Enzymes for Isothermal Amplification

Robust amplification for critical samples

Isothermal amplification is a robust method of exponential nucleic acid amplification at constant temperature, eliminating the need for thermal cycling. It is an ideal technique for amplification of limited amounts of DNA and where sensitivity as high as, or greater than, that of benchmark PCR-based methods is required. Isothermal methods of nucleic acid amplification continue to grow in applications like blood screening, infectious disease diagnostics, cancer research, and more.

RT-LAMP for rapid detection of SARS-CoV-2

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is a fast and sensitive technique that can be used to screen for viral pathogens, including the RNA virus SARS-CoV-2. The workflow is easy to set-up, with a fast turnaround time, and only requires a simple heat source to maintain a constant temperature. A positive RT-LAMP reaction can be visualized by several methods (e.g. change in color and via agarose gel electrophoresis).

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Advantages of isothermal amplification over PCR

  • Low cost or no equipment needed
  • Reduced chance of contamination
  • Lower sensitivity to reaction inhibitors
  • Can be used in point-of-care tests

EquiPhi29 and Bsm DNA polymerases are two enzymes that enable sensitive and robust isothermal amplification in two major applications: loop-mediated isothermal amplification (LAMP) and whole genome amplification (WGA).

LAMP is a rugged, low-cost method for specific DNA detection, with a visual readout. It is especially useful in field settings for rapid diagnosis of plant pathogens or infectious disease agents.

LAMP is based on use of six primers (rather than two for PCR), allowing you to include multiple genome sequence regions as specificity targets (Figure 1). The increased number of starting points for DNA synthesis delivers the improved specificity and sensitivity under isothermal conditions. As synthesis begins, pairs of primers form loops to facilitate each round of amplification.

Loop-mediated isothermal amplification (LAMP) reaction primer design based on six primers for increased specificity and sensitivity
Figure 1. LAMP reaction primer design.

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. It has strong strand displacement activity and an optimum temperature of 60°C. Amplification by Bsm DNA polymerase is very efficient copying DNA targets 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.

To increase the amount of limited DNA targets, isothermal whole genome amplification (WGA) is the most efficient technique.[1] 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.[2] 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 offers an improved EquiPhi29 DNA polymerase, which is a proprietary Phi29 DNA polymerase mutant developed through in vitro protein evolution.[3] 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 1 compares classical Phi29 and EquiPhi29 DNA polymerases.

Table 1. Comparison of Phi29 and EquiPhi29 DNA polymerases with supporting data.

  Phi29-type DNA Polymerases EquiPhi29 DNA Polymerase
Processivity/strand displacement High (up to 70 kb) High (up to 70 kb)
Optimal amplification temperature 30-37 °C 42-45 °C
Reaction time Slow – up to 12h Slow – up to 3h
Proofreading 3′→5′ (low error rates) 3′→5′ (low error rates)
Accuracy High (low error rates) High (low error rates)
Yield High Very high
Sequence bias (preference) Low bias, uniform amplification of long fragments (whole genome) Very low bias, including GC and AT rich (data valid for 0.5 ng starting material)

EquiPhi29 DNA Polymerase also enables a shorter and faster multiple displacement amplification (MDA) protocol when compare with other WGA commercial kits.  Hands-on time is roughly 30 minutes, with a total protocol time of about 2 hours (Figure 2).

EquiPhi29 DNA Polymerase

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 3) or whole genomic DNA (Figure 4) within 2 hours.

EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes

Figure 3. EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases (EquiPhi29 DNA Polymerase is indicated in yellow).

EquiPhi29 DNA Polymerase delivered high genomic DNA yields with faster reaction times than other suppliers’ products

Figure 4. EquiPhi29 DNA Polymerase delivered high genomic DNA yields with faster reaction times than other suppliers’ products. Amplification of 0.5 ng of human genomic DNA was carried out using EquiPhi29 and Phi29 DNA polymerases as well as DNA polymerases from other suppliers. The DNA products were purified using magnetic beads and quantified using the Qubit dsDNA BR Assay Kit. The recommended reaction temperature for EquiPhi29 DNA Polymerase is 42°C; however, higher yields can be obtained after a 4 hr incubation at 30°C.

