A new solid-phase chemical DNA sequencing method which uses streptavidin-coated magnetic beads.
To simplify the chemical DNA sequencing protocol, we developed a new solid-phase method which uses streptavidin-coated magnetic beads. This method is based on the finding that the biotinylated DNA-streptavidin complex was stable under the conditions for some chemical sequencing reactions. The 5'-biotinylated DNA generated by the polymerase chain reaction was first captured by streptavidin-coated magnetic beads and then subjected to a set of simplified chemical sequencing reactions on the beads at room temperature. Followed by the piperidine cleavage reaction, the products were resolved by gel electrophoresis, transferred onto a nylon membrane and visualized by chemiluminescent detection. As a consequence, high-quality sequencing ladders were obtained, due to complete removal of contaminating chemicals, without the time-consuming precipitation/centrifugation steps used in the conventional chemical sequencing protocol.
Ohara
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R
Laboratory of DNA Technology, Kazusa DNA Research Institute, Chiba, Japan.
Ohara
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O
D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D008280:Magnetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D001710:Biotin; D019809:Streptavidin