Guide to Selecting a PCR Method

PCR has transformed biological research, and its widespread use prompted the development of varieties of PCR as scientists adapted the method in pursuit of their own research goals. “The simplicity and flexibility of PCR assays make them useful for a wide variety of applications,” says Kamini Varma, Vice President of Research and Development for Genetic Testing Solutions at Thermo Fisher Scientific. “For example, in cancer research, investigators might want to know how expression levels of a set of genes change in response to cell development, stage of disease progression, or response to therapeutic agents.” As in clinical research, PCR is vital in all applications. “Conventional PCR, qPCR, and multiplex PCR continue to be ubiquitous in everyday research,” adds Steven Chiu, Product Marketing Manager for DNA Amplification at NEB. “In all methods, researchers are seeking products that offer simplicity with performance.” This article reviews the most common PCR methods, as well as their benefits and applications.

Conventional/endpoint PCR

Conventional PCR provides an endpoint result, and is relatively inexpensive and straightforward to conduct with a thermal cycler, followed by gel electrophoresis for sequence detection. Conventional PCR is best when testing for the presence or absence of a DNA sequence in a sample (e.g., genotyping, colony screening). “Endpoint PCR [is] useful when you want confirmation that a target exists or does not exist in your sample,” says Adam Waite, Product Manager for PCR Technologies at Promega. “You can get a yes/no answer in minutes with some endpoint setups.”

Quantitative PCR

Quantitative PCR (qPCR), also known as real-time PCR, differs from conventional PCR in two important ways: qPCR allows you to quantify the amount of starting material, and its reaction can be monitored in real time using either fluorescent probes (e.g., TaqMan probes) or a fluorescent intercalating dye (e.g., SYBR Green). In qPCR, a real-time thermal cycler records the fluorescent signals during each PCR cycle. “Because a signal is generated only when the fluorescent probe is degraded, TaqMan-based qPCR assays provide very high sequence specificity,” says Stephen Jackson, Associate Director for Product Applications in Genetic Sciences at Thermo Fisher Scientific. “This makes it the method of choice for most translational research, clinical research, and other investigations where high sensitivity and specificity are needed.”

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 that is referred to as RT-qPCR. The sensitivity and dependability of RT-qPCR quickly made it the gold-standard in coronavirus testing. “In recent years, quantitative PCR has made a strong push to replace endpoint as the gold standard in PCR technology,” says Waite. “The ability to measure sample yields in real time and multiplex targets has made it a key strategy in targeted disease tracking and control.”

In many research areas today, qPCR is used to measure gene expression levels and to determine genotypes. “Genotypes are determined using TaqMan-based qPCR assays in inherited diseases, pharmacogenomics, crop sciences, environmental monitoring, understanding normal phenotypic variation, and detecting epigenetic changes in the genome,” says Varma.

Multiplex PCR

Multiplex PCR uses multiple sets of primers, allowing researchers to interrogate multiple targets simultaneously in the same PCR reaction (either conventional or quantitative PCR). “Multiplex PCR reduces the amount of sample needed by detecting multiple targets in a single reaction, increasing throughput and saving time,” says Chiu. “It is a cost-effective way to gain more information from a single test run compared to running several independent PCR experiments.”

Multiplexing can be advantageous because it saves time and reagents, but requires advance planning to minimize nonspecific binding and cross reactivity of primers, and to coordinate the melting temperatures of the different primers. “TaqMan-based qPCR assays can be used for multiplex target detection since different fluorescent dyes can be coupled to different probes,” says Jackson. “A single PCR reaction can therefore generate abundance measurements for different targets based on the choice of dyes and the capabilities of the instrument.” Multiplexing can be applied to nearly any PCR assay to increase throughput and reduce the amount of sample required. For example, genotyping with multiplex qPCR can be used to identify the strains of viruses or bacteria present in a single sample.

Digital & digital droplet PCR

Digital PCR or digital droplet PCR (dPCR or ddPCR) relies on the principle of partitioning a DNA sample into many compartments, such that each compartment is most likely to contain one sequence (most compartments will contain one, while some will contain zero or two, and rarely three). Because the sequences are distributed among compartments in a binary manner, the presence or absence of DNA in a compartment can be analyzed using the Poisson distribution. This analysis provides an estimate of the amount of starting material in the original DNA sample.

Digital PCR is not only highly sensitive and reliable, but it’s also less affected by PCR inhibitors, and by the presence of closely related sequences. “Digital PCR is a great tool when you need to work around inhibition, are seeking out rare targets or variants, and want very precise measurements beyond what qPCR can offer,” explains Waite.

Although less common than other PCR methods, dPCR is increasingly used in applications that rely on rare or complex samples. “Clinical researchers might want to detect a rare cancerous allele or monitor residual disease using liquid biopsies,” says Varma. “Digital PCR approaches are often used for these analyses due to their precision and sensitivity in mixed genotypic samples.”

Advances in digital microfluidics technology may make dPCR accessible to more researchers, and could increase the ease and throughput of all types of PCR. “The types of applications that are enabled by PCR are really only limited by the researcher’s imagination,” says Varma.