Description:

Supreme NZYProof DNA polymerase is an engineered highly accurate, fast and sensitive variant of NZYProof DNA polymerase displaying a hot-start like PCR capacity. This feature is achieved by a novel hot-start technology, which inhibits both polymerase and 3’→5’ exonuclease activities and thus avoids extension of non-specifically annealed primers or primer-dimers, as well as the degradation of primers and template DNA during PCR reaction setup. Thus, Supreme NZYProof DNA polymerase generates higher specificity, sensitivity and yield during the accurate amplification of DNA. Supreme NZYProof DNA polymerase was also engineered for higher processivity, thus allowing fast PCR reactions of longer PCR products.

This highly robust version of NZYProof DNA polymerase is a broad range enzyme suitable for a variety of applications, including amplification of longer (≤10 kb) and difficult PCR products, as well as site-directed mutagenesis. Supreme NZYProof DNA polymerase possesses 3´→5´ exonuclease proofreading activity that enables the polymerase to amplify DNA with increased accuracy. The error rate of Supreme NZYProof DNA polymerase is similar to that of Pfu and Kod DNA polymerases and significantly lower than the error rate of Taq DNA polymerases. It generates blunt-ended PCR products that are suitable for cloning with NZYTech´s NZY-blunt PCR cloning kit (MB121).

Features:
– High sensitivity
– Eliminates nonspecific amplifications and minimizes primer-dimers
– High processivity/ Fast amplification (15-30 sec/kb)
– Proofreading activity

 Applications:
– High-fidelity PCR
– Hot-start PCR
– Amplification of difficult-to-amplify targets (e.g. high GC-contents)
– Site-directed mutagenesis
– Generation of products for blunt-end cloning

High specificity hot-start PCR 
Supreme NZYProof DNA polymerase was engineered to exhibit extreme specificity, enabling accurate amplification of a broad range of DNA templates with different sizes and concentrations. During reaction setup, Supreme NZYProof DNA polymerase is inactivated through a novel hot-start technology, which inhibits both polymerase and 3’→5’ exonuclease activities of the enzyme at room temperature, as well as on ice. Supreme NZYProof DNA polymerase does not require a separate activation step during the PCR cycling, since its activity is immediately restored at high temperatures.

Three E. coli genomic DNA fragments of different lengths (1, 2.5 and 5 kb) were amplified with Supreme NZYProof DNA polymerase (2.5 U). Three 4-fold gDNA dilutions ranging from 40 to 2.5 ng were used as starting template to amplify each DNA fragment. NC: No template control. Lane M: NZYDNA Ladder III (MB044). Results demonstrate amplification in all the conditions tested with high yields and specificity.

Specifications:

Components:
– Supreme NZYProof DNA polymerase (2.5 U/μL)
– Reaction buffer (5x)

1. What to do when there is no product amplification or low yield?
This can be due to the following situations:
a) Inadequate annealing temperature. The reaction mix composition may affect the melting properties of primers and DNA. Adjust the annealing temperature to accommodate the primer with the lowest melting temperature (5 ° to 10 °C lower than T_m).
b) Presence of PCR inhibitors. Some DNA isolation procedures, particularly genomic DNA isolation, can result in the co-purification of PCR inhibitors. Reduce the volume of template DNA in reaction or dilute template DNA prior to adding to the reaction. Diluting samples even 1:10,000 has been shown to be effective in improving results, depending on initial DNA concentration.

2. How should I store my Supreme NZYProof DNA polymerase?
The product should be stored at -20 ºC, in a constant temperature freezer.

3. I’d like to clone a fragment amplified with Supreme NZYProof DNA polymerase. How should I proceed?
Supreme NZYProof DNA polymerase has 3´→ 5´ exonuclease activity and will generate blunt-ended products. So, blunt-end cloning is recommended, by using NZY-blunt PCR cloning kit (MB121) or other similar cloning kit. Nevertheless, if TA cloning is required, the generated blunt-ended PCR fragments can be modified by adding 3´A-overhangs with a different polymerase, such as Taq DNA polymerase (see the A-tailing protocol in the product brochure of NZY-A PCR cloning kit – MB053). Due to the strong proofreading activity of Supreme NZYProof DNA polymerase, we recommend immediately proceed with cloning after this procedure, in order to avoid lost of the 3´A-overhangs during storage. Alternatively, the PCR products can be cloned using the innovative NZYEasy Cloning & Expression Systems in a rapid, precisely and ligase-independent manner. For optimal cloning efficiencies, silica-column purification of the PCR product using NZYGelpure kit (MB011) or other similar kit is highly recommended. This procedure will remove dNTPs, unused primers and other impurities. Spin-column purification is sufficient as long as the product is >90% pure. Gel-extraction should be performed in case non-specific amplifications and/or primer-dimers are formed.

4. Will Supreme NZYProof DNA polymerase incorporate dUTPs?
No. Supreme NZYProof DNA Polymerase will not incorporate dUTPs, which maximizes the efficiency of high-fidelity PCR.