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 & 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:
Figure 1 (Right): Difference Plot of a human 121 bp region in NAT2 that was amplified from 4 replicates of 24 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.
Mutation Scanning Experimental Workflow
A critical first step in setting up mutation scanning experiments using HRM analysis is to design PCR primers to amplify overlapping segments of the genomic region of interest. Important considerations for the primer design step include designing appropriately sized amplicons and observing well established conventions for designing high-quality PCR primers.
HRM analysis works well with amplicons in the ~120–300 bp size range; this size corresponds to 80–250 bp genomic DNA targets amplified using ~20–40 nt PCR primers. Amplicons in this range are sufficiently short to yield simple, easily analyzed melt profiles, yet long enough to minimize the number of individual amplicons needed to cover the target region. Large sequence variations may be detectable in amplicons longer than ~300 bp; however, more subtle variations such as A/T class 4 SNPs, are typically detectable only in smaller amplicons.
Recommended Products
 | Primer Express Software |
During the HRM stage of the melt curve, double-stranded amplicons denature as the reaction temperature is slowly increased, releasing bound dsDNA-binding dye. The real-time PCR instrument measures this decrease in fluorescence signal, and the HRM software plots fluorescence signal as a function of temperature.
Recommended Products
 | MeltDoctor™ HRM Reagents | |
 | Real-Time PCR Instruments
| |
| High Resolution Melt (HRM) Software | |
| MeltDoctor HRM Calibration Kits & Standard |
PCR products with variant HRM melt curves may contain mutation(s), and can be further analyzed via Sanger sequencing. PCR products from HRM analysis need only be diluted (at least 1:20 dilution) prior to sequencing; typically no purification is needed. This dilution reduces the concentration of leftover PCR primers and dNTPs to negligible levels in terms of the sequencing reaction.
Recommended Products
 | 3730 DNA Analyzer Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. | |
 | 3500 Genetic Analyzer | |
 | BigDye XTerminator® Purification Kit | |
 | BigDye® Terminator v3.1 Cycle Sequencing Kits |
A critical first step in setting up mutation scanning experiments using HRM analysis is to design PCR primers to amplify overlapping segments of the genomic region of interest. Important considerations for the primer design step include designing appropriately sized amplicons and observing well established conventions for designing high-quality PCR primers.
HRM analysis works well with amplicons in the ~120–300 bp size range; this size corresponds to 80–250 bp genomic DNA targets amplified using ~20–40 nt PCR primers. Amplicons in this range are sufficiently short to yield simple, easily analyzed melt profiles, yet long enough to minimize the number of individual amplicons needed to cover the target region. Large sequence variations may be detectable in amplicons longer than ~300 bp; however, more subtle variations such as A/T class 4 SNPs, are typically detectable only in smaller amplicons.
Recommended Products
| Primer Express Software |
During the HRM stage of the melt curve, double-stranded amplicons denature as the reaction temperature is slowly increased, releasing bound dsDNA-binding dye. The real-time PCR instrument measures this decrease in fluorescence signal, and the HRM software plots fluorescence signal as a function of temperature.
Recommended Products
| MeltDoctor™ HRM Reagents | |
| Real-Time PCR Instruments
| |
| High Resolution Melt (HRM) Software | |
| MeltDoctor HRM Calibration Kits & Standard |
PCR products with variant HRM melt curves may contain mutation(s), and can be further analyzed via Sanger sequencing. PCR products from HRM analysis need only be diluted (at least 1:20 dilution) prior to sequencing; typically no purification is needed. This dilution reduces the concentration of leftover PCR primers and dNTPs to negligible levels in terms of the sequencing reaction.
Recommended Products
| 3730 DNA Analyzer Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. | |
| 3500 Genetic Analyzer | |
| BigDye XTerminator® Purification Kit | |
| BigDye® Terminator v3.1 Cycle Sequencing Kits |
Literature
Application Note
High Resolution Melt Analysis for Mutation Scanning Prior to Sequencing
Quick Reference Card
HRM Mutation Scanning Experiment
Product Bulletin
MeltDoctor™ High Resolution Melt Reagents