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DNA as a Genetic Template02:05

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Two structural features of the DNA molecule provide a basis for the mechanisms of heredity: the four nucleotide bases and its double-stranded nature. The Watson-Crick model of double-helical DNA structure, proposed in 1952, drew heavily upon the X-ray crystallography work of researchers Rosalind Franklin and Maurice Wilkins. Watson, Crick, and Wilkins jointly received the Nobel Prize in Physiology or Medicine for their work in 1962. Franklin was, controversially, excluded from the prize for...
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High resolution melt analysis, DNA template quantity disparities and result reliability.

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    High Resolution Melt (HRM) analysis is reliable despite significant variations in starting DNA template quantity for PCR. Adequate PCR amplification quality is key to reproducible HRM results in genotyping.

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    Area of Science:

    • Molecular Biology
    • Genetics
    • Biotechnology

    Background:

    • Variations in DNA template quantity in PCR can lead to inaccurate results.
    • High Resolution Melt (HRM) analysis is a reproducible genotyping method, unaffected by reaction volume or DNA concentration.

    Purpose of the Study:

    • To assess the reproducibility and reliability of HRM analysis with varying amounts of starting DNA templates in upstream PCR.

    Main Methods:

    • Serial dilutions of DNA templates (amplicon-enriched and FFPE) were amplified.
    • PCR products underwent High Resolution Melt (HRM) analysis using ABI 7500 Fast Real Time PCR machine and ABI HRM software.

    Main Results:

    • HRM analysis demonstrated reproducibility with a two- to one thousand-fold difference in starting DNA template.
    • Reproducibility was maintained provided the starting DNA template quality and quantity were sufficient for adequate PCR amplification.

    Conclusions:

    • HRM analysis is highly reliable even with substantial variations in starting template amounts for upstream PCR.
    • The adequacy of PCR amplification is the primary determinant of HRM reproducibility.