PCR and qPCR
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DNA mutations and copy number alterations identified in FFPE tumor samples suggest potential therapeutic targets

Cani AK, Hovelson DH, et al. (2015) Next-gen sequencing exposes frequent MED12 mutations and actionable therapeutic targets in phyllodes tumors. Mol Cancer Res, 13(4):613–619.

Phyllodes tumors are rare breast tumors that involve both stromal (connective) and glandular tissue. They are classified as benign (~65% of cases), borderline (~25% of cases), or malignant (~10% of cases) based on histopathology, but this classification often does not predict disease progression. Phyllodes tumors, regardless of classification, usually grow quickly and can recur. Malignant tumors can also metastasize (to the lung, bone, heart, and liver), which decreases survival rates. Current treatment guidelines for all patients with phyllodes tumors require wide surgical resection margins and continued follow-up care, but efficacious treatment options for phyllodes tumors that progress to metastatic disease are lacking. These tumors do not respond to hormone therapy and do not respond well to radiation therapy or the chemotherapy drugs normally used for breast cancer.

In the featured article, Cani et al. used genomic sequencing and mutational profiling to characterize phyllodes tumors, which will help refine diagnoses and discover potential druggable targets for malignant disease. More specifically, they used targeted next generation sequencing to analyze 126 genes for somatic molecular alterations in 15 formalin-fixed, paraffin-embedded (FFPE) phyllodes tumors (5 of each classification). Sanger sequencing was used to validate somatic variants [for example, mutations in the mediator complex subunit 12 (MED12), p53, retinoblastoma, and neurofibromin 1 genes], and PrimeTime® qPCR Assays that included ZENTM Double-Quenched Probes were used to validate copy number alterations (for example, copies of the IGF1R and EGFR genes).

Product focus

For their copy-number experiments, Cani et al. used the IDT PrimerQuest® Tool to design PrimeTime® qPCR Assays that included ZEN™ Double-Quenched Probes. Here are some links and information to help you use these tools and reagents in your qPCR experiments:

  • The PrimerQuest Tool is a powerful, customizable design engine for creating primers and probes for PCR, qPCR, or sequencing. It enables you to spend less time designing and analyzing primers and probes, and more time on experiments.
    Learn the basics of using this free, online software here.
  • PrimeTime qPCR Assays are available with various combinations of dyes (for example, FAM, HEX™, TET™, and Cy® 5 dyes) with quenchers (for example, ZEN™/Iowa Black® FQ, TAMRA, and Iowa Black RQ quenchers). Double-quenched probes that include ZEN or TAO Internal Quenchers improve assay sensitivity and precision by reducing background fluorescence and increasing fluorescent signals.

Additional reading

  • Discriminating highly similar transcripts using rhPCR—Read about the uses of RNase H-dependent PCR, a technology developed by IDT to increase PCR specificity and eliminate unwanted interactions between primer sets (e.g., primer-dimers, etc.). An example is provided in which it is used to distinguishing highly similar alternatively spliced sequences. This technology can also be useful in genotyping applications, in highly multiplexed qPCR assays, library construction for Next Generation DNA Sequencing, and for rare allele detection, where the added specificity provided by the blocked-cleavable primers enables detection of a rare mutant allele in a background of large amounts of wild type DNA.

  • Developing onsite genotyping of Antarctic penguins—This research profile highlights the increasing need for mobile qPCR testing using an example from conservation biology. Read how PrimeTime® Custom qPCR Assays have been designed to distinguish genetic variants of Adélie penguins.

  • Increase sensitivity and precision in your qPCR experiments—Learn how you can use double-quenched probes to decrease background, and increase sensitivity and precision in your qPCR experiments.

  • Double-quenched probes increase sensitivity of qPCR assay detecting viral load—Citation summary: Find out how the use of a ZEN™ Double-Quenched Probe results in a marked decrease in background fluorescence compared to an identical TaqMan® probe containing only a single quencher. The data suggest that such double-quenched probes may be a better approach for other qPCR probe-based assays.

Author: Maureen Young is a scientific writer at IDT.

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