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Related Experiment Video

Updated: Jun 22, 2026

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
10:27

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Published on: November 10, 2015

Multiplex amplicon genotyping by high-resolution melting.

Michael T Seipp1, Jacob D Durtschi, Karl V Voelkerding

  • 1ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, Utah 84108, USA. seippmt@aruplab.com

Journal of Biomolecular Techniques : JBT
|July 2, 2009
PubMed
Summary

High-resolution melting analysis simplifies multiplex genotyping using a single instrument. This method accurately identifies thrombophilia mutations, reducing analysis time and costs.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • High-resolution amplicon melting is a genotyping method using PCR and DNA dyes.
  • Previous multiplex genotyping required two instruments, limiting throughput due to manual steps and slow melting rates.

Purpose of the Study:

  • To evaluate a single instrument combining real-time PCR and high-resolution melting for multiplex amplicon genotyping.
  • To assess the efficiency and accuracy of this integrated system for common thrombophilia mutations.

Main Methods:

  • Utilized the LightScanner-32 (LS-32) instrument for integrated rapid-cycle real-time PCR and high-resolution melting.
  • Developed a homogeneous assay for genotyping F5, F2, and MTHFR mutations (1298A>C, 677C>T) with internal controls.
  • Optimized melting rate to 0.3°C/s and reduced sample volume to 10 µL.
Keywords:
F2F5LightScanner-32MTHFRPCRSNPThrombophilia

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Last Updated: Jun 22, 2026

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria
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Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Published on: November 10, 2015

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Published on: February 5, 2014

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05:53

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

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Main Results:

  • Achieved complete concordance with standard assays in a blinded study of 109 samples (436 genotypes).
  • Successfully genotyped the rare MTHFR 1317T>C variant.
  • Reduced analysis time to 9.2 minutes for 40 temperature cycles and 2.2 minutes per capillary, a 3x increase in melting speed.

Conclusions:

  • The LS-32 instrument simplifies multiplex amplicon genotyping by integrating PCR and melting analysis.
  • This approach significantly reduces hands-on time, analysis duration, and reagent costs.
  • The system maintains high resolution for accurate genotyping of common and rare variants.