Fast, manual, nonradioactive method for DNA sequencing.
We describe a protocol that allows nonradioactive detection of sequencing products after manual, direct, solid-phase sequencing of polymerase chain reaction-amplified DNA. The amplified DNA fragment to be studied is biotinylated at the 5' end of one of the two oligonucleotide primers used for amplification, allowing coupling to streptavidin-coated magnetic beads. The immobilized double-stranded DNA is then separated into single strands by alkaline treatment. A 5'-biotinylated sequencing primer is used after saturating with a biotin solution any possible remaining affinity sites on the streptavidin-coated magnetic beads. Sequencing is performed by using T7 DNA polymerase, and the sequencing products are electrophoresed in denaturing polyacrylamide sequencing gel. After transfer of the products to a nylon membrane, the sequencing pattern is revealed by chemiluminescence. Biotinylated alkaline phosphatase is bound to the 5' end of the sequencing primer via a streptavidin bridge and catalyzes the reaction by cleaving a phosphate group from a chemiluminescent substrate. The emitted photons are detected by exposing the membrane to x-ray film. This method is simple, rapid, and consistently successful and reproducible.
Debuire
B
B
Service de Biochimie, Hôpital Paul Brousse, Villejuif, France.
Chabli
A
A
Frenoy
N
N
D001483:Base Sequence; D001710:Biotin; D004273:DNA, Neoplasm / Q000737:chemistry*; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000235:genetics; D008163:Luminescent Measurements; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
D004273:DNA, Neoplasm; D001710:Biotin; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase