A simple method for direct automated sequencing of PCR fragments.
A simple and rapid method for direct sequencing of PCR-generated fragments has been developed for use on Applied Biosystems 373A Automated DNA Sequencer utilizing the DyeDeoxy terminator chemistry. Standard PCR conditions are used to generate a DNA fragment, which is subsequently gel-purified to remove excess primers and unwanted PCR products. The sequencing reactions are carried out in a thermal cycler using the purified product as template DNA and the Dye-Deoxy terminators. The sequence of 500-bp region in the bacteriophage lambda genome and a 320-bp fragment of the human genomic erythropoietin gene were sequenced with greater than 99% accuracy using this method.
Tracy
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Dept. of Molecular and Cellular Biology, R.W. Johnson Pharmaceutical Research Institute, Raritan, NJ 08867.
Mulcahy
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D001331:Automation; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000302:isolation & purification; D004921:Erythropoietin / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012689:Sequence Homology, Nucleic Acid
D004921:Erythropoietin; D004247:DNA