Isothermal Amplification

Isothermal amplification methods provide detection of a nucleic acid target sequence in a streamlined, exponential manner, and are not limited by the constraint of thermal cycling. Although these methods can vary considerably, they all share some features in common. For example, because the DNA strands are not heat denatured, all isothermal methods rely on an alternative approach to enable primer binding and initiation of the amplification reaction: a polymerase with strand-displacement activity. Once the reaction is initiated, the polymerase must also separate the strand that is still annealed to the sequence of interest. Isothermal amplification chemistry has been applied to diagnostics with great success and is utilized in several commercial molecular diagnostic platforms, serving large testing centers and point-of-care markets.

The COVID-19 pandemic has seen isothermal methods being adopted as the foundational technology used to detect SARS-CoV-2 RNA in the clinical and home testing market. Two examples of colorimetric LAMP in COVID-19 diagnostics can be seen in the Color Genomics SARS-CoV-2 Diagnostic Assay and NEB’s internal CLIA-certified onsite testing program utilizing saliva. Recent preprints and publications communicate the utility of isothermal amplification methods such as NASBA and RTF-EXPAR in this area:

• INSIGHT: A population-scale COVID-19 testing strategy combining point-of-care diagnosis with centralized high-throughput sequencing
• Sub-5-minute Detection of SARS-CoV-2 RNA using a Reverse Transcriptase-Free Exponential Amplification Reaction, RTF-EXPAR

Isothermal methods typically employ unique DNA polymerases for separating duplex DNA. DNA polymerases with this ability include Klenow exo-, Bsu large fragment, and phi29 for moderate temperature reactions (25–40°C) and the large fragment of Bst DNA polymerase for higher temperature (50–65°C) reactions. To detect RNA species, a reverse transcriptase compatible with the temperature of the reaction is added (except in the NASBA/TMA reaction) to maintain the isothermal nature of the amplification.

Isothermal DNA Amplification Technologies

Loop-Mediated Isothermal Amplification (LAMP)

• Reaction temperature: 65°C
• Amplicon size: <250 nt
• DNA product: long, branched

LAMP is compatible with multiple detection methods via the WarmStart^® Multi-Purpose LAMP/RT-LAMP 2X Master Mix (with UDG). For fluorescent detection to support high-throughput testing workflows, the WarmStart Fluorescent LAMP/RT-LAMP Kit (with UDG) is recommended. Colorimetric detection by changes in pH is enabled by the WarmStart Colorimetric LAMP 2X Master Mix with UDG.

LAMP primers can be challenging to design manually, and software programs are strongly recommended for both ease of design and likelihood of reaction success. We recommend using the NEB LAMP Primer Design Tool.

Multiple Displacement Amplification (MDA)

• Reaction temperature: 30-42°C
• Amplicon size: ~40 bp to >30 kb
• DNA product: long, branched

Multiple Displacement Amplification (MDA) utilizes the strand-displacement activity of DNA polymerases such as phi29 DNA Polymerase, engineered phi29-XT DNA Polymerase, or Bst DNA Polymerase for isothermal amplification of an entire genome or circular templates. As a processive polymerase that can extend for tens of thousands of nucleotides in a single binding event, as well as increased proofreading activity, phi29-XT is ideal for accurate amplification of long or circular templates. Two common methodologies use the MDA mechanism: Whole Genome Amplification (WGA) and Rolling Circle Amplification (RCA).

WGA utilizes the strand-displacement activity of DNA polymerases such as phi29 or Bst DNA Polymerase to enable robust, universal amplification of an entire genome. WGA has become an invaluable approach for utilizing limited samples of precious stock material or to enable sequencing of single-cell genomic DNA. Products of the reaction are extremely long (>30 kb) and highly branched through the multiple displacement mechanism. The phi29-XT WGA Kit contains all components needed to amplify your sample genome, including exonuclease-resistant random primers, neutralization buffer, dNTPs, and phi29-XT DNA Polymerase.

RCA is a robust and highly sensitive approach for continuously amplifying circular DNA to generate long, repetitive copies of the circular template. The polymerase extends from the 3' end of a primer and continues around the circular template, displacing the nascent strand as it continuously utilizes the initial template. The run-off strands are then primed and exponential amplification proceeds to completion, typically around 2 hours. RCA can be used to amplify plasmids for downstream long-read or Illumina sequencing or for amplifying assembled constructs for cell-free protein expression. The phi29-XT RCA Kit contains all components needed to amplify your circular template, including exonuclease-resistant random primers, buffer, dNTPs, and phi29-XT DNA Polymerase.

Strand Displacement Amplification (SDA)

• Reaction temperature: 60°C
• Amplicon size: <100 nt
• DNA product: short, discrete

SDA, or a similar approach, Nicking Enzyme Amplification Reaction (NEAR), relies on a strand-displacing DNA polymerase, typically Bst DNA Polymerase, Large Fragment or Klenow Fragment (3’-5’ exo–), to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. The nicking site is regenerated with each polymerase displacement step, resulting in exponential amplification. NEAR is extremely rapid and sensitive, enabling detection of small target amounts in minutes. SDA and NEAR are typically utilized in clinical and biosafety applications.

Helicase-Dependent Amplification (HDA)

• Reaction temperature: 65°C
• Amplicon size: <150 nt
• DNA product: short, discrete

HDA employs the double-stranded DNA unwinding activity of a helicase to separate strands, enabling primer annealing and extension by a strand-displacing DNA polymerase. Like PCR, this system requires only two primers. HDA has been employed in several diagnostic devices and FDA-approved tests.

Recombinase Polymerase Amplification (RPA)

• Reaction temperature: 37°C
• Amplicon size: <1,000 nt
• DNA product: short, discrete

RPA uses a recombinase enzyme to help primers invade double-stranded DNA. T4 UvsX, UvsY, and T4 Gene 32 Protein, a single stranded DNA binding protein, form D-loop recombination structures that initiate amplification by a strand-displacing DNA polymerase. RPA is typically performed at ~37 °C and, unlike other methods, can produce discrete amplicons up to 1 kb.

Nucleic Acid Sequences Based Amplification (NASBA)

• Reaction temperature: 40-55°C
• Amplicon size: <150 nt
• DNA product: short, discrete

NASBA and Transcription Mediated Amplification (TMA) are both isothermal amplification methods that proceed through RNA. Primers are designed to target a region of interest; one of the primers must include the promoter sequence for T7 RNA polymerase at the 5’ end. NASBA and TMA reactions are utilized in a range of clinical diagnostics.