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Related Experiment Videos

PCR and DNA sequencing.

U B Gyllensten1

  • 1Department of Medical Genetics, University of Uppsala, Sweden.

Biotechniques
|July 1, 1989
PubMed
Summary
This summary is machine-generated.

Polymerase chain reaction (PCR) amplifies specific DNA segments for sequencing templates. This method can be automated for efficient DNA sequencing, generating single-stranded DNA templates directly within the reaction.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genomics

Background:

  • Polymerase chain reaction (PCR) enables enzymatic amplification of specific DNA segments.
  • Generating sequencing templates from cloned inserts or genomic DNA is a key application of PCR.
  • Reassociation of linear DNA strands can complicate sequencing reactions.

Purpose of the Study:

  • To describe methods for producing single-stranded DNA (ssDNA) templates for direct sequencing.
  • To present a combined PCR and direct sequencing approach.
  • To highlight the potential for full automation of DNA sequencing.

Main Methods:

  • Enzymatic amplification of DNA using PCR.
  • Production of ssDNA templates via in-PCR methods, enzymatic treatment, electrophoresis, or affinity purification.

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  • Direct sequencing of amplified DNA, with potential for same-vial processing.

    • Main Results:

    • PCR allows up to millionfold amplification of target DNA sequences.
    • Various methods effectively generate ssDNA templates, preventing reassociation issues.
    • Combining PCR with direct sequencing streamlines the process.

    Conclusions:

    • The described methods facilitate efficient generation of sequencing templates.
    • Integrating PCR amplification and direct sequencing offers a streamlined approach.
    • The use of fluorescent labels enables complete automation of the DNA sequencing procedure.