:: 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.
HRM technical note
Sean Taylor is a Field Applications Scientist at Bio-Rad Laboratories and the primary author of this tech note. Click here for other technical resources from Sean including his video entitled “A Practical Approach to MIQE for the Bench Scientist,” and the article A Simple Solution to Chromatography for High-Purity Protein Preparations: The Modular Approach
:: 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.
For more information and to register, see the official announcement on Genetic Engineering & Biotechnology News
* 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
:: Posted by American Biotechnologist on 07-12-2010
Double stranded DNA (dsDNA) melts at a temperature that is determined by its length (i.e. the number of base pairs) and nucleotide sequence composition. In general, longer strands of DNA and strands consisting of more “G”s and “C”s melt at higher temperatures than shorter strands and strands consisting of “A”s and “T”s. As such, every dsDNA molecule has a unique melting fingerprint which can be used to differentiate one strand of DNA from another. Over the past 10-15 years great advances have been made in utilizing this feature in DNA-based research. One example is the discrimination of Single Nucleotide Polymorphisms (SNP) which occurs when a single nucleotide differs between members of a species or paired chromosomes in an individual. SNPs play an important role in the development of disease and can serve as significant biomarkers under various conditions. Because SNPs involve the substitution of a single nucleotide within a DNA fragment, the resulting DNA fragment has a different melting temperature then its native form. When DNA melting analysis is coupled with a technique such as real-time PCR the result is a powerful tool for detecting SNPs from small amounts of starting material.
Precision Melt Analysis software imports and analyzes data files generated from Bio-Rad Laboratories’ CFX96 or CFX384 real-time PCR detection system to genotype samples based on the thermal denaturation properties of double-stranded DNA. The software can be used for a variety of genotyping applications, including scanning for new gene variants, screening DNA samples for SNPs, identifying insertions/deletions or other unknown mutations, and determining the percentage of methylated DNA in unknown samples. Use the default analysis settings to automatically normalize data and assign a genotype to each sample based on its melt characteristics — there is no need to include genotype controls to assign cluster labels.
Precision Melt Analysis software saves analysis time by assigning sample genotypes automatically based on cluster analysis, or manually using multiple data view options to tailor the software to the appropriate analysis. Use the normalized melt curves plot feature to generate a basic representation of the different clusters based on curve shifting (for homozygotes) and curve shape change (for heterozygotes). Difference curve plots of a sample fluorescence versus a selected control at each temperature transition provide a convenient visual aid to interpret the data.
Precision Melt Analysis software enables data comparison between multiple file runs by combining data into a single Melt Study. Develop a standard library of melt-curve runs to analyze an unlimited number of melt experiments without having to export data.
Precision Melt Analysis software makes it easy for you to:
* Streamline your data analysis using the customizable default analysis settings
* Utilize the multiple data view options to manually assign sample genotypes by tailoring the software to the appropriate analysis
* Examine results from a number of melt files, without having to export data, using the Melt Study module
* Analyze multiple experiments from a single plate using the Well Groups feature
* Publish your data in several formats by easily exporting data to Microsoft Excel or as an image
For more information, check out the Precision Melt Analysis Software Flier and the Precision Melt Analysis Software instruction manual or contact your sales rep at 1-800-4-BIO-RAD (1-800-424-6723) for details.
:: Posted by American Biotechnologist on 01-19-2010
High-Resolution Melting Analysis (HRMA) is rapidly becoming a very attractive tool to not only to screen patients for pathogenic variants but also for presequence screening, single nucleotide polymorphism (SNP) typing, methylation analysis, quantification (copy number variants), an alternative to gel-electrophoresis and clone characterization. The ease of use, simplicity, flexibility, low cost, nondestructive nature, superb sensitivity, and specificity of HRMA has brought the technique to the forefront of life science research.
I did some literature searching and have attached a recent review that covers the topic very well. The article was written prior to Bio-Rad’s launch of our own HRMA software module integrated into the CFX96 and CFX384 instruments so I have also attached a flyer on the topic which will show you the accessibility to HRMA through these instruments, associated software and SsoFast EvaGreen Supermix.
High Resolution Melt Analysis – Review Article 2009
Precision Melt Analysis Software Bulletin_5798A
Sean Taylor, pHD
Field Applications Specialist
:: Posted by American Biotechnologist on 12-29-2009
Genomics is rapidly expanding beyond merely identifying the genetic makeup of organisms to encompass studies of how variations in genotype impact physiological functions. See the article below which was published in the October 15th edition of Genetic Engineering and Biotechnology news to learn more about high-res met analysis as an alternative to probe-based genotyping.
High-Resolution Melt Analysis Applications