GEN Exclusives

More »

Assay Tutorials

More »
May 1, 2012 (Vol. 32, No. 9)

Digital PCR: Improving Nucleic Acid Quantification

Precision, Accuracy, and Sensitivity Are Among the Benefits Reported by Researchers

  • Copy Number Variation Determination

    Click Image To Enlarge +
    Figure 1. Copy number variation analysis using droplet digital PCR: Determination of MRGPRX1 copies per genome of seven different HapMap samples (Coriell). MRGPRX1 copy number values were normalized to the RPP30 reference gene using standard hydrolysis probe based assays. Error bars represent 95% confidence intervals.

    CNVs include deletions, insertions, duplications, and complex amplifications. The accelerated discovery of CNVs has increased the need for high-throughput, low-cost options for validation and follow-up studies.

    Traditional high-throughput technologies such as comparative genomic hybridization and single nucleotide polymorphism (SNP) arrays lack resolution and the ability to discriminate copy number differences of less than 50%. Quantitative PCR experiments require dozens of technical replicates for each target and reference gene in order to statistically discriminate between higher order variations.

    The high resolution made possible by sample partitioning and digital analysis in ddPCR provides the precision necessary to resolve higher-order copy number states, making the QX100 ddPCR system the ideal solution for CNV validation while delivering the necessary throughput and cost efficiency.

    Employing ddPCR, researchers have been able to completely resolve CNVs, distinguish less than 50% differences in gene copy, and accurately count genes that differ by only one nucleotide. For the HapMap MRGPRX1 sample, copy number states from 1 up to 6 were completely resolved (Figure 1).

  • Rare Event Detection

    Click Image To Enlarge +
    Figure 2. Droplet digital PCR detection of BRAF V600E rare mutant allele. Serial dilutions of mutant cell line ATCC HT29 in the presence of wild-type DNA using standard hydrolysis probe assays. High level of partitioning permits significant enrichment effects allowing detection of rare mutants at levels 1,000 times lower than with qPCR.

    Rare events include single nucleotide mutation, alteration of copy number, and deletion or insertion of nucleotides. These genetic variant molecules are difficult to detect due to their dilution by normal cells from either tissues or bodily fluids such as blood. Early detection of rare events can make all the difference in the outcome of cancer patients, as well as lead to more sensitive and less invasive diagnostics.

    During qPCR, rare and normal variants of DNA molecules are amplified at an equivalent rate. If the normal gene is initially present in 100-fold abundance over the mutated gene, at the end of the reaction, 1% of the total amplification product will be the mutated gene amplicon. Its detection will be extremely difficult as the total signal generated will be 1% of that generated by the wild-type gene amplicon.

    For rare event detection, sample partitioning increases sensitivity by distributing both mutant and normal genes into a large number of isolated reaction compartments. In each one of these isolated droplets, the rare mutant molecules are now at a more favorable ratio compared to the wild-type, and thus become detectable within that individual droplet. The QX100 can detect amplifications even in highly heterogeneous matrices where only a fraction of the cells are affected. This precision enables the detection of somatic copy number alteration—the hallmark of many cancers.

    The detection of point mutations requires a high degree of sensitivity. Droplet Digital PCR allows the detection of 0.001% mutation fractions, as demonstrated in a duplex PCR reaction using TaqMan probes targeting the BRAF 600E mutation (Figure 2). This detection limit is more than 1,000 times lower than qPCR.

  • Looking Ahead with the McCarroll Lab

    When McCarroll’s lab acquired the QX100 ddPCR system, they immediately began using the instrument to study structurally complex regions of the genome—regions that have historically been influenced by many different structural mutations. In such regions, ddPCR has allowed them to quantitate copy numbers in large populations, which is important in both population genetic analysis and disease analysis. Now they can analyze these regions in the same high-quality, high-precision way that is the standard for genetic analysis of simpler kinds of variation such as SNPs.



Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...

Unable to get Jobs Listings.

More »

GEN Poll

More » Poll Results »

Biosimilars

Compared to the original biologics, do you think biosimilars run the risks of being less effective and causing more side effects?