DESCRIPTION  

TOROIVD^® 5G DNA polymerase is an engineered Tth DNA polymerase for improved processivity, sensitivity and inhibitor tolerance through a process of directed evolution.The anti-Tth DNA polymerase antibodies was used in the enzyme solution to inhibit polymerase activity for Hot Start PCR. Like wild-type Tth DNA polymerase, The enzyme has a reverse transcriptase activity in addition to a 5’→3’ polymerase activity and a double strand specific 5’→ 3’ exonuclease activity in the presence of Mn²⁺ ions. Therefore, this enzyme also enables "1-step RT-qPCR" including the reverse transcription and PCR steps.

The enzyme is supplied with three reaction buffers, qPCR Buffer GB, qPCR Buffer T and RT-qPCR Buffer R. 2×5G qPCR Buffer GB is specifically formulated for  high sensitivity qPCR or 1-step RT-qPCR with MMLV Reverse transcriptase, while 2×5G qPCR Buffer T is recommended when template samples are contaminated with PCR inhibitors. 2×5G RT-qPCR Buffer R is recommended for 1-step RT-qPCR in the presence of Mn²⁺ ions.  

Features

-Rapid and highly sensitive
  With 2×5G qPCR Buffer GB, this polymerase can achieve the rapid and highly sensitive quantification of a low-copy targets with probes and be suitable for the quantification of DNA viruses or mRNA expressed at a low level.

-Optimized for multiplexing

  With 2×5G qPCR Buffer GB, this polymerase has been validated for multiplexing up to five targets simultaneously, allowing for additional targets and/or controls to be run simultaneously for efficiency or quality control purposes.

-Inhibitor tolerant

  With 2×5G qPCR Buffer T,the unique properties of this polymerase allows robust performance even in the presence of substances that can normally inhibit PCR, such as heparin, hematin, or EDTA, increasing your confidence when working with a variety of   complex clinical samples.

-Be flexible.
  With 2×5G RT-qPCR Buffer R, TOROIVD^® 5G DNA polymerase enables amplification of both RNA and DNA targets in the presence of Mn²⁺ ions. Due to high temperature reverse transcription at +60 to +65°C and improved RNA processivity, low-copy RNA targets amplification can be achieved reliably.

Components

Storage Buffer

 
10 mM Tris-HCl (pH7.0), 50 mM KCl, 50% Glycerol, etc.

Reaction Buffer

Notes: ASF-101GB and ASF-101T contains 0.4mM dA/C/G/UTP, 5mM Mg²⁺, PCR buffer and stabilizer，etc.).

ASF-201R contains 0.4mM dA/C/G/UTP, PCR buffer and stabilizer.

Quality control

Each batch of TOROIVD^® 5G DNA polymerase is confirmed to contain <2% contaminating protein by SDS-PAGE. TOROIVD^® 5G DNA polymerase are subjected to stringent quality control tests, are free of contaminating exo- and endonuclease activity, and meet strict requirements with respect to DNA contamination levels.

Protocol

(1)  qPCR Protocol for DNA amplification  

Step 1: Prepare the reaction mixture 
• Ensure that all reagents are properly thawed and mixed.
• Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.
• Calculate the required volumes of each component based on the following table:

 
• Gently mix the reaction solutions and spin down in microcentrifuge.

Step 2: Run the qPCR
• Perform PCR with the following cycling protocol:    

(2) 1-Step RT-qPCR Protocol I  

 
Step 1: Prepare the reaction mixture

• Ensure that all reagents are properly thawed and mixed.
• Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.
• Calculate the required volumes of each component based on the following table:

• Gently mix the reaction solutions and spin down in microcentrifuge.

Step 2: Run the RT-qPCR
      • Perform PCR with the following cycling protocol:

(3) 1-Step RT-qPCR Protocol II

Step 1: Prepare the reaction mixture

• Ensure that all reagents are properly thawed and mixed.
• Prepare a PCR master mix containing the appropriate volume of all reaction components common to all or a subset of reactions to be performed.
• Calculate the required volumes of each component based on the following table:

• Gently mix the reaction solutions and spin down in microcentrifuge.

