PCR and qPCR
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Validating sequencing results of a repetitive element with digital droplet PCR

White TB, McCoy AM, et al. (2014) A droplet digital PCR detection method for rare L1 insertions in tumors. Mobile DNA, 5(1):30 DOI 10.1186/s13100-014-0030-4

Long interspersed element 1 (L1) is a human retrotransposon with over 500,000 copies per genome. Most L1 elements are nonfunctional, meaning they are immobile because of truncations and mutations. Although normal cells can inhibit L1 retrotransposition, ~100 L1 elements are potentially active in germline and somatic tissues. These retrotransposition-competent L1 elements are ~6 kb in length and encode 2 proteins essential for mobilization. L1 insertion, through a mechanism called target-primed reverse transcription, often results in target-site duplications.

Not surprisingly, L1 mobilization has resulted in de novo cases of human disease, for example, diabetes and hemophilia, and is associated with various cancers, including some breast, colon, lung, prostate, liver, renal, bladder, and ovarian cancers. L1-mediated gene expression or retrotransposition may drive tumorigenesis in some cases or may contribute to aberrant gene expression during tumor development.

High throughput sequencing technology has been instrumental in detecting L1 insertion events. However, validation of these de novo insertions can be problematic, due to tumor heterogeneity and the high copy number of L1 elements. In this study, White et al. describe how they designed primers and PrimeTime® qPCR Probes from IDT to maximize PCR amplification of the L1 of interest. They then used droplet digital PCR (ddPCR) to detect and quantify the targeted L1. In each reaction well of a ddPCR experiment, a 5′-nuclease PCR assay is partitioned into ~20,000 droplets as a water-in-oil emulsion. Ideally, each droplet contains 0 or 1 copy of the target DNA, so that the number of fluorescent droplets indicates the number of targets in the reaction. This paper shows how ddPCR allows detection of rare L1 insertion events (as low as an L1 event in 1 in 10,000 cells) and how ddPCR can be used to distinguish heterozygous loci from homozygous loci.

Product focus: Assays, probes, and tools for dPCR

PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies
  • Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime. For assistance with assay design, contact our scientific application specialists at applicationsupport@idtdna.com.


Double-Quenched Probes

ZEN™ and TAO™ Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.


Free tools for qPCR and PCR assay design

Explore IDT free, online tools for qPCR probe design and analysis. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm, and provide the very best qPCR assay designs.

Additional reading

Digital PCR (dPCR)—What Is It and Why Use It? If you are new to digital PCR, read this article to learn the fundamentals of how this method works.

Digital PCR—Simplifying Quantitative PCR. Learn more about why digital PCR makes gene expression quantification easier and more accurate. RainDance Technologies, a leader in this field, uses ZEN™ and LNA probes to increase their signal-to-noise ratio and rare allele detection.

Modification Highlight: ZEN™ Internal Quencher. This short article describes several unique properties of the ZEN™ Internal Quencher, including how it can improve the function of probes in 5′-nuclease qPCR assays.

Review other DECODED Online newsletter articles on qPCR advice and applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.


Author: Maureen Young, PhD, is a scientific writer at IDT.

© 2015, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

 


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