PCR Methods Finetuned by Pandemic Demands

A backbone among lab techniques, PCR is a reliable method of amplifying target DNA. Technological advances mixed with human creativity have produced many types of PCR, each with its benefits and challenges. This article will review some widely used PCR methods amidst a pandemic, and give some examples of new PCR tools.

Conventional PCR

This endpoint assay is relatively inexpensive, using a straightforward protocol and equipment present in virtually every research center around the world. Conventional PCR is useful when you just need to test for the presence or absence of a DNA sequence (detected by subsequent gel electrophoresis), for example in genotyping or colony screening. Because it is an endpoint assay, it lacks the quantitative information that can be gleaned from qPCR.

Quantitative/real-time PCR

Quantitative PCR (qPCR), also known as real-time PCR, has seen remarkable growth over the last 1–2 years. The “real-time” moniker refers to the distinction between conventional PCR and qPCR. The reaction progress of qPCR can be monitored in real time as indicated by fluorescent probes (e.g. TaqMan probes) or fluorescent intercalating dye (e.g. SYBR Green). A real-time thermal cycler is equipped with a light source and camera to record the fluorescent signal per PCR cycle as the reactions proceed. A qPCR reaction allows you to quantify the amount of starting material (commonly used to quantify gene expression) and is also relied upon for its sensitivity and dependability.

Sometimes reverse transcription (RT) is used prior to qPCR to translate the RNA of a retrovirus into cDNA for starting material for qPCR, a process is referred to as RT-qPCR. “The COVID-19 pandemic has highlighted the utility of PCR methods, namely RT-qPCR, in molecular diagnostic tests for virus detection,” says Steven Chiu, product marketing manager for DNA amplification at New England Biolabs^® (NEB). “RT-qPCR in this application has seen increased adoption, whether in surveillance or individual testing protocols.”

PCR Biosystems also reports that interest in qPCR rose during the COVID-19 pandemic. “We find there is a huge demand for quantitative PCR kits and reagents, particularly probe-based quantitative real-time PCR, as that is the method of choice for clinical confirmation of COVID-19 infection, recommended both by the WHO and the CDC,” says Constantine Garagounis, marketing specialist at PCR Biosystems. They also launched the qPCRBIO Probe 1-Step Virus Detect kit specifically for COVID RNA detection, both for mass testing and for COVID research applications. PCR Biosystems’ 1-step qPCR kits allow you to add RNA directly without first converting to cDNA.

Digital droplet PCR

Interest in digital droplet PCR (ddPCR) is quickly growing. It relies on the principle of partitioning a DNA sample into compartments such that each compartment contains zero or very few copies of a sequence. The binary signal of presence or absence of a sequence, along with occasional multiples in a compartment, can be analyzed using the Poisson distribution, which allows you to estimate the amount of starting material in the original DNA sample. Digital PCR can rival qPCR for reliability and sensitivity, but is less prevalent in part because it requires the purchase of new equipment.

Multiplex PCR

In multiplex PCR, multiple targets are run in the same PCR reaction (conventional or quantitative), using more than one probe simultaneously. This is advantageous in that it saves time and reagents, but it takes some advance planning. Primer design is especially important to minimize nonspecific binding and cross reactivity; also, it’s important to use primers with melting temperatures within a few degrees. Multiplex PCR is used in many applications to increase throughput; forensic scientists also use multiplex qPCR to detect multiple genetic markers in human identification.

PCR buffers and enzymes optimized for multiplexing can be very helpful. For example, Promega’s ProNex^® DNA QC Assay uses multiplexed, probe-based qPCR to evaluate the quality and quantity of genomic DNA extracted from potentially degraded DNA sources, such as formalin-fixed, paraffin-embedded (FFPE) samples. It is particularly valuable for expensive sequencing applications that are sensitive to sample quantity, quality, and PCR inhibition. “The ProNex^® DNA QC Assay is designed for evaluating sample PCR amplifiability and quantity, key factors for downstream success with next-generation or massive parallel sequencing [MPS],” says Oberley. “It can save a lot of time and money to do some sample evaluation up front, before proceeding into expensive downstream assays such as MPS.”

Hot-start PCR

Hot-start PCR uses a modified DNA polymerase that remains inactivated at room temperature, usually by association with an antibody, aptamer, or chemical modifier. This prevents nonspecific amplification, mis-priming, and primer-dimer formation prior to the beginning of the reaction, during set up. The PCR reaction begins upon heating (hence, ‘hot-start’), which releases the modifier from the enzyme and allows it to be active. Hot-start PCR is particularly helpful in setting up high-throughput work when reactions may need to remain at room temperature for longer.

NEB’s new enzyme, the Q5U^® Hot Start High-Fidelity DNA Polymerase, is a modified version of their Q5^® High Fidelity DNA Polymerase. The new Q5U enzyme contains a mutation that allows it to read and amplify templates with uracil and inosine bases. “It is an ideal product for amplifying bisulfite-converted, deaminated, or damaged DNA, preventing carryover contamination in PCR when used with dUTP and UDG, and in USER cloning methods,” says Chiu.

Long or GC-rich DNA

Amplifying challenging DNA, such as long or GC-rich sequences, is easier with DNA polymerases that have high fidelity and processivity. These proof-reading enzymes bind the template more tightly during extension and have a higher amplification efficiency. To meet demand for high-fidelity enzymes, PCR Biosystems recently released the PCRBIO HS VeriFi^TM Polymerase, a hot-start, proof-reading polymerase for conventional PCR. “It’s particularly reliable with long (>17 kb) or tough, high-GC content templates, it allows unbiased multiplexing and has a high processivity, which allows for shorter amplification times and hence faster PCR completion,” says Garagounis.

Pandemic advances

Since the advent of COVID, technologies have adapted to make PCR more responsive to solving pandemic-related problems. For example, Promega shortened their qPCR workflow to speed diagnostic results and adapt to a global shortage of nucleic acid extraction reagents. “We rapidly developed a method to perform direct amplification of viral samples that did not require RNA extraction,” says Knox. “The XpressAmp™ Direct Amplification Reagents were introduced in August 2020 to help global laboratories respond to the pandemic.”

Direct amplification enables faster diagnostic COVID testing. “In situations where quick turnaround is critical, such as COVID testing, direct amplification of nasal swab or saliva samples using XpressAmp, and a multiplex RT-qPCR assay using our GoTaq^® Probe 1-Step RT-qPCR System, can tremendously increase sample throughput,” says Chris Oberley, supervisor, technical services scientist at Promega.

A new product from NEB, the Luna^® Probe One-Step RT-qPCR 4X Mix with UDG, is designed to increase the sensitivity of RT-qPCR. “It includes both dUTP and thermolabile UDG to prevent carryover contamination, where the unintended product of a previous amplification can serve as the substrate of a subsequent reaction,” says Chiu. “Minimizing DNA contamination is important given the sensitivity of RT-qPCR, and this product offers that capability embedded in the master mix.”

The recent innovations in PCR methods and tools are likely to persist as scientists continue to fight the pandemic. The progressive rise and fall of myriad COVID variants continues to rely on RT-qPCR for genetic testing, monitoring variant levels to gauge the threats they pose around the world.