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:: Posted by American Biotechnologist on 01-10-2011
High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis.
In the following 20 minute tutorial presented by Sean Taylor, Field Application Specialist, Bio-Rad Laboratories, you will learn the basics of high resolution melt analysis and how to practically use it in your research. The video contains information on:
what is high resolution melt analysis, how does it work and why it is useful
various applications for high resolution melt analysis including single nucleotide polymorphism (SNP) analysis and DNA methylation analysis
the difference between a standard melt curved performed in routine QPCR and HRM
the difference between non-saturation dyes such as SYBR green and saturation dyes such as evagreen
the definition of the four class types of SNPS
critical factors for good experimental design
what technique you should be using for your SNP analysis (it is not one size fits all…think HRM VS Sequenome VS multiplex array
:: Posted by American Biotechnologist on 10-28-2010
High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis. The technique relies on quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and in combination with qPCR permits the identification of genetic variation in nucleic acid sequences by the controlled melting of a double-stranded PCR amplicon. As opposed to standard melt curves which are run for routing qPCR experiments, HRM melt curves involve the collection of melt data in 0.2 degree C increments. Furthermore, in order to identify small nucleotide changes it is essential to eliminate background fluorescence from any HRM analysis. Recent advances in real time PCR equipment, software and reagents (including fluorescent dyes) has turned HRM into a robust analytical technique capable of detecting a small proportion of variant DNA in a background of wild-type sequence at sensitivities approaching 5%. Perhaps one of the most astounding accomplishments of HRM analysis is the ability to detect class IV SNPs (A>T or T>A mutations) which are extremely rare (they occur at a frequency of approximately 7% withing the Human Genome) and difficult to identify due to their small melt curve temperature shift.
The attached technical note from Bio-Rad Laboratories will provide you with a fantastic overview of HRM analysis and a detailed list of things to consider prior to embarking on HRM analytical experimentation. More specifically, the technical note will cover:
- important features required for HRM compatible instrumentation
- key features for HRM compatible software
-experimental design considerations for successful HRM analysis including: HRM-compatible saturating dyes, primer design and amplicon length and PCR reaction optimization
At the tech note mentions, HRM is a low-cost, readily accessible technique that can be used to rapidly analyze multiple genetic variants. Careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. Following the attached guidelines will assist in the development of such assays.
:: Posted by American Biotechnologist on 08-02-2010
High-resolution melt (HRM) analysis is rapidly gaining in popularity as a cost-effective and faster alternative to traditional post-PCR genotyping methods such as single-stranded conformation polymorphism, denaturing high-performance liquid chromatography, and restriction fragment length polymorphism.
In this webinar you will gain an overview of the fundamentals of HRM and learn techniques for success through appropriate experimental design, assay optimization, and data analysis. You will also learn about specific applications from scientists using HRM technology for basic microbiological genotyping research of pathogens as well as in clinical studies, detecting receptor gene mutants linked to cancer and identifying epigenetic differences in double-stranded DNA.
The webinar will take place Wednesday August 11 at 1pm EST.
* Kim De Leener, Ph.D., Center for Medical Genetics, University of Ghent, Belgium
* Jonas Winchell, Ph.D., Chief, Response and Surveillance Laboratory, International Emerging Infections Program, Centers for Disease Control and Prevention
* Adam McCoy, Ph.D., Senior Scientist, Gene Expression Division, Life Science Group, Bio-Rad Laboratories