Advancements in Isothermal Amplification Techniques: Exploring the Potential of 37°C as an Optimal Reaction Temperature for Molecular Detection

Compared to the temperature cycling PCR, isothermal amplification techniques are more diverse, with the diversity of enzymes used determining the variations in the principles of isothermal amplification. Although these methods are collectively referred to as isothermal amplification techniques, the optimal reaction temperature for each method varies. However, several isothermal amplification techniques that we will introduce today share a common characteristic, which is that their initial optimal reaction temperature is around 37°C.

Recombinase Polymerase Amplification (RPA)

Introduction

Recombinase Polymerase Amplification (RPA) is a nucleic acid amplification technique that is based on the DNA replication mechanism of T4 bacteriophage. It uses the action of recombinase protein (T4 UvsX Recombinase), single-stranded DNA-binding protein (T4 Gene 32 Protein), and DNA polymerase (Bsu DNA Polymerase) to achieve isothermal amplification of nucleic acids in vitro. The principle of RPA is as follows:

1. In the presence of ATP, recombinase T4 UvsX binds to the upstream and downstream primers to form a recombinase-primer complex.
2. This complex scans the double-stranded DNA template and recognizes the target sequence homologous to the primers.
3. After positioning to the homologous target sequence, the recombinase opens the double-stranded structure and promotes strand exchange between the primers and the template, forming a D-loop structure. At the same time, single-stranded DNA-binding protein T4 gp32 binds to the displaced single-stranded DNA, stabilizing the D-loop structure.
4. ATP hydrolysis provides energy, causing conformational changes in the recombinase-primer complex, leading to exposure of the 3' end of the primer, and DNA polymerase Bsu binds to the primer, initiating DNA amplification.
5. During DNA polymerase extension, the displaced single-stranded DNA binds to the single-stranded DNA-binding protein, stabilizing the single-stranded structure. The upstream and downstream primers react synchronously, eventually resulting in the formation of a complete amplicon.

The RPA system also contains auxiliary factors (T4 UvsY Protein) and crowding agents (Carbowax20M), which can regulate the reversible reaction process of dissociation and reassembly of the recombinase-primer complex, favoring the direction of RPA. If reverse transcriptase is added to the RPA system, it can also be used for RNA detection.

The incorporation of fluorescent probes and nucleases into the RPA system allows for real-time detection and visualization. Different types of fluorescent probes, along with their corresponding nucleases, are designed differently, resulting in three types of RPA probes: Exo probes, Fpg probes, and LF probes, each with their own detection principles as follows:

• Exo probes: Exo probes, when combined with exonucleases (exo), can be used for real-time detection. These probes introduce a modification site of tetrahydrofuran abasic-site mimic (THF) internally, which is coupled with fluorescent and quenching groups on either side of the modification site. When the probe hybridizes with the target sequence to form double-stranded DNA, exo recognizes the THF modification site and cleaves at that position, separating the fluorescent and quenching groups, resulting in a fluorescent signal.
• Fpg probes: Fpg probes, when combined with Fpg nuclease, can also be used for real-time detection. These probes have a quenching group modified at the 5' end, and a fluorescent group connected to the probe base in the vicinity of the quenching group as C-O-C(dR group). When the probe hybridizes with the target sequence to form double-stranded DNA, Fpg nuclease recognizes the dR group and cleaves at that position, releasing the fluorescent group and generating a fluorescent signal.
• LF probes: LF probes, when combined with endonucleases (such as nfo) and lateral flow chromatography, can achieve visual detection of RPA results. These probes are modified with a fluorescent group at the 5' end, and also introduce a tetrahydrofuran (THF) modification site internally. When the probe hybridizes with the target sequence to form double-stranded DNA, nfo recognizes the THF de-modification site and cleaves at that position, generating a free 3'-OH group that can serve as a primer for further extension and amplification. Simultaneously, the downstream primer modified with biotin is also amplified, resulting in amplicons with fluorescent and biotin labels at both ends. These amplicons, when combined with lateral flow chromatography, can be visually observed for detection results.

