[Research of restriction display technique in cDNA microarrays preparation for detecting of HCV].
The cDNA microarrays for HCV detection was prepared. With the restriction display technique (RD), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and amplified by RD-PCR. We separated the differential genes through polyacrylamide gel electrophoresis and sliver staining. Single bands were isolated which were cut out from the polyacrylamide gel. The third-round PCR could be performed by using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vectors. The recombinant plasmids were extracted from positive clones and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting PCR products to the surface of amido modified glass slides by the robotics. We validated the detection of microarray by the hybridization and the results of sequence analysis. A total of 24 different cDNA fragments ranging from 200 to 800 bp were isolated and sequenced, which were the specific gene fragments of HCV. These fragments could be further used as probes in the microarray preparations. From the results of hybridization and sequence date analysis, the specificity, sensitivity, accuracy, reproducibility and linearity in detecting HCV RNA were satisfactory. RD technique is of great value in obtaining a large number of size-comparable gene probes, which provide a swift protocol in generating probes for the preparation of microarrays, and the optimized microarray is sensitive and effective in clinical diagnosis of HCV.
Sun
Zhao-hui
ZH
Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, China. zhaohui2@fimmu.com
Zheng
Wen-ling
WL
Peng
Yi-fei
YF
Zhang
Bao
B
Ma
Wen-Li
WL
D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood*; D012680:Sensitivity and Specificity; D016622:Silver Staining
D015342:DNA Probes; D012367:RNA, Viral