Application of restriction display (RD) technique in the preparation of the HCV probes for HCV cDNA microarray / 解放军医学杂志

ABSTRACT

Objective To investigate the applieation of restriction display (RD) technique in the preparation of HCV probes of clinical genotyping microarray. Methods Restriction enzyme Sau3A Ⅰwas chosen to digest the full-length HCV cDNAs of three distinct subtypes, i.e.1a, 1b and 2a. The resultant restrictive fragments were then ligated with universal adapters. PCR primers were designed to match the universal adapters but with one "nesting" base overhanging at the 3′- end. The PCR reactions were performed by ten pairs of different primer combinations. The differential genes were separated through polyacrylamide gel electrophoresis and silver staining. The second-round PCR was performed using the isolated bands as PCR templates. The purified PCR products were then cloned into T-vectors. The recombinant plasmids were extracted from positive recombinant clones and the target gene fragments were sequenced. Results The target HCV gene fragments ranging from 200 to 900bp were isolated and sequenced, which were correlated precisely with the RFLP (Restriction Fragment Length Polymorphism) prediction. A total of 66 different fragments were obtained, averaging about 22 for each subtypes. These fragments could be further used as probes in HCV microarray preparations. Conclusion RD technique is of great value in obtaining a large number of equal sized gene probes, which provide a swift protocol in generating DNA probes for the preparation of microarrays.