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- W4298382617 startingPage "1682" @default.
- W4298382617 abstract "Abstract 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." @default.
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- W4298382617 date "1993-08-01" @default.
- W4298382617 modified "2023-10-11" @default.
- W4298382617 title "Fast, manual, nonradioactive method for DNA sequencing" @default.
- W4298382617 doi "https://doi.org/10.1093/clinchem/39.8.1682" @default.
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