AK Taq DNA Polymerase V2 is developed based on the wild-type Taq DNA polymerase. Through genetic engineering modification, its amino acids are mutated to enhance the binding ability with DNA templates. This modification significantly improves its anti-interference capability against impurities in PCR templates, enabling it to amplify target fragments normally even under conditions where conventional wild-type Taq polymerase fails to do so. Meanwhile, this enzyme retains the 5’→3’ exonuclease activity of Taq polymerase and can be used in probe-based quantitative real-time PCR (qPCR) detection. Additionally, two Taq monoclonal antibodies are added to modify the original enzyme, which enhances the blocking effect on different active sites of Taq polymerase and improves specificity.
Before high-temperature heating, the Taq monoclonal antibody targeting the 5’-3’ polymerase active site of Taq binds to Taq polymerase to inhibit its polymerase activity, thereby suppressing non-specific amplification caused by non-specific annealing of primers or primer dimers under low-temperature conditions. The monoclonal antibody targeting the 5’-3’ exonuclease active site of Taq binds to Taq polymerase to inhibit its exonuclease activity, reducing the degradation of primer-probes by Taq polymerase in the pre-mixed system and ensuring the stability of the pre-mixed system.
When the amplification reaction system is heated to 95°C for 2.5 minutes, the polymerase activity is restored due to the denaturation of the anti-Taq monoclonal antibodies. Therefore, no special inactivation treatment is required, and the enzyme can be used under conventional PCR reaction conditions. When used in conjunction with the improved buffer, it can be activated quickly, effectively increasing the amount of reaction products and improving the sensitivity and specificity of PCR reactions. It can be applied in rapid PCR and amplification experiments of samples containing relatively high levels of interfering substances, such as blood, soil, plant sections, and sputum.
Scope of Application
Hot-start PCR amplification, direct whole blood amplification, anti-interference amplification, rapid PCR
Precautions
Primer Design
1. The last base at the 3' end of the primer is preferably G or C.
2. Continuous mismatches should be avoided in the last 8 bases at the 3' end of the primer.
3. Hairpin structures should be avoided at the 3' end of the primer.
4. It is optimal that the Tm value difference between the forward primer and the reverse primer does not exceed 1°C, and the Tm value is preferably adjusted to 55–65°C (Primer Premier 5 is recommended for calculating the primer Tm value).
5. The additional sequences of the primer (i.e., sequences not paired with the template) should not be involved in the calculation of the primer Tm value.
6. The GC content of the primer should be controlled between 40% and 60%.
7. The overall distribution of A, G, C, and T in the primer should be as uniform as possible, and regions with high GC or AT content should be avoided.
8. Complementary sequences of more than 5 bases should be avoided within a single primer or between two primers, and complementary sequences of more than 3 bases should be avoided at the 3' ends of the two primers.
9. After primer design, the NCBI BLAST function should be used to check the primer specificity to avoid non-specific amplification.
Other Precautions
1. Excessive template DNA is likely to lead to the production of non-specific PCR products.
2. For rapid amplification of qualitative long fragments (≥1 kb), it is recommended to use 5×AK Taq Fast Buffer (without Mg²⁺).
Usage Method
Preparation before Reaction System Configuration
1. Dissolve and mix all solutions required for the reaction at room temperature or 4°C, then place them on an ice bath or in an ice box. It is recommended to aliquot the reaction solutions for use to avoid repeated freezing and thawing.
2. Refer to the table below to set up the quantitative real-time PCR (qPCR) reaction system. It is recommended that the preparation of the qPCR reaction system be carried out on an ice bath or in an ice box:
Component
| Volume Added
| Final Concentration
|
|---|
| 5×AK Taq Buffer（without Mg²⁺） | 10μL | 1× | | dNTP（25mM each） | 0.4μL | 0.2mM | | Forward Primer（10 μM） | 1μL | 0.2μM | | Reverse Primer（10 μM） | 1μL | 0.2μM | | Probe（10 μM） | 0.5μL | 0.1μM | | AK Taq DNA Polymerase V2（5 U/μL） | 0.5μL | 0.05U/μL | | 100mM MgCl₂ | 1μL | 2mM | | Template DNA | 10μL | — | | ddH₂O | To 50μL | — | | 终体积 | 50μL | — |
|
3. The working concentration of MgCl₂ can be adjusted within the range of 1–3 mM; excessively high Mg²⁺ concentration may lead to an increase in non-specific products.
4. Recommendations for the use of two types of templates are as follows:
Mammalian genomic DNA: 0.1–1 μg
E. coli genomic DNA: 10–100 ng
5. When used for quantitative real-time PCR (qPCR), the volume of whole blood added is recommended to be ≤ 5% (v/v) of the reaction volume; excessive addition of blood samples will affect the collection of fluorescent signals.
6. For rapid qualitative PCR amplification of long fragments, especially for fragments ≥ 1 kb, it is recommended to use 5×AK Taq Fast Buffer. However, 5×AK Taq Fast Buffer is not suitable for qPCR, as it will cause a decrease in the fluorescence signal of the amplification curve.
Reaction Procedure
1. Conventional Qualitative PCR
| Step Name | Temperature | Time | Number of Cycles |
|---|
| Heat Shock | 95°C | 2min30s | 1 | | Denaturation | 95°C | 10s | 30 - 35 | | Annealing | 55°C | 20s | 30 - 35 | | Extension | 72°C | 20s | 30 - 35 | | Extension | 72°C | 7min | 1 | | Storage | 12°C | ∞ | 1 |
|
2. Rapid PCR (Taking the Amplification of 1kb λDNA as an Example)
| Step Name | Temperature | Time | Number of Cycles |
|---|
| Heat Shock | 95°C | 2min30s | 1 | | Denaturation | 98°C | 1s | 30 - 35 | | Annealing | 55°C | 5s | 30 - 35 | | Extension | 72°C | 5s | 30 - 35 | | Extension | 72°C | 5min | 1 | | Storage | 12°C | ∞ | 1 |
|
3. Fluorescent Quantitative PCR：
| Step Name | Temperature | Time | Number of Cycles |
|---|
| Heat Shock | 95°C | 2min30s | 1 | | Denaturation | 95°C | 15s | 35 - 40 | | Annealing + Extension | 55°C | 40s | 35 - 40 |
|
3.1 The settings of the PCR reaction need to be determined according to different conditions such as the template, primers, length of the PCR product, and GC content. Different PCR reaction conditions include temperature, time, number of cycles, etc.
3.2 The extension time needs to be set according to the length of the PCR product. Usually, the fastest extension time for each kb of product is 5 seconds. For example, if the length of the PCR product is 1 kb, the extension time can be set to 5 seconds; if the length of the PCR product is 2 kb, the extension time can be set to 10 seconds. If the result is not satisfactory, it can be appropriately extended to 15 seconds, and so on for fragments of other lengths.
Experimental Examples
Extension Rate:
AK Taq V2 shows stronger extension performance. When the extension time is 5 seconds and the enzyme amount is as low as 0.15625 U, it can still amplify a 1 kb fragment.
5’-3’ Exonuclease Activity Blocking:
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Under different temperature conditions, AK Taq V2 has a better blocking effect on 5’-3’ exonuclease activity and can effectively reduce the degradation of primer-probes.
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