While PCR (polymerase chain reaction) has long been the star of molecular biology, there’s a promising newcomer—isothermal amplification (see Table 1 for summary of methods). This robust method can amplify nucleic acids exponentially at constant temperature, eliminating the need for thermocycler equipment. Isothermal amplification is an ideal technique for pathogen monitoring, and for amplifying limited amounts of DNA where analyte sensitivity as high or greater than that of benchmark PCR-based methods are required.
Table 1. Summary of isothermal amplification methods.
|NASBA||Nucleic acid sequence-based amplification is a method used to amplify RNA.|
|LAMP||Loop-mediated isothermal amplification is a single tube technique for the amplification of DNA. It uses 4-6 primers, which form loop structures to facilitate subsequent rounds of amplification.|
|HDA||Helicase-dependent amplification uses the double-stranded DNA unwinding activity of a helicase to separate strands for in vitro DNA amplification at constant temperature.|
|RCA||Rolling circle amplification starts from a circular DNA template and a short DNA or RNA primer to form a long single stranded molecule.|
|MDA||Multiple displacement amplification is a technique that initiates when multiple random primers anneal to the DNA template and the polymerase amplifies DNA at constant temperature.|
|WGA||When MDA is used to amplify DNA from a whole genome of a cell it is called whole genome amplification. (Other methods of WGA include MALBAC, LIANTI, DOP-PCR.)|
|RPA||Recombinase polymerase amplification is a low temperature DNA and RNA amplification technique.|
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.
|Amplification||Cycles through 3 temperature steps:
||Works under a constant temperature usually between 60-65°C|
|Denaturation||High temperature required for separation of strands, enabling primer binding||Denaturation step is performed by strand displacing polymerase|
|Equipment||Requires thermocycler||Doesn’t require dedicated thermocycler; can use a simple water bath|
|Reaction time||At least 90 minutes to results||Results are typically ready in less than 30 minutes|
|Sensitivity||Detects targets starting at nanogram levels||Detects targets starting at femtogram levels|
|Specificity||Requires careful primer design to avoid primer dimer or non-specific amplification||Tolerates (works well with) multiple primer combinations for greater specificity|
|DNA visualization||DNA visualization only possible after gel electrophoresis||Immediate visualization of DNA by colorimetry/visual turbidity|
|DNA template preparation||Requires purification or special handling for high sensitivity and specificity||Tolerates inherent impurities and inhibitors common to field samples with highest sensitivity and specificity|
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.
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. 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. 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. 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.
|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)|
|Sequence bias (preference)||Low biased, uniform amplification of long fragments (whole genome)||Very low bias, including GC and AT rich (data valid for 0.5 ng starting material)|
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.
Figure 1. 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).
Figure 2. EquiPhi29 DNA Polymerase delivered high plasmid DNA yields with faster reaction times than other suppliers’ products. Amplification of 0.5 ng of pUC19 plasmid DNA was carried out using EquiPhi29 DNA Polymerase, Phi29 DNA Polymerase, and 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, delivering the highest yield after 2 hr of incubation.
Figure 3. 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.
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.
For Research Use Only. Not for use in diagnostic procedures.