Heterogeneity of hepatitis B virus C-gene sequences: implications for amplification and sequencing.
Occasionally direct sequencing of amplified hepatitis B virus DNA leads to weak signals on autoradiograms. Using amplified C-gene sequences we investigated whether this is due to sequence heterogeneity of virus populations and use of inappropriate primers for direct sequencing. High C-gene sequence heterogeneity (point mutations, stop codons and a one codon deletion) was observed in HBV genomes from serum of a chronic carrier who underwent interferon treatment. The type of C-gene mutations detected by direct sequencing depended on the type of primers used. Cloning and sequencing of amplified C-gene sequences demonstrated that this was due to mutations in the region complementary to the sequencing primer. These data demonstrate the existence of novel HBV C-gene mutants and imply that multiple or degenerate sequencing and amplification primers are essential for accurate evaluation of the extent of HBV C-gene heterogeneity. Based on comparative sequence analysis of all available completely or incompletely sequenced C-genes, guidelines for optimal primer design are proposed for similar studies.
Miska
S
S
Max-Planck-Institut für Biochemie, Martinsried, Munich, Germany.
Günther
S
S
Vassilev
M
M
Meisel
H
H
Pape
G
G
Will
H
H
D000328:Adult; D000595:Amino Acid Sequence; D001483:Base Sequence; D002353:Carrier State; D005260:Female; D005784:Gene Amplification; D005814:Genes, Viral; D006509:Hepatitis B / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009154:Mutation