Step 2: Run the RT-qPCR
• Perform PCR with the following cycling protocol:

Important Parameters:

(1) Primer/probe design and optimize

-Design of primers

Primer length: 18–25bp; Tm of primer: 60–65°C; GC content: 40–60%;

Target length: 70–200 bp; Larger targets (>200 bp) tend to reduce the efficiency and specificity of amplification.

Purification grade: OPC or HPLC grade;

-Design of probes

Probe length: 20–30bp; Tm of probe: 65–70°C; GC content: 40–60%;

Purification grade: HPLC.

-Checking the performance of primers and probes：

Prepare a dilution series with five or more dilutions of template DNA or RNA.

 Perform qPCR assay using the diluted template with the newly designed primers and probe, and draw a standard curve.

Confirm that the PCR efficiency is between 90% and 110% and R² is equal to or greater than 0.99. If the PCR efficiency or R2 are outside of these ranges, the reaction conditions should be optimized. If this does not improve the result, the primers and probe should be redesigned.

- Optimizing the concentration：

The primer concentration should be optimized between 0.2-0.8 µM and the concentration of TaqMan^® probe should be optimized between 0.1-0.4 µM. Increasing the primer concentration can improve the PCR efficiency in cases in which the annealing of primers is insufficient, and decreasing may improve the efficiency in cases with primer-dimer formation. Similarly, increasing the TaqMan^® probe concentration can increase the sensitivity in cases in which the annealing of TaqMan^® probes is insufficient and a lowconcentration of TaqMan^® probe ofless than 0.1 µM decreases the sensitivity because of low fluorescence intensity.

(2) Components optimize

- Polymerase concentration：TOROIVD^® 5G DNA polymerase concentration can be optimized between 0.6-1.2U/20µL reaction.

- MgCl₂  concentration：2.5mM MgCl₂ final concentration have been added in this reaction mixture. But for the direct qPCR to crude template DNA, the MgCl₂ concentration may need to be optimized between 2.5-8 mM.

- About ROX：-The 50× ROX is not supplied with this kit. It is recommended that you use 50×ROX (ROX-050) optionally. 50× ROX reference dyes are used for analyses with instruments that correct for cross-talk between wells, such as the real-time PCR instruments by Applied Biosystems and Agilent Technologies. 0.8ul 50×ROX Reference Dye was added for a total 40ul reaction volume in when using the following instruments, Applied Biosystems 7300/7700/7900HT, StepOnePlus, etc. And 0.08ul was added for using the following instruments, Applied Biosystems 7500/7500Fast StepOne Plus, Agilent Technologies AriaMx, etc. No ROX Reference Dye is required when using other brand instruments, such as LightCycler 96 /LightCycler 480 system (Roche), CFX96 Real-Time PCR Detection System (Bio-Rad), Smart Cycler System (Cepheid), etc.

- About UNG: Reacrion Buffer contains dUTP, so the rate of false-positive detection can be reduced by adding uracil-N-glycosylase (UNG) (not supplied). The added volume and the indicated reaction condition are according to the instructions of the manufacturers.     

(3) Cycling conditions optimize

-The indicated initial denaturation temperature can be optimized 95-98°C,

and time between 10sec-5min.

-The indicated RT temperature of TOROIVD^®III  Reverse transcriptase can be optimized between 50-60°C, and time between 5-15min.

-The indicated denaturation temperature can be optimized 95-98°C,

and time between 3sec-10sec.

-The indicated Extension /Annealing temperature can be optimized 60- 65°C, and time between 5sec-31sec.

 
Thermal stability

TOROIVD^® 5G DNA polymerase maintained all the performance at 37°C for one week.

STORAGE

This reagent can be stored at 4°C for 1 months. For longer storage, this reagent should be kept at -20°C.