the enzyme's terminal transferase activity adds a few additional nucleotides,primarily deoxycytidine, to the 3 'end of the cDNA. The SMART IV Oligo, which has an oligo(G) sequence at its 3'end ,base-pairs with the deoxycytidine stretch, creating an extended template. The resulting full-length single-strand cDNA contains the complete 5'end of the mRNA. The double-strand cDNA was amplified through LD-PCR (long-distance PCR) and then digested by Sfi Ⅰ ( Ⅰ A & Ⅰ B) restriction enzyme. After cDNA size fractionation through CHROMA SPIN-400 column, the double-strand cDNA was ligated into the Sfi Ⅰdigedted λ TripIE×2 vector and then was packaged in vitro. Results Theunamplified human lung cancer cell cDNA library consists of 1.356×10~6 independent clones and the percentage of recombinant clones is about 95%. The titer of the amplified cDNA library is 6.5×10~9pfu/ml and the exogenous inserts of therecombinants is 750-2500bp. Conclusions The human lung cancer cell cDNA library has an excellent quality and lays solid foundation for screening and cloning new tissue-specific genes of human lung cancer.