The Long and Short of Isothermal Amplification

Table 1. Summary of isothermal amplification methods.

Instead of melting DNA strands apart at high temperatures, isothermal amplification takes advantage of DNA polymerases with high strand displacement activity, like Bst or Phi29 DNA polymerases. Essentially, such enzymes can push their way in and directly unzip the DNA as they synthesize complementary strands. These polymerases can amplify a target in less than an hour, and in some cases in as little time as 10 minutes. Isothermal amplification systems can use sequence-specific primers to detect target genes, or random primers for whole genome amplification.

Loop-mediated isothermal amplification (LAMP) is a rugged, low-cost method for specific DNA detection, with a visual readout. LAMP is especially useful in field settings for rapid diagnosis of plant pathogens or infectious disease agents like malaria, Zika, or tuberculosis. Table 2 summarizes the differences between LAMP and PCR.

Table 2. Comparison of PCR and LAMP.

LAMP is based on use of six primers (rather than two for PCR), allowing you to include multiple genome sequence regions as specificity targets. The increased number of starting points for DNA synthesis delivers the improved specificity and sensitivity compared to most PCR-based detection assays. As synthesis begins, pairs of primers form loops to facilitate each round of amplification.

The enzyme often used for LAMP is Bsm DNA polymerase, a portion of DNA polymerase of Bacillus smithii, and an equivalent to Bst DNA polymerase Large Fragment. Bsm has strong strand displacement activity and an optimum temperature of 60°C. Amplification is very efficient with DNA being copied a billion-fold in as little as 15 minutes. The enzyme is highly resistant to inhibitors in complex samples, so plant tissue, blood, urine, or saliva can be assayed with minimal processing.

The LAMP reaction produces magnesium pyrophosphate as a by-product, which precipitates from solution and makes it cloudy. You can measure turbidity or use a dye to monitor the reaction. Hydroxynaphthol blue (KNB) will change color from violet to blue. Calcein, quenched by manganese, will turn from orange to yellow-green when magnesium is present and which it prefers. SYBR Green, EvaGreen® (Biotium), and berberine are intercalating nucleic acid-specific dyes that emit fluorescent signal under UV light. This colorimetric readout is extremely simple and fast but does not offer precise target quantification. You can choose an optimal detection method depending on the screening applications.[2]

Most cells contain only one or a few copies of their genome, constituting picograms of DNA, which is not enough for direct analysis with current sequencing technologies. Assaying single cell DNA for diagnostics, which can require multiple experiments with limited sample, also demands rapid unbiased DNA amplification. The research on ancient DNA, which is often fragmented, poses yet another type of challenge. These are areas where isothermal amplification technology is used to increase DNA material needed for downstream analysis.

To increase the amount of limited DNA targets, isothermal whole genome amplification (WGA) is the most efficient technique.[3] This is particularly useful in genetic disease research, where many repetitions are required. DNA amplified by WGA is used in downstream next-generation sequencing, Sanger sequencing, genotyping with microarrays, and single nucleotide polymorphism (SNP) genotyping.[4] Various WGA techniques have been developed that differ both in their protocols and ease of use.

Phi29 DNA polymerase is the main enzyme of choice for WGA. Thermo Scientific also offers an improved EquiPhi29 DNA polymerase, which is a proprietary Phi29 DNA polymerase mutant developed through in vitro protein evolution.[5] This enzyme is significantly superior over Phi29 in protein thermostability, reaction speed, product yield, and amplification bias. Moreover, it retains all the benefits of the wild-type enzyme, including high processivity (up to 70 kb), strong strand displacement activity, and 3'–5' exonuclease (proofreading) activity. For this reason, exo-resistant random primers are recommended. Table 3 compares classical Phi29 and EquiPhi29 DNA polymerases.

Table 3. Comparison of Phi29 and EquiPhi29 DNA polymerases with supporting data.

In studies comparing other commercially available versions of Phi29 DNA polymerases, EquiPhi29 DNA polymerase demonstrated the lowest bias when amplifying targets with GC-rich content (Figure 1) and delivered the highest yield of a target sequence whether from DNA plasmid (Figure 2) or whole genomic DNA (Figure 3) within 2 hours. 

Phi29-type polymerases produce the best template for downstream assays from long intact DNA stretches. Therefore, it is important to denature DNA carefully but completely. The two most common methods include heat denaturation at 95°C and alkaline DNA denaturation. Heat denaturation carries the risk of DNA breakage, whereas alkaline denaturation may be incomplete and inconvenient. Intact but well-separated stretches of starting DNA ensure lower bias and higher yields. The more double-stranded DNA in a sample, the lower the performance of the WGA reaction.

Using the Phi29-type polymerases, the genome is amplified during multiple displacement amplification (MDA) reaction, which starts by binding of random primers to multiple sites of denatured DNA. The polymerase amplification involves strand displacement, therefore additional priming events occur on each displaced strand yielding a branched DNA product of up to 70 kb. The reaction takes place at a constant temperature.

Phi29-type polymerases are capable of replicating DNA from minute starting amounts without dissociating from the genomic DNA template (the average product length is greater than 10 kb). This feature makes it a great candidate for whole genome amplification from single cells. The larger the amount of DNA, and therefore the copy number of the genome, the more likely a specific locus will be detected after whole genome amplification.

Isothermal amplification methods offer important alternatives to lab-based methods that depend on expensive equipment and protocols for sequential cycling to amplify of a target of interest. Among some of the more common or well-studied isothermal amplification methods (Table 1), LAMP and WGA have an increasingly important role in scientific research expanding our toolbox beyond PCR-based methods in the lab to critical analytical work in the field.

1. Li J et al. (2019) Review: a comprehensive summary of a decade development of the recombinase polymerase amplification. Analyst 144(1):31-67.
2. Fischbach J et al. (2018) Shining a light on LAMP assays' A comparison of LAMP visualization methods including the novel use of berberine. BioTechniques 58(4):189-94.
3. Zhang L et al. (1992) Whole genome amplification from a single cell: Implications for genetic analysis. PNAS 89(13):5847-51.
4. Dean FB et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. PNAS 99(8):5261-6.
5. Povilaitis T et al. (2016) In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Eng Des Sel 29(12):617-628.
6. Stepanauskas R et al. (2017) Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles. Nature Communications 8(1):84.