Construction and identification of a cDNA library of human normal kidney tissues / 西安交通大学学报(医学版)

ABSTRACT

Objective To construct a full-length cDNA library of human normal kidney tissues and identify the quality of the library. Methods By using the template-switching mechanism at 5′end of mRNA technique to construct the library, a powerscript reverse transcriptase was used to transcribe, and a 5′-oligo fragment as an extended template was added to 5′ end of mRNA to enrich full-length cDNAs. After amplification, the ds cDNAs digested by sfi I and size-fractionated by columns were recombined into ?TripIEx 2 vectors. After package, the recombinant vectors were titered and the recombinant rate (blue/white) was determined,then the library was amplified. We identified the library using PCR reaction to determine the size of the inserts. Results The titer of cDNA library was 2.6?10 6pfu?mL -1, the rate of recombinant was above 95%, and the titer of amplified library was 9?10 11pfu?mL -1. The insert size ranged from 0.7 to 2 kb. Conclusion The cDNA library of human normal kidne we constructed is a highly efficient one and can be used for screening by probe and antibody to find the genes related to kidney diseases.