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Related Experiment Videos

High-resolution genotyping by amplicon melting analysis using LCGreen.

Carl T Wittwer1, Gudrun H Reed, Cameron N Gundry

  • 1Department of Pathology, University of Utah Medical School, Salt Lake City, UT 84132, USA. carl.wittwer@path.utah.edu

Clinical Chemistry
|May 27, 2003
PubMed
Summary

This study presents a new closed-tube method for genotyping and mutation scanning using high-resolution amplicon melting analysis without labeled primers. The technique efficiently distinguishes DNA sequence variants, offering a promising approach for genetic analysis.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • High-resolution amplicon melting analysis (HRM) is a closed-tube genotyping method.
  • Existing HRM requires fluorescently labeled primers, limiting mutation detection.
  • A novel HRM approach was developed to eliminate the need for labeled oligonucleotides.

Purpose of the Study:

  • To develop a closed-tube system for genotyping and mutation scanning.
  • To create a method that does not require labeled oligonucleotides.
  • To assess the efficacy of HRM with a generic double-stranded DNA dye.

Main Methods:

  • Studied polymorphisms in HTR2A, beta-globin, and cystic fibrosis genes.
  • Utilized PCR with the double-stranded DNA dye LCGreen.

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  • Obtained high-resolution amplicon melting curves and analyzed for sequence alterations.

    • Main Results:

    • Successfully distinguished six common beta-globin genotypes (AA, AS, AC, SS, CC, SC).
    • Genotyped HTR2A single-nucleotide polymorphism in a 544-bp fragment.
    • Achieved amplification and analysis in 10-20 minutes using rapid cycling.

    Conclusions:

    • High-resolution melting analysis with LCGreen identifies heterozygous and homozygous sequence variants.
    • The technique uses unlabeled primers and a generic DNA dye for amplicon genotyping.
    • This method is a promising tool for mutation screening and genetic analysis.