Posts Tagged ‘qPCR’

What can be more accurate than real time PCR?

 :: 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

For more information read “Third Generation PCR: A Novel Way to Accurately Measure Copy Number Variation in the Human Genome.”

More Quantitative Than qPCR

 :: 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.

Check out Digital PCR: Improving Nucleic Acid Quantification.

Great video explanation of droplet digital PCR

 :: 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.

Related posts:
Introducing the 3rd Generation of PCR
Video Introduction to Droplet Digital PCR
An Advanced Look at Droplet Digital PCR

Introduction to High Resolution Melt Analysis

 :: 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!

More on Bio-Rad’s QX100 Droplet Digital PCR System

 :: Posted by American Biotechnologist on 02-13-2012

The QX100 Droplet Digital PCR system from Bio-Rad Laboratories provides an absolute measure of target DNA molecules with unrivaled precision and accuracy. The QX100 system partitions each sample into 20,000 individual nanoliter-sized droplets. PCR-positive and PCR-negative droplets from every sample are then counted to provide absolute target quantification in digital form. The QX100 system provides a revolutionary approach to target DNA quantification.

The QX100™ ddPCR™ system lets you:

  • Detect rare target sequences with unmatched sensitivity for cancer and viral research
  • Measure small differences in target copy number variations (down to 1.2x differences)
  • Determine absolute gene expression levels without a standard curve or the ΔΔCq method

Click here to download the latest product information, or here to see performance data, copy number variation data, gene expression data and rare mutation detection data.

Related posts:
Introducing the 3rd Generation of PCR
Video Introduction to Droplet Digital PCR
An Advanced Look at Droplet Digital PCR