The 3' ends of these three types of probes need to be blocked to prevent probe extension. If probes with different fluorescent labels are used, it is possible to achieve multiplex detection.

Technical Features

• RPA is a multi-enzyme reaction system that requires the cooperative action of multiple enzymes to achieve amplification. Different manufacturers have established their own amplification technologies based on different sources of key enzymes.
• RPA can be performed at a wide temperature range of 25~42°C without the need for thermal cycling, with the optimal reaction temperature being around 37°C. This temperature range overlaps with the reaction temperature range of the CRISPR/Cas system, which has also facilitated the development of next-generation molecular diagnostic technologies.
• RPA has a fast reaction speed, with detection levels achieved within 5~30 minutes, making it suitable for point-of-care testing (POCT) applications.
• The primer length of RPA is typically 3035bp, and the length of the fluorescent probe is 4652bp. Compared to other nucleic acid detection technologies, both the primer and probe of RPA are relatively long, requiring more optimization and screening during testing. At the same time, the longer primer length also limits the ability of RPA to detect short nucleic acid sequences.

Further Reading: BodyIAmp™ Technology

BodyIAmp™ technology is a very unique technology of SBS Genetech, which is very similar to RPA technology in principle. The difference is that the enzymes used come from different species and are engineered to make the reaction faster. BodyIAmp™ technology is developed by advanced molecular design, directed evolution, affinity maturation, and other technical means in the field of antibody medicine to modify and mutate specific tool enzymes derived from bacteria, viruses, and bacteriophages and screen their functions, obtaining a new rapid isothermal amplification reaction system of nucleic acid through combinatorial optimization. Under constant temperature conditions, it only takes 7-10 minutes to complete the specific amplification of target DNA fragments hundreds of millions of times. Compared with similar technology products, its low-temperature adaptability and sensitivity have made a new breakthrough, reaching the world's industry-leading level.

Rolling Circle Amplification (RCA)

Introduction

Rolling Circle Amplification (RCA) is a nucleic acid detection method that mimics the natural process of circular DNA rolling circle replication. The DNA polymerase used in RCA is phi29 DNA polymerase, which has strong strand displacement activity. RCA can be performed in linear amplification mode or exponential amplification mode, as illustrated as follows:

• For circular templates, a single primer complementary to the template is added, and after primer-template hybridization, rolling circle amplification is initiated, resulting in the synthesis of repetitive linear single-stranded DNA sequences, which is a linear amplification process.
• For linear target sequences, padlock probes can be designed with ends complementary to the target sequence, and under the action of a ligase, the padlock probes are ligated into a circular form, which can then serve as templates for rolling circle amplification.
• In addition to the linear amplification triggered by a single primer, a reverse primer sequence can be added, which hybridizes with the single-stranded DNA amplification product, and under the action of DNA polymerase, multiple branching amplification reactions can occur due to the strand displacement activity of phi29 DNA polymerase, resulting in exponential amplification of the product.

Technical Features

• RCA requires single-stranded circular templates for detection. If the sample does not meet this requirement, denaturation and annealing treatment may be needed for circularization.
• The linear amplification product of RCA is a large amount of repetitive linear single-stranded DNA that is complementary to the circular template. This allows for direct probe binding for signal amplification, and the continuous amplification products can avoid signal diffusion, making it suitable for in situ hybridization detection.
• The efficiency of linear RCA can reach up to 10^5-fold, and the efficiency of exponential RCA can reach up to 10^9-fold, allowing for single-molecule detection.
• For RNA detection, there is no need for reverse transcription of RNA prior to RCA. Only ensuring the recognition sequence of the probe is complementary to the target sequence is sufficient for RCA detection.
• Based on the principle of complementary pairing between the 5' and 3' recognition sequences of padlock probes, this method can be used for SNP detection.

