A common source of non-specific amplification is the extension of mismatched sequences by DNA polymerase and the formation of primer dimers. Although DNA polymerases are less active at low temperatures and non-specific amplification can be reduced by preparing PCR reaction mixtures on ice; however, during the reaction setup, DNA polymerases can catalyze the extension of mismatched primers or the formation of primer dimers, thus resulting in non-specific amplification that interferes with normal amplification and interpretation of results. Therefore, a more effective way to improve the specificity of PCR is to use hot-start DNA polymerase. Among them, Taq antibody-modified hot-start DNA polymerase have been widely used because of their fast hot-start speed and high sensitivity.
How Taq antibody-modified hot-start DNA polymerase works?
After Taq DNA polymerase binds to its antibody (Taq antibody), its activity is efficiently inhibited. During the initial high-temperature denaturation step (e.g., >90℃), the bound antibodies are degraded, activating the Taq DNA polymerases. By using Taq antibody to inhibit the activity of Taq DNA polymerase at room temperature, non-specific amplification caused by primer mismatch can be avoided and amplification specificity can be improved.
Novoprotein provides high-quality and strictly quality-controlled Taq antibody (Cat. No.: Z087) to help you obtain highly specific PCR results. In addition, we also provide glycerol-free Taq antibody (Cat. No.: Z087-03) to assist the development of lyophilized reagents.
Features
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Good activity inhibition effect, high-efficiency affinity Taq DNA polymerase, precise inhibition of enzyme activity.
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Fast hot-start, release Taq DNA polymeraseactivity at the same time of DNA initialdenaturation.
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High stability, no effect on the activity inhibition effect of Taq DNA polymeraseafter placed at 37℃for 7 days.
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Strict quality control, no residues of endonuclease, exonuclease, RNase, DNase, etc.
Performance
1. Specificity verification
The specificity of amplification was significantly improved by the Taq antibody-modified Taq DNA polymerase compared with the unmodified Taq DNA polymerase.
The Taq antibodies from company c and Novoprotein were incubated with Taq DNA polymerase to obtain hot-start Taq DNA polymerase for target gene amplification. The amplification performance was compared with the finished hot-start Taq DNA polymerase (Control). The hot-start Taq DNA polymerase incubated with Novoprotein Taq antibody had better specificity.
2. Repeatability verification
The hot-start Taq DNA polymerase incubated with Novoprotein Taq antibody (Test2) has better repeatability than the finished hot-start Taq DNA polymerase (Control) and the control antibody (Test1).
3. Amplification efficiency verification
The amplification efficiency of hot-start Taq DNA polymerase incubated with Novoprotein Taq antibody (Test2) was better compared with that of the finished hot-start Taq DNA polymerase (Control) and the control antibody (Test1).
4. Multiplex amplification validation
Compared with the finished hot-start Taq DNA polymerase (Control), the hot-start Taq DNA polymerase incubated with Novoprotein Taq antibody is able to perform 10-20 plex PCR amplification well with as low as 1ng of template.
5. Probe-based duplex qPCR performance verification(glycerol-freeTaq antibody)
Glycerol-containing Taq antibody and glycerol-free Taq antibody have the same sensitivity in probe-based duplex qPCR detection.
6. Stability test
The Taq antibody was placed at 37°C for 7 days and then was incubated with Taq DNA polymerase for amplification, there was no difference in the results.
7. RNase residue detection
The addition of RNase Inhibitor in the amplification system did not affect the amplification Ct value, proving that there was no RNase contamination.
8. DNase residue detection
1U, 2U, and 10U Taq antibody were added to the 100bp DNA Ladder, respectively. After incubation at -20℃ and 37℃ respectively for 2 hours, the integrity of the DNA Ladder was detected by electrophoresis, and the results showed that there was no DNase contamination.
Thanks to the customer for kindly providing some of the data plots.