Rare Target Detection using Digital PCR

Digital PCR is ideally suited to detect and quantify rare targets

Combinati’s emulsion-free Digital PCR (dPCR) enables accurate and precise quantitation of nucleic acids without relying on standard curves. By splitting a bulk PCR reaction into thousands of micro-reactions, individual target molecules can be counted accurately. This is particularly advantageous for applications searching for low concentration targets – or when you are looking for a “needle in a haystack”.
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Accurate measurement of nucleic acid is impossible when only a small portion of the original sample is analyzed. For many molecular diagnostic applications such as detection of circulating tumor DNA in liquid biopsy, both accuracy and precision are required. The Absolute Q is the only platform that can analyze more than 95% of each loaded sample by utilizing the proprietary Microfluidic Array Partitioning (MAP) technology. Read more about how the Absolute Q dPCR platform is being used for rare target detection.

Rare target quantification in liquid biopsy

Applications for digital PCR in oncology are myriad, from biomarker discovery to early detection, liquid biopsy research as well as monitoring recurrence and response to treatment.

Specifically, identification and tracking of cancer-causing mutations via liquid biopsy can be used for early detection, measuring therapeutic response, quantifying residual tumor burden, and monitoring emerging resistance to targeted therapies. Here, we demonstrate compatibility with Applied Biosystems™ TaqMan™ Liquid Biopsy assays, performing robust detection of 5 different hotspot mutations down to 0.1% allelic frequency. See the TaqMan protocol here.

Precision cancer monitoring

For applications in translational cancer research, the Absolute Q digital PCR platform brings 4-color multiplexing capabilities, improved sensitivity, and a one-step, walkaway workflow to your research. This publication (Scientific Reports), demonstrates the Absolute Q’s capability to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards.
Additionally, this publication highlights how the Absolute Q was used to validate the complete molecular remission of a patient diagnosed with juvenile myelomonocytic leukemia (JMML) by tracking a tumor specific (CML) fusion gene (BCR-ABL1) with a custom assay. The assay which was validated down to 0.01% mutant allele frequency was used to precisely track the quantity of the cancer mutation over time.

Sensitive and precise detection of treatment-relevant cancer mutations with an existing 2-enzyme PCR chemistry

Differentiation of single nucleotide polymorphisms (SNPs) or single nucleotide variants (SNVs) using PCR can be challenging due to a myriad of factors including sequence specificity and the possibility of primer-dimer formation. In this application note, IDT’s rhAmp technology was used to design a point mutation detection assay for the hotspot cancer mutation EGFR p.T790M – detecting both the wild-type and mutation allele in a single digital PCR reaction.

Tyrosine kinase inhibitors are a cornerstone of precision medicine and are a standard of care for those harboring oncogenic mutations. Unfortunately, drug-resistant mutations are a common result of this treatment. Sensitive and precise detection of these variants are critical to make well-informed treatment decisions. Read more about how theAbsolute Q was used to enable digital PCR quantification of this challenging genomic region quantifying EGFR p.T790M mutations down to 5 copies per reaction – all without the need for a standard curve.
 

Luminex & Absolute Q

Luminex uses the Combinati Absolute Q platform to identify underlying mechanisms of drug resistance in metastatic breast cancer with Luminex multiplex PCR chemistry.

Highlights of this webinar include:

  • Introduction to Microfluidic Array Partitioning (MAP) technology for digital PCR
  • Demonstration of a multiplex assay to identify and quantify 11 single nucleotide polymorphisms (SNPs) within the ESR1 gene in a single sample
  • Utility of using the Absolute Q, a 4-color digital PCR platform with a walkaway workflow, in translational oncology research

Ready to learn more?

Find out more about harnessing the
power of Digital PCR for your research.