Application Of RD-PCR Technology In The Preparation Of HCV Microarray

Hepatitis C virus(HCV) can be divided into six phylogenetically distinct groups as clades 1 to 6, consisting mainly 6 genotypes and dozens of subtypes. Genotype classification of HCV helps to direct the treatment of hepatitis C, to develop effective HCV vaccine and to the identification of the epidemic sources. Unfortunately, the existing methods of gettype classification of HCV are far from satisfaction. DNA chip(microarray) technique was applied in this study together with an unique technique of DNA fragmentation restriction display PCR (RD-PCR). It was used to isolate the necessary probes being used in DNA chip preparations. Restriction enzyme Sau3A I was chosen to digest the full-length HCV cDNAs of three distinct subtypes, i.e. la, lb and 2a. The resulted restrictive fragments were then ligated with universal adapters. PCR primers were designed to match the universal adapters but with one esting?base overhanging at the 3? end. The PCR reactions ~were divided into ten subgroups for ten different primer combinations. The second- or third-turn PCR was performed using the single bands as the templates which were cut out from the agarose gel. The purified PCR products were then cloned into T-vectors. The positive clones were propagated and plasmids were extracted. The target HCV gene fragments ranging from 0.2 to 1 kb were isolated and sequenced which were correlated precisely with the RFLP(Restriction Fragments Length Polymorphism) prediction. A total of 62 different fragments were obtained, averaging about 20 for each subtype. The necessary amount of probes were then amplified using the T-vector subclones as the templates. RD-PCR was initially developed by Dr. Zheng Wenlin and Dr. Ma Wenli to reduce the false positive results in the procedure of classical differential display PCR(DD-PCR). When applied, the corresponding primers can be designed to match the size of any genome, large or small, whether its sequence is revealed or not. Then the amplified restriction fragments can be effectively divided into certain number of subgroups. Through the regular agarose gel electrophoresis those fragments then can be easily separated. The proper amount of these fragments, i.e. DNA probes, can also be obtained by amplification in normal PCR condition. It's obvious that RD-PCR technique is of great value in obtaining a large number of size-comparable probes which are fit for DNA chips manufacture in a relatively short period. It proves to be an effective and practicable method for expediting the isolation of known or unknown gene fragments.