Nonspecific amplification of Taq DNA polymerase is a common issue in PCR experiments, which directly affects the interpretation of detection results and can lead to a decrease in amplification sensitivity, and even a reduction in the yield of the target fragment. Utilizing enzyme modifiers to seal the active center of Taq DNA polymerase at room temperature, thereby inhibiting the DNA polymerase activity of Taq enzyme at room temperature, is currently the most effective method to suppress nonspecific amplification. The dual-sealing antibodies developed by Yeasen not only seal the polymerase activity of Taq DNA polymerase but also simultaneously seal the exonuclease activity of Taq DNA polymerase. This dual approach effectively prevents nonspecific amplification caused by mispairing or primer dimers and also prevents the generation of nonspecific signals due to material degradation, thereby doubly enhancing the specificity of the reagent. This enables detection reagents to cope with transportation or room temperature use with ease. Features 1.Ultra-high amplification specificity—The dual-sealed antibody can simultaneously seal both polymerase and exonuclease activities, effectively avoiding nonspecific amplification and enhancing specificity. 2.Exceptional detection sensitivity—The hot-start formulation ensures effective detection of low-abundance genes, offering higher sensitivity. 3.Outstanding product stability—Stable in performance when placed at 37°C for 5 days or at 4°C for 10 days. 4.Baseline stability and good linearity—Addressing the issue of baseline drift, resulting in excellent linearity. 5.Rapid enzyme activity release—Over 95% of enzyme activity can be released by heating at 95°C for 20 seconds. 6.Broad applicability of enzymes—Suitable for both wild-type and mutant Taq enzymes, with each mg of antibody capable of sealing 4-10 KU of Taq enzyme. 1 Blocking of Taq DNA polymerase exonuclease activity Synthetic primer probes were respectively incorporated into the reaction mixtures of Taq DNA polymerase that were either unsealed or sealed with dual-closure antibodies, and the reactions were conducted at 40°C. The fluorescence signals of both were detected, and it was found that the dual-closure antibodies can effectively seal the exonuclease activity of Taq DNA polymerase. Figure 1. Detection of the sealing efficiency of double-sealed antibody-endonuclease activity. 02 Detection sensitivity verification Respectively using Yeasen's double-sealed antibody and T Company's antibody to seal Taq enzyme, followed by detection of positive samples through ARMS-PCR technology, it was found that the Taq enzyme sealed by Yeasen's double-sealed antibody was accurate in ARMS-PCR amplification with higher sensitivity. Figure 2. Amplification curve of ARMS-PCR. Red:YEASEN antibody+Taq; Blue:supplier A antibody+Taq. 03 Stability Verification (4°C for 10 days, 37°C for 5 days). Using traditional closed antibodies and double-closed antibodies to respectively block Taq DNA polymerase, the polymerase is then formulated into a complete premix solution containing primers and probes. This solution is left at 4°C for 10 days or at 37°C for 1, 3, and 5 days, and then used to amplify 10,000 copies of the ASF plasmid. It was observed that there were no significant changes in the amplification curves and Ct values . Figure 3. Amplification curves of 10,000 copies of ASF plasmid after Taq polymerase was blocked with traditional blocking antibody (A) and double-blocked antibody (B), amplify by the system of qPCR. 04 Baseline Detection In certain conditions where specific primers are used in conjunction with particular buffers and instruments, a phenomenon known as baseline drift may occur. However, the use of double-closed antibodies can effectively address the issue of baseline drift, thereby facilitating a more stable baseline. Figure 4. Amplification curves of SARS-CoV-2 N gene (A) and ACT gene (B) in qPCR analysis. 05 Linearity Verification The reaction mixture sealed with double-closed antibodies was used to amplify 100,000, 10,000, 1,000, and 100 copies of the ASFV/ACT dual plasmid to generate a standard curve. As can be seen from Figure 7, both exhibit good linearity. Figure 5. Standard curve graphs for ASFV gene and ACT gene generated using a double-block reaction mixture. 06 Enzymatic Activity Release Assay Using the double-closed antibodies (Cat#31303) from Yeasen to seal Taq enzyme, and subjecting it to a thermal shock at 95°C for 20 seconds, the enzymatic activity was detected. It can be observed that after a 20-second thermal shock at 95°C, 31303 can release more than 95% of the enzymatic activity, which is comparable to the antibodies from Company T. Table 1. Heat shock at 95°C for 20 seconds of the enzyme. 07 Multiplex Taq Polymerase Detection Yeasen's double-closed antibodies (Cat#31303) were used to seal three types of Taq polymerases (wild-type + two mutant types), with a sealing ratio of 1μg to 5U, and the fluorescence values and sealing efficiency were measured. It was found that for different types of Taq polymerases, Yeasen's double-closed antibodies (Cat#31303) exhibited good sealing effects. Table 2. Blocked efficiency of different types of Taq polymerase. Documents: Manuals 31303_Manual_HB230110 Related blog: The Double-Block anti-Taq DNA Polymerase Antibody to Help Reducing Nonspecific Amplification
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