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- W2364304913 abstract "Objective To investigate the Restriction Display(RD) technique in the preparation of HCV probes of clinical genotyping microarray. Methods Restriction enzyme Sau3A Ⅰ was chosen to digest the full-length HCV cDNAs of three distinct subtypes.i.e.1a,1b and 2a. The resulted 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 divided into ten subgroups for ten different primer combinations. The differential genes through polyacrylamide gel electrophoresis and sliver staining were separated. Single bands were isolated and were cut out from the polyacrylamide gel. The second-round PCR could be performed by using the single bands as PCR template. 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 fragment 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 fragment were obtained, averaging about 22 for each subtype. These fragments could be used further as probes in the microarray preparations.Conclusion RD technique is of great value in obtaining a large number of size-comparable gene probes, which provide a swift protocol in generating DNA probes for the preparation of microarrays." @default.
- W2364304913 created "2016-06-24" @default.
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- W2364304913 date "2004-01-01" @default.
- W2364304913 modified "2023-09-25" @default.
- W2364304913 title "Application of restriction display technique in the preparation of the HCV probes for the preparation HCV microarray" @default.
- W2364304913 hasPublicationYear "2004" @default.
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