[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.