The Long and Short of Isothermal Amplification

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.

Table 1. List of the various isothermal amplification methods.

The widely used isothermal techniques are LAMP, MDA, and RCA.

LAMP

LAMP is a fast and simple DNA amplification method under isothermal conditions that utilizes a DNA polymerase with strong strand displacement activity. One of the most popular modifications of LAMP is RT-LAMP which incorporates reverse transcriptase to create a cDNA template from RNA. LAMP is especially useful in field settings for rapidly diagnosing plant pathogens or infectious disease agents like malaria, Zika, or tuberculosis.

MDA-WGA and RCA

MDA-WGA and RCA are the most efficient techniques to increase the amount of DNA from limited sample input [2]. MDA is an isothermal amplification technique which starts by binding 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. WGA focuses on amplifying the entire genome for situations with limited DNA. This is particularly useful in genetic disease research, where many repetitions are required. DNA amplified by MDA-WGA can be used in downstream applications such as next-generation sequencing, Sanger sequencing, genotyping with microarrays, and single nucleotide polymorphism (SNP) genotyping [3].

RCA is an isothermal amplification of circular DNA templates. It could be performed with random primers, resulting in a branched DNA product, or using specific primers that increase specificity and help ensure that only the intended DNA sequences are amplified. RCA products can be used in downstream applications such as NGS, Sanger sequencing, SNP detection, DNA enrichment, and as a template for cell-free protein expression [4].

The enzyme often used for LAMP is Bst DNA Polymerase, an enzyme derived from the large fragment of Bacillus stearothermophilus DNA Polymerase I. Bst DNA Polymerase contains 5´→3´ DNA polymerase activity and strong strand displacement activity but lacks 5´→3´ exonuclease activity. Bst DNA Polymerase works at an optimal 65 °C temperature and can amplify target DNA in as little as 10 minutes. The enzyme is highly resistant to inhibitors in complex samples, so plant tissue, blood, urine, or saliva can be assayed with minimal processing.

Another 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.

phi29 DNA Polymerase is the main enzyme choice for MDA-WGA and RCA technologies. This enzyme is derived from Bacillus subtilis phage phi29 (Φ29) and is a highly processive polymerase featuring strong strand displacement activity. phi29 DNA Polymerase possesses a 3'→5' exonuclease (proofreading) activity but lacks 5´→3´ exonuclease activity. Because of its proofreading activity, exo-resistant random primers are recommended [5].

phi29-type polymerases can replicate 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 single-cell whole genome amplification. 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 essential alternatives to lab-based methods that depend on expensive equipment and protocols for sequential cycling to amplify 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. Table 2 summarizes the advantages of LAMP over PCR.

Differences between LAMP and PCR

Table 2. Comparison of PCR and LAMP.

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• Zhang L et al. (1992) Whole genome amplification from a single cell: Implications for genetic analysis. PNAS 89(13):5847-51.
• Dean FB et al. (2002) Comprehensive human genome amplification using multiple displacement amplification. PNAS 99(8):5261-6.
• Zibluski DL et al. (2022) HyperXpress: Rapid single vessel DNA assembly and protein production in microliterscale. Frontiers in Bioengineering and Biotechnology 10: 832176.
• 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.