Analysis of S. gene nucleotide sequence of hepatitis B. virus cenotypes and determining them with RFLP methods

2.SUMMARY Analysis of S gene nucleotide sequence of hepatitis B virus genotypes and determining them with RFLP methods Key Words : HBV, genotype, PCR, RFLP Although hepatitis B virus (HBV) infections are a major health problem in Turkey, there is a little information on the genotype distribution of the virus. In this study, DNA sequencing and RFLP methods were used to genotype 23 HBV-DNA positive samples originating from Turkey. HBV-DNA was extracted by proteinase k / phenol-chloroform method and 541 nucleotide fragment between positions 256 and 796 was amplified using 'S' gene specific primers by single-step PCR. The analytical sensivity of the procedure was 19 copy/mL. PCR products purified by ethanol precipitation were then used in sequencing reactions utilizing "Sequitherm Excel II DNA Sequencing" kit and a LI-COR sequencing apparatus. A 420 bp fragment of the S gene between nucleotide positions 356 and 775 was sequenced and aligned with a data set of 19 HBV S gene sequences available from Gen-Bank. Phylogenetic trees were reconstructed using a neighbour-joining method. All samples were clustered with genotype D sequences. A RFLP based genotyping assay using Mva I, Rsa I and Hinf I enzymes were developed as an easier and less expensive alternative to sequencing. The results of the RFLP analysis of the samples were consistent with the phylogenetic analysis.