Method for detection of epsilon-secondary structure in the precore region of human hepatitis B virus DNA using a fluorescence-based polymerase chain reaction-single-strand-conformation polymorphism technique with capillary electrophoresis.
A portion of the precore region of the human hepatitis B virus (HBV) genome is the signal sequence with an epsilon secondary structure, which plays a role in the encapsidation of HBV pregenome RNA. To determine the genetic mutations which occur in the precore region of HBV, we have devised a typing method using a fluorescence-based polymerase-chain-reaction-single-strand conformation polymorphism technique with automated capillary electrophoresis (CE-FSSCP). Using the cloning sequencing method, we analyzed serum samples from 10 patients with hepatitis B, and detected three types of HBV-DNA including two mutants which are crucial to the function of the encapsidation sequence: position 1896 G (guanine) to A (adenine, stop codon), position 1899 G to A, and wild-type. We performed CE-FSSCP analysis of these three types of HBV-DNA and described conditions for determination of the mutations which play roles in the encapsidation of the HBV pregenome. The two types of epsilon mutants and wild-type DNA were identified as separate individual peaks respectively. The observed migration times of the three types of DNAs agreed fairly well with estimates obtained from total RNA secondary structure energy.
Katsuragi
K
K
Diagnostics Division, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan.
Kitagishi
K
K
Mizuguchi
T
T
Nagashima
T
T
Kinoshita
M
M
Kumada
H
H
D001483:Base Sequence; D002021:Buffers; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000097:blood / Q000737:chemistry* / Q000235:genetics; D003911:Dextrans / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods*; D005990:Glycerol / Q000737:chemistry; D006515:Hepatitis B virus / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D012367:RNA, Viral / Q000737:chemistry; D017422:Sequence Analysis, DNA; D013050:Spectrometry, Fluorescence
D002021:Buffers; D017931:DNA Primers; D004279:DNA, Viral; D003911:Dextrans; D012367:RNA, Viral; D005990:Glycerol