Further Reading: phi29 HT DNA Polymerase

phi29 HT DNA polymerase is specially engineered enzyme of SBS Genetech, which is an updated version of phi29 DNA polymerase. In addition to the strong strand displacement and continuous synthesis (> 70kb) activity of phi29 DNA polymerase, phi29 HT DNA polymerase can continuously synthesize DNA at 42°C, while the activity of phi29 DNA polymerase is very low at this temperature. In addition, phi29 HT DNA Polymerase still has a strong 3 '- 5' exonuclease proofreading function and the fidelity of the synthesized DNA fragments is high. The exonuclease activity of this enzyme is strong, so the primer needs 3'- end thio-modification in the process of synthesis to reduce the cleavage effect of the exonuclease activity on the primer.

The high-temperature reaction characteristic of phi29 HT DNA Polymerase has the following advantages:

• In the next generation sequencing (NGS), the enzyme has stronger amplification activity for complex templates such as high GC content and palindrome structure, which makes the coverage of NGS more uniform and reduces the depth required for sequencing.
• High-temperature reaction conditions improve the synthesis of WGA products of genomic DNA and can be used for variable temperature amplification.
• The gap region in sequencing is reduced, which can improve the quality and integrity of the data from single-cell sequencing.
• Reduce non-specific amplification products.
• Improve the amplification performance and specificity of MDA/RCA and other experiments.

Strand Displacement Amplification (SDA)

Introduction

Strand Displacement Amplification (SDA) is a temperature-dependent amplification technique that relies on the cleavage of DNA restriction enzyme recognition sites (such as HincII) by restriction endonucleases, and the extension of the cleavage site by DNA polymerases (such as exo-Klenow), followed by displacement of downstream DNA fragments. The original principle of first-generation SDA is as follows:

1. In the first-generation SDA, amplification is performed using a pair of primers containing the recognition sequence of the restriction enzyme (such as HincII recognition site 5'-GTT//GAC-3'). Prior to amplification, the template is first cleaved by the restriction enzyme, and then annealed to the primers through heat denaturation.
2. In the presence of a DNA polymerase with no exonuclease activity (such as exo-Klenow), extension occurs to form double-stranded DNA, generating a cleavage site recognized by the restriction enzyme. However, due to the addition of a chemically modified dNTP (such as dATPαS) in the reaction system, HincII will only cleave the unmodified DNA strand at the cleavage site (such as 5'-GTT//GAC-3' or 5'-GTT//GsAC-3'), while the other strand containing a phosphorothioate modification will not be cleaved, resulting in the formation of a nick with a exposed 3' end. This nick serves as a primer for DNA polymerase extension to initiate amplification, resulting in the formation of amplification products with cleavage sites in a partially modified state.
3. In addition, during the extension process of DNA polymerase, the downstream DNA single strand is displaced, and the displaced DNA single strand can anneal to the primers to initiate further amplification, resulting in the formation of double-stranded DNA with cleavage sites in a partially modified state. The restriction endonuclease then recognizes and cleaves the cleavage site again.
4. This process of cleavage by the restriction enzyme, DNA polymerase-mediated amplification, and strand displacement reaction cycles, leading to a cyclic amplification process.

The first-generation SDA requires the use of a restriction endonuclease to cleave the target DNA, generating templates that can be used for amplification. This not only increases the difficulty of the procedure but also limits the choice of target sequences. The second-generation SDA has improved upon this by simplifying the process and using two pairs of primers instead of one. The outer primers (B1 and B2) are conventional specific primers, while the inner primers (S1 and S2) are similar to those used in the first-generation SDA, with a restriction endonuclease recognition sequence at the 5' end. The reaction principle is as follows:

1. The target sequence is denatured by heat, and during the annealing process, the inner primers (S1 and S2) and the outer primers (B1 and B2) bind to the target sequence, initiating the amplification. The synthesis chain initiated by the outer primers can displace the synthesis chain of the inner primers.
2. The single-stranded DNA continues to anneal and extend with the inner primers, ultimately forming double-stranded DNA molecules with restriction endonuclease recognition sites, which serve as the main templates for entering the SDA amplification cycle.
3. The restriction endonuclease recognizes and cleaves the unmodified DNA strand at the restriction site, creating a nick. DNA polymerase extends from the nick and displaces the downstream DNA strand.
4. The cycling process formed in this way is similar to the first-generation SDA.

