Traditional reverse transcriptases cannot tolerate high temperatures (the optimal temperature for MMLV activity is 37-50°C, and AMV is 42-60°C). The more complex viral RNA cannot be effectively reverse transcribed into cDNA at low temperatures, resulting in detection efficiency The reduction. Traditional RT-qPCR generally requires the participation of two key enzymes (reverse transcriptase and DNA polymerase), which makes it impossible to simplify the operation of the reaction system and reduce the cost. Here we will introduce two high-temperature-resistant reverse transcriptases, TtH and RevTaq. These two enzymes also have the function of DNA polymerase, so they are called bifunctional enzymes.

TtH DNA polymerase

You must have heard about TtH, which is derived from the thermophilic bacterium Thermus thermophilus HB8. In the presence of divalent cations such as Mg2+, it has DNA polymerase activity. It is widely used in PCR reactions like Taq enzyme, but it has higher heat resistance than Taq enzyme, so it also has a better effect on PCR with high GC content templates.

· This enzyme basically has no 3′→5′ exonuclease activity and 5′→3′ exonuclease activity, so it can also be used for dideoxy sequencing.

· This enzyme has RTase activity. In the presence of Mn2+, RTase activity will be enhanced. Using this feature, it can be used to perform reverse transcription reaction and PCR reaction in the same tube, that is, one-step RT-PCR. However, in the presence of Mn2+, the accuracy of RT-PCR is not high. RT activity has nothing to do with rnaase H activity.

· The increased activity of Tth-DNA polymerase (pH9, optimum +55℃～+70℃, maximum +95℃) overcomes the problems caused by RNA secondary structure. The resulting cDNA can be amplified by PCR with the same enzyme in the presence of Mg2+ ions.

· The ability of Tth-DNA polymerase to perform reverse transcription and DNA amplification at high temperatures makes this enzyme useful for quantitative RT-PCR, cloning, and gene expression analysis of cellular and viral RNA.
· Tth-DNA polymerase is used for RT-PCR to amplify RNA up to 1kb.

Features and advantages

Tth DNA polymerase:

• Ensure the optimized polymerase chain reaction (PCR) product size, at least 1000 bp in the RT-PCR reaction

• Accept modified deoxyribonucleoside triphosphate as a substrate

• Not related to RNase H activity

• Has high thermal stability to overcome problems, usually related to the high secondary structure present in RNA

RevTaq RT‐PCR DNA polymerase

A heat-resistant DNA polymerase with reverse transcriptase activity

RevTaq-RT-PCR-DNA polymerase is an engineered, highly heat-resistant, dual-functional enzyme with reverse transcriptase and DNA polymerase activities obtained through directed and artificial evolution.

· The half-life of RevTaq RT-PCR DNA polymerase at 95°C is greater than 40 minutes.

· RevTaq RT-PCR DNA polymerase allows high-temperature reverse transcription directly from the RNA template, and the reverse transcription step can be repeated multiple times to generate more cDNA templates.

· RevTaq RT-PCR DNA polymerase allows “zero-step” RT-PCR (no isothermal reverse transcription step), because in the cyclic PCR extension step, reverse transcription and DNA amplification occur simultaneously. This also promotes the reverse transcription reaction at high temperatures, thereby minimizing the problems encountered in melting the strong secondary structure in RNA at high temperatures.

· Due to the aptamer-based hot-start formula, RevTaq RT-PCR DNA polymerase will produce better results when the annealing and extension temperature is higher than 57°C.

· Since the enzyme is heat-resistant, it is recommended to design primers and probes with very high melting points (>60°C).

· It is recommended to optimize the temperature of the annealing/extension step through a temperature gradient during the reaction setup process.

· The higher the temperature, the higher the specificity of PCR. The reverse transcription cycle is usually performed at a higher temperature than the PCR cycle, because DNA Primer:RNA Template hybridization usually has a higher melting point than DNA Primer:cDNA Template duplex.

· RevTaq RT-PCR DNA polymerase is genetically engineered and optimized, and the amplicon size is between 60-300 bp.

RevTaq RT-PCR DNA polymerase detection limit reaches 4 copies/

The optimized reaction system (the establishment of high melting point primers) is shown in the figure. RevTaq RT-PCR DNA polymerase-driven RT-PCR shows better sensitivity than TaqPath 1-step RT-qPCR master mix, and lower detection Sample dilution gradient.

More advantages:

Quick start function → The initial thermal denaturation step can be skipped.

Hot-start aptamer formula → Provide 100% enzyme activity immediately and prevent non-specific amplification at low temperatures (<57°C).

Cleavage function → The RNA extraction step is omitted, because RevTaq RT-PCR DNA polymerase can also process crude reaction samples. It can immediately destroy the cell membranes of eukaryotes, bacteria and viruses in a hot RT-PCR cycle.

IVD raw material level → high quality standards and extremely competitive prices