[Construction and characterization of cDNA library for lung cancer cell line YTMLC-90 isolated from Gejiu, Yunnan province].

AIM: To construct the cDNA library for Yunnan Gejiu human lung cancer cell line YTMLC-90. METHODS: The total RNA was extracted from YTMLC-90 cells and the first-strand cDNA was synthesized by reverse transcription with a modified oligo(dT) primer (containing Sfi I B digestion site). Simultaneously, the SMART oligonucleotide (contained Sfi I A digestion site) was utilized as a template that the first-strand of cDNA could be extended out the 5' terminal of mRNA. The ds cDNA was amplified by LD-PCR (long-distance PCR) and then digested with Sfi I(IA & IB). After fractionation of cDNA through CHROMA SPIN column, the ds cDNA was cloned into lambdaTripIEx2 vector which was then packaged. RESULTS: The unamplified cDNA library for human lung cancer cells consisted of 1.01 x 10(9) pfu/L independent clones in which the recombinant rate was about 93.2%. The clone number in the amplified cDNA library reached 5.24 x 10(12) pfu/L and the length of inserted exogenous cDNA sequence was 750-3 000 bp. CONCLUSION: The constructed cDNA library for YTMLC-90 cells has an excellent quality, which lays a solid foundation for further screening and cloning novel tissue-specific genes of human lung cancer.