Technical Features

• SDA requires a heat denaturation step to open the double-stranded DNA before isothermal amplification. If the enzyme used is not thermostable, stepwise addition may be required.
• SDA reaction system requires the addition of chemically modified nucleotides to ensure that the restriction endonuclease only forms a nick at the restriction site. The inclusion of non-standard nucleotides may reduce the amplification efficiency.
• Similar to SDA, another isothermal amplification technique called Nicking Enzyme Amplification Reaction (NEAR) uses a nicking endonuclease as the restriction enzyme, which cleaves only one strand of the double-stranded DNA to form a nick, allowing amplification with regular nucleotides. The enzyme used in NEAR is thermostable, and the reaction temperature can reach around 54°C.

Helicase-Dependent Amplification (HDA)

Introduction

Helicase-Dependent Amplification (HDA) is an in vitro isothermal amplification technique that mimics the DNA replication mechanism in animals. It uses components such as helicase and single-stranded DNA binding protein (SSB) to replace the temperature cycling process of denaturation-annealing-extension in PCR. The reaction principle is as follows:

1. Helicase unwinds the double-stranded DNA, and SSB binds to the single-stranded DNA, stabilizing the single-stranded state.
2. Primers bind to the single-stranded target sequence, and new double-stranded DNA is synthesized by DNA polymerase.
3. The newly synthesized double-stranded DNA can serve as a template for the next round of amplification by repeating the above steps.

Technical Features

• Compared to other isothermal amplification techniques, the amplification process of HDA is simple, akin to a thermocycling version of PCR.
• The amplification process requires the coordinated action of helicase and polymerase, with a need to balance their reaction rates to favor synthesis of new strands.
• The initial reaction temperature of HDA is 37°C, but through screening, adjustment of key components, and genetic engineering modifications, a thermostable HDA has been developed, with a reaction temperature of around 65°C.

Summary and Prospect

37°C, the temperature within human touch, is the basis for molecular detection methods that are most imaginative in future applications. Just imagine, a molecular detection product that can provide results simply by warming it up with your hand... Of course, an ideal molecular detection product cannot be achieved solely by optimizing nucleic acid amplification methods. Considerations need to be given to sample pre-processing, result presentation, convenience and safety of product use before and after, and more.

Moreover, the requirement for reaction equipment is low when it comes to a reaction temperature around 37°C, at least from the perspective of energy consumption. With the product performance reaching its peak, it may not even need a device powered by batteries. Just imagine, in the future, you can perform molecular detection anywhere, anytime with a simple gesture, without worrying about energy supply, how convenient and high-tech that would be!

In fact, looking back at the development history of some amplification technologies, the past trend was to establish an in vitro nucleic acid amplification method under moderate to low temperature conditions, and then further improve the application value of the technology by seeking heat-stable components to optimize the reaction system. However, with the continuous advancement of technology and the expansion of application scenarios, will the iteration strategy of technology change in the future? Will the constant temperature for reactions approach the human body temperature of 37°C? This is an exciting question to anticipate. Just imagine, in the future, we could perform molecular detection at a comfortable temperature without the need for additional heating devices, creating a perfect fusion of technology and the human body experience.

At the current stage, the temperature of 37°C leaves us with boundless imagination, and we look forward to more surprises and breakthroughs in the future of technological development. Let us continue to explore and expand the boundaries of molecular detection technology, creating a more astonishing future.