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Mutation Scanning

Difference Plot of a human 121 bp region

Difference Plot of a human 121 bp region in NAT2 that was amplified from 4 replicates of 24 samples.

Analyzing Genetic Variation Using HRM Mutation Scanning Followed by Sanger Sequencing

Mutation scanning strategies seek to quickly and efficiently scan DNA samples from many individuals for minor genetic variations to identify candidates with genetic variations for full sequencing analysis.

HRM Analysis for Fast, Accurate, and Reliable Mutation
Use high resolution melt (HRM) analysis to scan large numbers of samples for genetic variation. With the introduction of brighter DNA binding dyes, real-time PCR instruments that collect fluorescence data at finer temperature resolution, and intuitive software platforms, high resolution melt (HRM) analysis is becoming the method of choice for scanning large numbers of samples for genetic variants.

Compared to other technologies for mutation scanning, such as pyrosequencing, denaturing HPLC (dHPLC), and denaturing gradient gel electrophoresis (DGGE), HRM offers:
  • A far easier and faster procedure that consumes significantly less reagent
  • Higher reproducibility
  • Reduced optimization and interpretation efforts
  • Better accuracy and sensitivity
  • A closed tube procedure reducing the risk of cross-contamination of samples

Non-Destructive and Well-Suited for Variant Scanning
Select samples with HRM melt profiles that are significantly different from those of wild-type samples for further study. For example, the PCR reaction product from HRM can be introduced directly into the Sanger sequencing reaction for DNA sequencing.

Sanger DNA sequencing remains the most straightforward, reliable method for determining the precise genetic sequence of DNA fragments.

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