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That is why we are excited to tell you about Biomed Central’s (BMC) topical series on Quantitative Real Time PCR normalization and optimization, Edited by Joshua S. Yuan. According to BMC, topical series bring together manuscripts published in BMC Research Notes that are associated with individual topics. Through the topical series, BMC Research Notes aims to highlight exciting topical areas of research and provide a home for concise articles that raise awareness and encourage discussion of the subjects covered.
:: Posted by American Biotechnologist on 09-08-2010
Food scientists have traditionally relied upon culture-based methods for food safety testing. Due to the proven reliability of these methods and their long history of use in food safety labs, inspectors have been reluctant to adopt more “modern” technologies such as real-time PCR into their food testing repertoire. The food industry is cognizant of the fact that standard microbiological techniques offer much slower time-to-results than can be obtained by real-time PCR, (culture-based Salmonella detection takes at least 4 d to complete whereas real-time PCR can detect the presence of bugs within an hour or two), and are always looking for ways to speed up the safety inspection process. Nonetheless, before safety inspectors jump on the real-time PCR bandwagon, they will require that the wrinkles inherent in some real-time PCR analysis kits (such as the detection of false positives) be ironed out before the they are used on a routine basis. Well, the time for switching techniques may be near.
In a study published in the Journal of Food Science Investigators from the Division of Food Systems and Bioengineering at the University of Missouri have employed the use of Ethidium bromide monoazide (EMA) to bind to DNA of dead cells and prevent its amplification by PCR thereby eliminating detection of dead cells when testing Salmonella levels in chicken and eggs. While PCR cannot differentiate between DNA from live and dead cells (thus posing a problem if used as a technique for quantifying live cells only in a mixture of live and dead cells), EMA staining step prior to PCR allows for the effective inhibition of false positive results from DNA contamination by dead cells.
Modifications to real-time PCR techniques such as the one described above will certainly enhance the uptake of PCR in food safety testing cutting down on manufacturers time to market thereby increasing their profits.
This news comes just in time for Sonya “black widow” Thomas the new buffalo wing eating champion who weighing in at just 105 pounds, beat out professional eating champion Joey Chestnut (231 pounds) by consuming 181 chicken wings in 12 minutes in the 9th annual National Buffalo wing Festival in Buffalo, NY. Faster food safety testing of chicken wings should translate into more wings available for Sonya to consume in the next gluttonous competition!
:: 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
:: 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
:: Posted by American Biotechnologist on 04-28-2010
If your biotechnology research requires you to be engaged in high-throughput Quantitative PCR analysis than you’ve probably made good use out of automated systems such as liquid handling robots and plate stackers. And what a relief they are. Imagine what your poor thumb would feel like if you had to pipette out tens of thousands of reactions. Or think about how much time you would have wasted standing around waiting for a run to end in order to load the next plate into the QPCR machine.
While automated handing systems are fantastic, they also bring with them the complicated question of sample stability. When samples are loaded into the automated system, they may be stored there at room temperature for hours. As such, it is important for the reaction mix to be stable at room temperature for the duration of the waiting period.
In this Bio-Rad Technical note, you will learn how Bio-Rad’s SsoFast EvaGreen supermix meets the challenge of providing reaction mix stability and can be used to acheive consistent results over a large dynamic range of templates with automated runs lastng up to 48 hours.