Choosing an Amplification Method: PCR vs Isothermal

Amplifying DNA samples prior to sequencing them is an important step that has become routine and sometimes even automated. PCR is the most common and widely known amplification method, but isothermal amplification is another option. Temperature changes are the main difference between isothermal and PCR amplification methods. In PCR, a thermal cycler changes the reaction temperatures repeatedly to affect the actions of the temperature-dependent reagents; in contrast, an isothermal amplification reaction occurs at a single temperature.

Isothermal amplification methods share the ability to amplify DNA at one temperature, as well as the use of strand-displacing DNA polymerases to “unzip” strands as they move along double-stranded DNA (rather than using heat to denature it as in PCR), allowing primers access to templates. Types of isothermal amplification methods include strand-displacement amplification (SDA), rolling-circle amplification (RCA), whole-genome amplification (WGA), loop-mediated isothermal amplification (LAMP), helicase-dependent amplification (HDA), and multiple displacement amplification (MDA), among others. This article looks at the advantages of isothermal and PCR amplification methods, and when one method might be preferable over another.

PCR vs isothermal amplification

Choosing an amplification method depends on the individual application and available equipment. While PCR is widely used for amplification, it requires access to a thermal cycler. This can be a disadvantage/deal-breaker for point-of-care and diagnostic applications. But if a thermal cycler is available, PCR amplification is a relatively straightforward process, and can be a better choice for rare transcripts. In contrast, isothermal amplification is ideal for situations in which there is no access to a thermal cycler (e.g., clinics, field work, home testing kits), and can sometimes be faster, simpler, and more cost-effective.

Sometimes isothermal amplification methods can have lower sensitivity and/or specificity when compared to PCR, but this can be ameliorated by optimizing reactions.¹ Madhuri Hegde, Senior Vice President and CSO at Revvity, agrees that isothermal amplification can be hampered by non-specificity. “For clinical use, a highly sensitive and specific assay is needed, and the same applies for research and discovery to avoid false positive results,” she says. However, isothermal amplification is quickly improving and driving innovation in COVID-19 testing (see below).

Indeed, there are some situations where PCR amplification is still preferable—it can be a simpler means of detecting a target of interest exclusively. This is in contrast to Tecan’s Single Primer Isothermal Amplification (SPIA), a whole-transcriptome approach that allows users to detect the target of interest as well as other relevant sample information, such as co-pathogens or host information. “The decision between SPIA and PCR amplification is dependent on the level of information that is required,” says Rupert Yip, Vice President of Marketing and Product Management at Tecan Genomics. “If only limited information is required, then PCR may be sufficient, but if in-depth information about the sample is required, then SPIA can provide a better solution.”

With SPIA, enzyme complexes land on cDNA and start making 100s to 1000s of copies of each molecule. SPIA is often used for amplifying low-input or degraded RNA samples, or detecting rare transcripts such as viral RNA. “It amplifies the whole transcriptome and is a very effective way to amplify the signal quickly,” says Yip. “The amplified cDNA can be used to test for the presence or absence of a particular virus or target of interest, using a number of detection methods from qPCR to NGS.”

Isothermal bridge amplification

A special type of isothermal method known as bridge amplification is incorporated into Illumina’s next-generation sequencing platforms prior to sequencing. It amplifies templates to generate millions of clusters of replicated sequences with minimal hands-on time. It differs from most isothermal methods in that it occurs in cycles, using denaturing agents to separate double-stranded DNA (similar to heat steps in PCR). However, “a disadvantage of this method is that each of the multiple amplification cycles requires the addition of fresh mixture containing polymerase, followed by a chemical denaturation step, making it cost much more than regular PCR,” says Hegde.

A type of in situ solid-phase isothermal amplification may provide an alternative to bridge amplification in next-generation sequencing. Based on the template walking mechanism of copying DNA, it uses low-melting temperature homopolymer primers (such as polyAs) on the solid surface, and low-melting temperature primers in solution. “The in situ solid-phase isothermal PCR approach generates more than one billion monoclonal colonies in a single lane of a 5500 flowchip without multiple cycles of amplification and denaturation,” says Hegde. “This simple approach can be fast, cost-effective, and capable of paired-end sequencing.”

Noise suppression

Tecan’s new SPIABoost allows for the suppression of unwanted signals that could otherwise drown out your signal of interest, by blocking the amplification of unwanted targeted sequences. For example, a saliva sample typically has a combination of human as well as viral transcripts, and SPIA enables you to block human rRNA sequences while amplifying viral RNA. This noise suppression technique can increase the possibility of finding rare transcripts. “For example, it can be used to search the microbiome for a specific species that is present with Crohn's disease,” says Yip. “Or if you're looking in blood samples for rare signals indicating cancer, you can use SPIA and noise suppression to reduce other signals and boost the cancer signal.”

Isothermal amplification applications

Yip notes increasing interest in using SPIA for rare transcript detection in microbiome and infectious disease applications. “We have one customer using SPIABoost with human rRNA suppression to identify early signs of meningitis infection in cerebrospinal fluid,” he says. Researchers at Imperial College London and the University of Leicester used an allele-specific LAMP assay to detect a common driver mutation in breast cancer.² Identifying this mutation early can aid clinicians and patients in making important treatment decisions.

Researchers are also using SPIA to study mosquito saliva, which is well known as a viral vector for Zika and dengue viruses. “Noise suppression can remove high background, such as mosquito rRNA, enhancing the viral information recovered from the mosquito saliva,” says Yip. “We can design SPIABoost to target practically any transcript from any species, to reduce the background and provide more informative reads.”

Researchers have also been applying isothermal amplification methods to innovations in COVID-19 testing. For example, a researcher from Portugal recently developed a RT-LAMP-based colorimetric COVID-19 test that can be performed in one tube within 30 minutes, using nasopharyngeal swab or saliva samples, and is suitable for point-of-care or low-resource settings as it uses resources unlikely to encounter bottlenecks.³ While not quite as sensitive as the RT-PCR COVID-19 test, the RT-LAMP test is sensitive enough for surveillance and screening of infectious people who are asymptomatic or presymptomatic. The ability to curb the epidemic while not relying on scarce lab supplies will be advantageous.

Another research group from the Broad Institute, MIT, and the University of Bonn developed a LAMP-based amplification method called LAMP-Seq, which adds molecular barcodes and amplifies nasal swap samples in one step.⁴ This allows for large-scale sample pooling for high-throughput analysis by next-generation sequencing. With high sensitivity and specificity, low material costs, and a testing time of less than 24 hours, new tests like LAMP-Seq may help to relieve the current demand for COVID testing in the face of the omicron variant.

References

1. Ozay, B., et al. A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions. Nov 2021. Sensors and Actuators Rep 3:100033

2. Kalofonou, M., et al. A novel hotspot specific isothermal amplification method for detection of the common PIK3CA p.H1047R breast cancer mutation. March 2020. Sci Rep 10:4553

3. Amaral, C., et al. Amaral, C., Antunes, W., Moe, E. et al. A molecular test based on RT-LAMP for rapid, sensitive and inexpensive colorimetric detection of SARS-CoV-2 in clinical samples. August 2021. Sci Rep 11, 16430.

4. Ludwig, K.U., et al. LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding. June 2021. Nat Biotechnol 39, 1556–1562.