Viral Hepatitis
Copyright ©2005 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 28, 2005; 11(48): 7579-7584
Published online Dec 28, 2005. doi: 10.3748/wjg.v11.i48.7579
Application of restriction display PCR technique in the preparation of cDNA microarray probes
Zhao-Hui Sun, Wen-Li Ma, Bao Zhang, Yi-Fei Peng, Wen-Ling Zheng
Zhao-Hui Sun, Bao Zhang, Yi-Fei Peng, Wen-Li MA, Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, Guangdong Province, China
Wen-Ling Zheng, Institute of Molecular Oncology, Guangzhou General Hospital of Guangzhou Military Area Command, Guangzhou 510010, Guangdong Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 39880032; Major Programs for Science and Technology Development of Guangzhou, No. 01-Z-005-01
Correspondence to: Professor Wen-Li Ma, Institute of Genetic Engineering, Southern Medical University, Guangzhou 510515, Guangdong Province, China.
Telephone: +86-20-61648210 Fax: +86-20-61647755
Received: April 14, 2005
Revised: July 10, 2005
Accepted: July 15, 2005
Published online: December 28, 2005

AIM: To develop a simplified and efficient method for the preparation of hepatitis C virus (HCV) cDNA microarray probes.

METHODS: With the technique of restriction display PCR (RD-PCR), restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs. The products were classified and re-amplified by RD-PCR. We separated the differential genes by polyacrylamide gel electrophoresis and silver staining. Single bands cut out from the polyacrylamide gel were isolated. The third-round PCR was performed using the single bands as PCR template. The RD-PCR fragments were purified and cloned into the pMD18-T vector. The recombinant plasmids were extracted from positive clones, and the target gene fragments were sequenced. The cDNA microarray was prepared by spotting RD-PCR products to the surface of amino-modified glass slides using a robot. We validated the detection of microarray by hybridization and sequence analysis.

RESULTS: 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 microarray preparation. The diagnostic capability of the microarray was evaluated after the washing and scanning steps. The results of hybridization and sequence analysis showed that the specificity, sensitivity, accuracy, reproducibility, and linearity in detecting HCV RNA were satisfactory.

CONCLUSION: The RD-PCR technique is of great value in obtaining a large number of size-comparable gene probes, which provides a speedy protocol in generating probes for the preparation of microarrays. Microarray prepared as such could be further optimized and applied in the clinical diagnosis of HCV.

Keywords: Restriction display PCR, HCV, Microarray, Probes