Choosing a DNA Amplification Method

There are many reasons to want to amplify DNA, and many ways to do so. Whether in a state-of-the-art GMP-certified lab, in a resource-constrained or time-sensitive point-of-care situation, or out in the field far from even basic infrastructure, researchers need to factor in parameters such as speed, cost, available equipment, ease of design, and ease of execution when deciding on the method to be used.

Biocompare’s readers are (presumably) all familiar with the polymerase chain reaction (PCR), yet it’s not the only way to amplify DNA. This article will look at the uses, pros, and cons of various DNA amplification methods—especially PCR and its iterations—including several popular isothermal protocols.

Why amplify?

Researchers will amplify DNA for two principal reasons. The first is to establish the presence, absence, or abundance of a genetic sequence. This might be in the context of diagnosing cancer or infectious disease, checking the accuracy of a CRISPR edit, or DNA fingerprinting (forensic science). “The aim is not to amplify the material, the aim is to detect what's in the material by means of amplification,” says Monika Seidel, Principal Investigator in Genomics, Diagnostics and Genomics, at Cytiva. RNA can also be amplified (after converting it to cDNA) for, for example, transcriptional analyses.

The other prevailing reason to amplify DNA is to make more of it for downstream purposes. These might include cloning and gene therapy, and amplifying plasmids for RNA vaccine manufacture. Next-generation sequencing (NGS) library preparation protocols—especially when dealing with single cell or cell-free DNA or RNA—generally call for one or more amplification steps in order to generate enough input material.

What is PCR?

PCR is the most common method for DNA amplification, notes Constantine Garagounis, Product Development and Marketing Specialist at PCR Biosystems. It is currently “still considered most reliable and versatile in terms of the number of application variants, its ability to offer accurate quantitative, real-time results, and its recognition by the scientific community.”

What distinguishes PCR from the other amplification methods discussed here is that it is an iterative process relying on a high temperature to denature the double-stranded DNA, followed by a lowering of the temperature to allow homologous primers to anneal. This is usually, but not always, followed by a slight rise in temperature to allow polymerase to extend the newly double-stranded DNA. And repeat.

In the most rudimentary form of PCR (as outlined in the paragraph above), the target sequence (the “product”) is typically detected by agarose gel, which reveals the size(s) of the amplified product(s). Given the makeup of the primers, size will reveal presence or absence of the gene, and sequence-specific probes can be used to assure greater specificity. The results are considered qualitative rather than quantitative. Product can be extracted from the gel for downstream applications.

Real-time PCR, in which increasing levels of amplified product can be seen as the reaction is progressing, affords relative quantitation—to query expression level changes resulting from addition of a drug, for example. And “as long as you are able to create a standard curve, you will be able to make an absolute quantification call,” explains Seidel.

Digital PCR (dPCR) divides the reaction into thousands of smaller reactions containing zero or one template, and thus giving a yes/no answer to the presence or absence of the product (and therefore the template). By counting these and applying Poisson statistics, absolute quantification can be achieved without the use of a standard curve. Other advantages to dPCR include the sensitivity to detect an extremely rare sequence—the proverbial needle in a haystack—and its greater tolerance of contaminants and other substances that may inhibit a bulk reaction.

In order to make this happen a thermocycler and perhaps other relatively expensive equipment are generally required, as is a reliable power source to run them.

How else can DNA be amplified?

Such equipment isn’t needed for isothermal amplification. The reason, says Seidel, is that the polymerases involved have “the ability to unwind the DNA and make the molecule single stranded without the need of the temperature cycling.”

For the most part, isothermal amplification techniques as a group have found niches in applications such diagnostic assays, agricultural testing, biosensing, and environmental monitoring. “Speed, simplicity, and the need for less sophisticated equipment make them ideal for point-of-care and field applications,” notes Garagounis.

Several protocols have been adopted that can operate without the need of thermocycling. Perhaps most prominent among these is loop-mediated isothermal amplification (LAMP), which relies on a single hybrid polymerase/helicase, and four to six primers, to deliver results in about ten minutes.

Another technique is rolling circle amplification (RCA), a “process which produces long single-stranded DNA copies and has been used to enable cost-effective RNA sequencing,” notes Angelica Olcott, Market Development Manager, Gene Expression Group, Bio-Rad. She also calls out “transcription-mediated amplification (TMA), used in the detection of RNA viruses, and helicase-dependent amplification (HDA), which uses helicase enzymes to separate DNA strands.”

In general, PCR is the method of choice for DNA amplification, a versatile method offering high sensitivity and specificity—except when cost, time-to-result, or infrastructure are limiting. But there are exceptions. Seidel points out that RCA is gaining traction in the amplification of plasmids used in the gene therapy and vaccine fields. And whole genome amplification (WGA) by a related technique called multiple displacement amplification (MDA) outperforms PCR-based methods for amplifying limiting amounts of genetic material—such as from single cells—for NGS analysis of single nucleotide variations (SNVs). (But PCR-based methods outperform MDA when it comes to NGS analysis of copy number variations.)

There are many factors to consider when looking to amplify DNA. Those mentioned above, such as sensitivity, specificity, infrastructure (equipment) requirements, costs, and time-to-results, and whether and how the product is to be used downstream are of course crucial. But to best match the technique to the application, it may also be important to keep in mind the complexity of designing and running the assay, fidelity and processivity of the enzymes, accessibility of reagents and expertise, as well as other considerations.

Table. DNA Amplification Methods