LAMP is a rugged, low-cost method for specific DNA detection, with a visual readout. It is especially useful in field settings for rapid diagnosis of plant pathogens or infectious disease agents.

LAMP is based on use of six primers (rather than two for PCR), allowing you to include multiple genome sequence regions as specificity targets (Figure 1). The increased number of starting points for DNA synthesis delivers the improved specificity and sensitivity under isothermal conditions. As synthesis begins, pairs of primers form loops to facilitate each round of amplification.

Loop-mediated isothermal amplification (LAMP) reaction primer design based on six primers for increased specificity and sensitivity
Figure 1. LAMP reaction primer design.

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. It has strong strand displacement activity and an optimum temperature of 60°C. Amplification by Bsm DNA polymerase is very efficient copying DNA targets 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.

To increase the amount of limited DNA targets, isothermal whole genome amplification (WGA) is the most efficient technique.[1] 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.[2] 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 offers an improved EquiPhi29 DNA polymerase, which is a proprietary Phi29 DNA polymerase mutant developed through in vitro protein evolution.[3] 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 1 compares classical Phi29 and EquiPhi29 DNA polymerases.

Table 1. Comparison of Phi29 and EquiPhi29 DNA polymerases with supporting data.

  Phi29-type DNA Polymerases EquiPhi29 DNA Polymerase
Processivity/strand displacement High (up to 70 kb) High (up to 70 kb)
Optimal amplification temperature 30-37 °C 42-45 °C
Reaction time Slow – up to 12h Slow – up to 3h
Proofreading 3′→5′ (low error rates) 3′→5′ (low error rates)
Accuracy High (low error rates) High (low error rates)
Yield High Very high
Sequence bias (preference) Low bias, uniform amplification of long fragments (whole genome) Very low bias, including GC and AT rich (data valid for 0.5 ng starting material)

EquiPhi29 DNA Polymerase also enables a shorter and faster multiple displacement amplification (MDA) protocol when compare with other WGA commercial kits.  Hands-on time is roughly 30 minutes, with a total protocol time of about 2 hours (Figure 2).

EquiPhi29 DNA Polymerase

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 3) or whole genomic DNA (Figure 4) within 2 hours.

EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes

Figure 3. EquiPhi29 DNA Polymerase demonstrated low GC bias when amplifying 3 bacterial genomes. A mixture of bacterial genomes with low-GC (S. aureus, 33% GC), moderate-GC (E. coli, 51% GC), and high-GC (P. aeruginosa, 68% GC) content was amplified using EquiPhi29 and Phi29 DNA polymerases as well as a DNA polymerase from another supplier. For each genome, the GC content of the reference genome, in 100 bp windows indicated in gray, was plotted versus the coverage normalized to the unamplified genome mix, indicated in green. In the absence of sequencing bias, all windows should be equally distributed close to the normalized coverage of 1, indicated in light blue. The normalized coverage obtained after amplification using different polymerases is shown. EquiPhi29 DNA Polymerase amplifies DNA with the lowest GC bias across all GC contents when compared to other DNA polymerases (EquiPhi29 DNA Polymerase is indicated in yellow).

EquiPhi29 DNA Polymerase delivered high genomic DNA yields with faster reaction times than other suppliers’ products

Figure 4. EquiPhi29 DNA Polymerase delivered high genomic DNA yields with faster reaction times than other suppliers’ products. Amplification of 0.5 ng of human genomic DNA was carried out using EquiPhi29 and Phi29 DNA polymerases as well as DNA polymerases from other suppliers. The DNA products were purified using magnetic beads and quantified using the Qubit dsDNA BR Assay Kit. The recommended reaction temperature for EquiPhi29 DNA Polymerase is 42°C; however, higher yields can be obtained after a 4 hr incubation at 30°C.

Lyo-Ready Formats

Lyo-Ready Formats

Both EquiPhi29 and Bsm DNA Polymerases are available in lyophilization-compatible (lyo-ready) OEM formats that provide assay flexibility, extended shelf life, and much more.

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