Differences between qPCR and dPCR 20th September 2022 – Tags: dPCR, qPCR
In this article we will discuss the differences between qPCR and dPCR for the sake of clarity. DNA amplification by PCR is now a fundamental laboratory technique, and innovations continue to expand its use in research laboratories and clinics. Quantitative PCR (qPCR) allows relative quantification of target DNA and is a reliable and well-established method for verifying the presence or absence of certain sequences (for example, most PCR-based tests for coronavirus use qPCR). Another form of PCR, digital PCR (dPCR) or digital droplet PCR (ddPCR), uses similar chemistry to detect DNA sequences.
What is Real-Time PCR (qPCR)?
Real-time or quantitative PCR (qPCR) uses fluorescent reporter molecules to quantify the amplified products. As in conventional PCR, a DNA, cDNA or RNA template is amplified, but at each cycle the fluorescent signals are monitored for relative or absolute quantification. This technique is useful for many research areas, such as gene expression analysis, genotyping, microRNA analysis, genetic variation analysis and protein analysis.
What is Digital PCR (dPCR)?
Digital PCR (dPCR) is becoming the complementary technology to real-time PCR for the accurate quantification of nucleic acids, as it can offer high sensitivity and robust performance against inhibitors. Digital PCR brings together a wide range of research applications, offering unmatched reliability for quantification of rare targets, copy number variations (CNVs), gene expression and more.
- Quantification of genes with low abundance or small differences
- Absolute quantification
- Gene copy number variation (CNV)
- Determination of vector copy number (CNV) and detection of replication competent retroviruses (RCR) or lentiviruses (LCL) in the context of adoptive cell therapy
- Detection of rare events, such as cancer gene mutations in tissues or liquid biopsies.
Differences between qPCR and dPCR
qPCR is a frequently used method for screening large populations of cells and can provide rapid genotyping by HRM. dPCR is recommended for analysing copy number variation as it can accurately detect copy variation with fewer replicates than qPCR.
Advantages qPCR
- Wide dynamic detection range
- Low experimental cost per sample
- High sample throughput
Advantages dPCR
- No dependence on standard curves and reference samples
- High tolerance to biological inhibitors and sample preparation
- Better performance for applications requiring higher sensitivity and accuracy
When there is a need to detect rare events
A particularly good application for dPCR is the detection of rare events in clinical trials. Biocompare spoke to Gary Lee, Senior Scientist at Sangamo, Inc. about the company’s adoption of dPCR in late 2011. Sangamo specialises in gene therapy and designs nucleases that bind DNA to zinc finger protein (ZFP), allowing for the customisation of gene expression. One of these therapies, currently in two Phase II clinical trials, could one day provide a functional cure for HIV, offering an alternative to conventional drug treatments.
RT-PCR or qPCR
Advances in biotechnology have led to the discovery of various ways to meet the demand for organ transplants over the years. In the past, a person who voluntarily donated an organ immediately after death to a patient in need could only trigger the transplant operation. Continued study and research by biologists led to the discovery of stem cells.
Stem cells are cells extracted from the DNA of an embryo, which will serve as a source for the regeneration of cells that will essentially be a clone of the organ from which the DNA was extracted. Although human rights activists are pushing for an end to these procedures, successful operations have demonstrated the effectiveness of stem cell advances in organ transplantation.
However, before stem cells can be fully appreciated and understood, it is necessary to become familiar with the different terminologies associated with this scientific discovery. Stem cell culture is about DNA and its coding. It is therefore crucial for students or anyone interested in this field to make the distinction between RT-PCR and qPCR.