[New developments of molecular genetic analysis on human mitochondrial DNA].
The use of magnet beads for the capture and purification of PCR product allows simple preparation of immobilized single-stranded template. This template is ideal for use in DNA direct sequencing. The end labeling of PCR product with biotin is achieved simply by using a biotinylated primer. Capture and purification are then accomplished by incubation with Streptavidin attached magnet beads and magnet separation. Once immobilized, the DNA is converted to a single-stranded template by elution with alkali and another magnetic separation. Immobilized single-stranded template prepared in this manner are suitable for manual and automated solid-phase DNA sequencing by the dideoxynucleotide chain termination method. A 100% single-stranded template is produced, without interference from primers, free nucleotides or the complementary strand. The solid phase approach provides reproducible sequencing results with high yields. The eluted complementary strand can also be sequenced after neutralization.
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D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004272:DNA, Mitochondrial / Q000235:genetics* / Q000302:isolation & purification; D005807:Genes, Overlapping; D008280:Magnetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D019809:Streptavidin
D001426:Bacterial Proteins; D004272:DNA, Mitochondrial; D001710:Biotin; D019809:Streptavidin