Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation

Figure 1 Polymerase chain reaction amplification and direct sequencing analysis of hepatitis B virus X region. A: Location of primer set for the nested PCR direct sequencing (1^st primer set; X-pro-F1 and X-pro-R1, 2^nd primer set; X-pro-F2 and X-pro-R2) and Fok-I based nested PRA (1^st primer set; X-pro-F1 and X-pro-R1, 2^nd primer set; X-pro-F2 and X-pro-R3) for the detection of HBxAg V5M mutations and the 3 polymorphisms [V5(GTG), M5(ATG), and L5(CTG or TTG)] of the 5^th codon of HBxAg; B: The amino acid and nucleotide sequences of the X region from six reference strains (Genotype A, B, C1, C2, and D) and 10 chronic hepatitis patients (five carriers and five HCC patients) were aligned.

Figure 2 Algorithm of Fok-I nested polymerase chain reaction-restriction fragment length polymorphism analysis method for the detection of V5M mutation in the HBV X antigen region used in the present study.

Figure 3 Application of Fok-I nested polymorphism analysis method (A) and direct sequencing analysis (B) into serum DNAs. A: The Fok-I PRA application of nested PCR products could produce three types of PRA pattern on an agarose gel (2.5%): an undigested 290-bp from the wild type, V5(GTG); two bands of complete digestion, 186-bp and 104-bp, from the V5M mutant and three bands, an undigested one of 290-bp from the wild type and 2 digested bands, 186-bp and 104-bp, from V5M mutant. B: Direct sequencing could also produce three types: wild type only [V5(GTG)], mutation only [M5(ATG)], and mixed types with the wild type V5(GTG) and M5(ATG). The mixed types showed the mixed peaks (G and A) at the first nucleotide of the 5^th codon of HBxAg (NTT). The results of the Fok-I nested PRA method were completely concordant with those obtained by direct sequencing analysis. Lane M, 100-bp DNA ladder marker.