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:: Posted by American Biotechnologist on 08-27-2012
Real-Time PCR can be tough. It requires careful planning and much optimization. When it works, you feel great. When it fails…fill in the blank. There are times in our research career when we feel like giving up. Nothing we do seems to yield positive results. Then…along comes a kit. Sure, at first we are wary about using a kit. After all, weren’t we put on this planet to troubleshoot and suffer through tortuous experiments? Alas, many of us quickly overcome that guilt and put our trust and faith in the hands of others. But how do we know that commercially available kits are indeed trustworthy? Perhaps they too will yield erroneous results and lead us down the dark path of non-publishable gobbledygook data. So what do we do? We troubleshoot. We troubleshoot the commercially available kit. The kit that we purchased to avoid troubleshooting! Curses! It’s one thing to troubleshoot my own experimental protocol, but to troubleshoot someone else’s? And one that I paid for nonetheless?
avoided secondary structures in primer annealing sites
avoided SNPs in target regions
maximized fraction of transcript isoforms being detected
designed intron-spanning assays whenever possible
used latest release of genome builds and annotation databases
Moreover, the kits have undergone wet-lab validation at the hands of PCR professionals so you don’t have to waste precious time validating and troubleshooting an assay that you spent money on acquiring.
:: Posted by American Biotechnologist on 08-01-2012
“People are different from each other in ways that are fascinating and medically important, and to understand the ways in which variations in our genome give rise to those differences presents an important and interesting set of questions,” says Dr Steve McCarroll, faculty member and principal investigator with the Department of Genetics at Harvard Medical School and the Broad Institute of Harvard and MIT. The McCarroll laboratory studies the biological effects of human genome polymorphism, seeking to define how genome variation influences gene expression and risk of disease. Their work requires them to accurately measure the copy number of a genomic sequence with integer precision in hundreds of people. While real-time PCR and CGH (comparative genomic hybridization) arrays are useful for measuring simple deletions and duplications, these techniques simply weren’t sufficient for obtaining single-molecule resolution of target sequences with extreme precision and accuracy. That’s where Droplet Digital PCR entered the story.
Learn how Dr. McCaroll’s lab, along with other labs across the globe utilize ddPCR in applications such as:
Absolute quantification of HIV proviral DNA and human genomic DNA in the same reaction
Absolute quantification of HIV viral RNA using two-step reverse transcription-ddPCR
Rare event detection of BRAF V600E, EGFR L858R and T790M, JAK-2, and cKIT (cancer mutation analysis and drug resistance mutation)
Absolute quantification of miRNA from circulating nucleic acids for biomarker identification
High-resolution copy number variation (CNV) analysis of human and mouse DNA’s
High-resolution CNV analysis of the MET gene, association study for lung cancer
Absolute quantification of next generation sequencing library
Rare event detection, mitochondrial mutagenesis
Absolute quantification of the BCR-ABL (Philadelphia chromosome) fusion gene and transcript, association with leukemia
Absolute quantification and sex determination of fetal DNA in circulating nucleic acid purified from maternal blood
:: Posted by American Biotechnologist on 05-09-2012
The introduction of real time PCR (qPCR) revolutionized the world of molecular biology and provided scientists with a tool for obtaining truly quantitative nucleic acid data. While qPCR remains one of the most robust tools available on the bench, its resolution power is limited to changes in DNA concentration that are higher than 50%. This limitation is mainly due to the compounding of errors derived from each step in the quantification process. That’s where Droplet Digital PCR enters the picture.
Droplet Digital PCR alleviates the compounding error effect by partitioning samples into 20,000 droplets such that each copy of DNA is encapsulated in its own bubble. Subsequent amplification reactions are then carried out individually which leads to reduce background noise and therefore a more sensitive measurement of low concentrations of nucleic acid that may not have been detectable using qPCR.
Bio-Rad Laboratories‘ Frank Bizouarn recently published an outstanding article in Genetic Engineering and Biotechnology News detailing the benefits of ddPCR including a real life example. The article covers how ddPCR works and its use in gene-expression analysis, copy number variation determination and rare event detection.
:: Posted by American Biotechnologist on 03-21-2012
The QX100 Droplet Digital PCR system from Bio-Rad Laboratories is the third generation of PCR technology. The system provides an absolute measure of target DNA molecules with unrivaled performance in precision, accuracy, and sensitivity for quantitative PCR applications. Watch the video below to learn more about this powerful technology.
:: Posted by American Biotechnologist on 03-05-2012
In our last post we told you about how Bio-Rad Laboratories very own Sean Taylor and Francisco Bizouarn were crowned the kings of MIQE. Today we’d like to bring you another classic from his majesty Frank. In the slideshow below, you will learn the basics of High Resolution Melt Analysis (HRM), applications, important considerations, assay design and optimization and analysis software. Enjoy. And all hail the king!