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Related Experiment Videos

Re-usable DNA template for the polymerase chain reaction

S N Sheikh1, P Lazarus

  • 1Department of Pathology and Laboratory Medicine, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140, USA.

Nucleic Acids Research
|September 1, 1997
PubMed
Summary

This study demonstrates that DNA covalently bound to nylon membranes enables repeated, successful Polymerase Chain Reaction (PCR) amplifications of various genes. This method provides a stable and reusable DNA template source for genetic analysis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Polymerase Chain Reaction (PCR) is a fundamental technique in molecular biology.
  • Efficient and repeatable DNA amplification is crucial for genetic analysis and diagnostics.
  • Storing and reusing DNA templates can be challenging.

Purpose of the Study:

  • To investigate the feasibility of using DNA covalently bound to nylon membranes for repeated PCR amplifications.
  • To assess the integrity and sequence accuracy of PCR products amplified from membrane-bound DNA.
  • To establish a stable and reusable DNA template system for PCR.

Main Methods:

  • Covalent binding of genomic and plasmid DNA to uncharged nylon membranes.
  • Performing multiple PCR amplifications using the same membrane-bound DNA template.

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  • Analyzing PCR products using restriction fragment length polymorphism (RFLP), single-strand conformational polymorphism (SSCP), and dideoxy sequencing.
  • Quantifying the minimum amount of DNA required for successful amplification from the membrane.

    • Main Results:

    • Successful PCR amplifications were achieved with both membrane-bound genomic and plasmid DNA.
    • Membrane-bound DNA templates were reused over 15 times with consistent amplification success.
    • Sequencing confirmed that PCR products from bound DNA were identical to those from free DNA.
    • PCR amplification was successful with as little as 5 ng genomic DNA or 4 fg plasmid DNA bound to the membrane.

    Conclusions:

    • DNA covalently bound to nylon membranes serves as a stable, reusable template for PCR.
    • This method offers a potentially unlimited source of DNA for sample-specific PCR amplifications.
    • The technique preserves DNA integrity, allowing for accurate genetic sequence analysis.