Microplate hybridization of amplified viral DNA segment.
We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the amplified DNA segment with a length of more than 300 bases is adsorbed to microplate wells in the presence of 1.5 M NaCl or 0.5 M ammonium sulfate; the immobilized DNA is hybridized with a biotin-labeled DNA probe; then, the hybridization signal is detected by streptavidin-conjugated beta-galactosidase or peroxidase. This method has several advantages over the conventional dot blot hybridization method: (i) radioisotopes are not used, (ii) synthetic oligonucleotide for the probe is not needed, (iii) the time required for washing of the solid phase is greatly reduced, and (iv) the baking and prehybridization procedures are eliminated. By this method, we were able to detect viral genomes in vesicle specimens from patients infected with varicella-zoster virus.
Inouye
S
S
Department of Microbiology, University of Tokyo, Japan.
Hondo
R
R
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D005784:Gene Amplification; D006562:Herpes Zoster / Q000175:diagnosis*; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction
D004279:DNA, Viral