[Rapid construction of directional cDNA library from human nasopharynx].

OBJECTIVE

To construct a directional cDNA library from human adult nasopharynx by SMART (switching mechanism at 5' end of RNA transcript) technique.

METHODS

The total RNA was separated from human adult nasopharynx epithelial tissue and the first-strand cDNA was synthesized through reverse transcription by a modified oligo(dT) primer(contained sfi IB site) while the SMART oligonucleotide(contained sfi IA site) was utilized as a template so that the first-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 (IA & IB) restriction enzyme. After cDNA size fractionation through CHROMA SPIN column, the double-strand cDNA was ligated into the sfi I-digested lambda TripIEx2 vector and then the recombinant DNA was packaged in vitro.

RESULTS

The unamplified human adult nasopharynx cDNA library consists of 1.5 x 10(6) independent clones in which the percentage of recombinant clones is about 100%. The titer of the amplified cDNA library is 3.8 x 10(9) pfu/ml and the average exogenous inserts of the recombinants is 1.5 kb.

CONCLUSION

These results shows that the human adult nasopharynx cDNA library has an excellent quality and lays solid foundation for screening and cloning new tumor suppressor genes of nasopharyngeal carcinoma(NPC) and tissue-specific genes of human nasopharynx.