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A novel primer-extension assay for the detection of a G to A mutation in the distal precore region of hepatitis B virus DNA

 J. P. Chung  ;  K. H. Han  ;  K. S. Kim  ;  B. I. Yeh  ;  S. T. Kim  ;  K. S. Lee  ;  C. Y. Chon  ;  Y. M. Moon  ;  J. K. Kang  ;  I. S. Park 
 Journal of Viral Hepatitis, Vol.6(4) : 305-313, 1999 
Journal Title
 Journal of Viral Hepatitis 
Issue Date
Adult ; Base Sequence ; DNA Primers* ; DNA, Viral/analysis ; Female ; Hepatitis B e Antigens/analysis ; Hepatitis B virus/genetics* ; Hepatitis B, Chronic/virology* ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Point Mutation* ; Polymerase Chain Reaction/methods ; Reproducibility of Results ; Sequence Analysis, DNA
The roles of genetic heterogeneity of the hepatitis B virus (HBV) precore gene in the pathogenesis of HBV infection are unclear. Various methods have been used to detect nucleotide (nt) 1896 precore mutants. We established a new primer-extension assay to facilitate the detection of these mutants. This assay is based upon the fact that there is no adenine in the distal precore region of wild-type HBV. Polymerase chain reaction (PCR)-amplified template DNA was denatured and annealed to the [gamma-32P]-labelled primer. During primer extension in the presence of DNA polymerase and dCTP, dGTP, dTTP and ddATP, the reaction terminates if there is a nucleotide A. When mixtures of different ratios of wild-type and nt 1896 precore mutants were analysed in the primer-extension assay, correlation between the percentage known amounts and the percentage measured amounts of nt 1896 precore mutants was excellent (r2=0. 9669). When the primer-extension assay and direct sequencing were compared in hepatitis B e antigen (HBeAg)-positive and -negative chronic active hepatitis B patients, the primer-extension assay detected a greater number of nt 1896 precore mutants than direct sequencing and thus most HBV infections were found to be mixed infections. In conclusion, the primer-extension assay is a reliable and sensitive method for the detection of nt 1896 precore mutants.
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Yonsei Authors
Kim, Seung Taek(김승택)
Lee, Kwan Sik(이관식) ORCID logo https://orcid.org/0000-0002-3672-1198
Han, Kwang-Hyub(한광협) ORCID logo https://orcid.org/0000-0003-3960-6539
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