Sequencing of PCR-amplified DNA.
Alternatives for sequencing of PCR products essentially fall into one of two categories; generation of single-stranded DNA for sequencing or the direct sequencing of double-stranded product. Of the two alternatives, sequencing of double-stranded PCR products is likely to be of greatest immediate significance in terms of general applicability and rapidity. Double-stranded sequencing allows the use of the PCR product for other purposes either prior to or subsequent to generation of sequence data. The single-stranded sequencing methods generally require some prior decision regarding sequencing of the product. Assisted by automated workstation development, sequencing of single-stranded DNA PCR products generated either during thermal cycling or following affinity-capture strand separation may have significant future utility, particularly in genome mapping and routine clinical diagnosis. Despite template type and protocol differences, in all situations the purity and concentration of PCR-amplified DNA template used remains the most critical factor determining the efficiency and reliability of nucleotide sequencing methods.
Bevan
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University Department of Clinical Biochemistry, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK.
Rapley
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Walker
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D001426:Bacterial Proteins; D001710:Biotin; D002846:Chromatography, Affinity; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods; D004277:DNA, Single-Stranded / Q000032:analysis; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000295:instrumentation / Q000379:methods*; D019809:Streptavidin; D019914:Taq Polymerase; D013698:Templates, Genetic
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase