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:
  • 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

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
Allows you to design your own primers and probes using TaqMan® and SYBR® Green I dye chemistries for gene quantitation and allelic discrimination (SNP) real-time PCR applications.

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
Provides all PCR components and the MeltDoctor™ HRM Dye required for high resolution melting analysis. The MeltDoctor™ HRM Master Mix, developed and optimized solely for HRM applications, contains all components (excluding template and primers) formulated for superior HRM performance across a wide range of genomic targets.

 

Real-Time PCR Instruments

  • ViiA™ 7 Real-Time PCR System: enables high-productivity qPCR through ultimate flexibility, performance, and the most advanced features available.
  • StepOne™ Real-Time PCR System: a remarkably simple 48-well system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
  • StepOnePlus™ Real-Time PCR System: a remarkably simple 96-well system designed with a user-friendly, yet powerful, interface for researchers of all experience levels.
  • 7500 Fast Real-Time PCR System: offers maximum performance in the minimum time. Fully optimized for Fast cycling, the 7500 Fast delivers high-quality results in as little as 30 minutes.
  • 7900HT Fast Real-Time PCR System: a real-time quantitative PCR system that combines 384-well plate compatibility with fully automated robotic loading—and also offers optional Fast real-time PCR capability.

 

 

High Resolution Melt (HRM) Software v3.0
High Resolution Melting (HRM) analysis is an alternative to dHPLC sequencing screening of new gene variants. The application does not require temperature shifting, which results in a greater likelihood of identifying new homozygous mutations than methods that require temperature shifting.

 

MeltDoctor HRM Calibration Kits & Standard
Ready-to-use MeltDoctor™ HRM Calibration Plates contain all the components required for pure dye and HRM calibration reducing the complexity of getting started with HRM experimentation. They provide thermal and optical calibration required for accurate and reproducible discrimination between melt curves. 

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
e 3500 8-capillary platform can run a wide variety of applications - including de novo sequencing and resequencing (mutational profiling) - as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP®, and SNP validation or screening.

 

BigDye XTerminator® Purification Kit
A fast, simple purification method for DNA sequencing reactions that removes unincorporated BigDye® terminators.

 

BigDye® Terminator v3.1 Cycle Sequencing Kits
Highly flexible chemistry and is ideal for de novo, resequencing, and finishing with PCR product, Plasmid, Fosmid, and BAC templates. 

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