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Fannie E. Rippel Biochemistry and Biotechnology Facility
Fox Chase Cancer Center

CEL I mutation detection Assays

Yeung screening veggies from Italian Market

Thank you for your interest in CEL I. We hope you will find it useful for your work. The enzyme has been licensed exclusively to Transgenomic Inc. Transgenomic Inc. and sold under the trade name of SURVEYOR as kits. I will put you in contact with the right person if you would send me a brief email. The SURVEYOR uses CEL II which is more mismatch-specific than CEL I. My lab now uses these kits exclusively for our mutation detection purposes.

Our laboratory discovered the CEL I family of mismatch endonuclease, purified the celery enzyme and cloned the celery gene. The role of CEL I in the cell may be to initiate the degradation of the chromatin at the early stages of cell death. In the test tube, however, it is an effective reagent for the detection of nucleotide mis­match. CEL I requires both Zn++ and Mg++, and has a neutral pH optimum. CEL I is a very stable enzyme that can be inactivated by removal of the Zn++.

We have published two CEL I mutation detection protocols. One uses the single-strand nicking activity of CEL I. The other uses double-stranded breaking activity of CEL I when the enzyme is present in much higher concentrations. Other users are developing interesting applications as well.

In a review article Enzymatic mutation detection technologies. BioTechniques?, 38:749-758, 2005. I discussed the parameters I think are important to the design of mutations detection in general as well as introduce the use of the Agilent Bioanalyzer for CEL I mutation detection.

It is necessary to pay attention to the quality of the PCR products, the size of the amplicon, and the method of fragment detection. Amplicons less than 500 basepairs are best. When using CEL I for mutation detection, the quality of your PCR reaction can be your biggest problem. Long-distance PCR products and bad PCR reactions can produce too many PCR generated mismatches that are cut by CEL I, and lead to non-specific DNA nicking. Fluorescence 5' end-labeled products allow visualization of only those fragments that retain the 5' tag of that strand. Fragment analysis methods that provide high dynamic range of signal detection allow detection of low abundance CEL I cut fragments over background noise. CEL I cuts provide single-nucleotide resolution of the mismatch cutting event, therefore fragment analysis methods that provide single-nucleotide resolution will minimize the amount of noise versus the CEL I signal.

When doing a CEL I assay, it is important to know that there is some exonuclease activity associated with CEL I. As a result, if you try to use excess CEL I to cut all the heteroduplexes, you may destroy the entire signal and generate high background. It is true that for the best-behaved mismatches, you can make CEL I work like a restriction enzyme. In practice, we normally cut only 2 to 20% of the heteroduplexes such that most of the full-length PCR product is untouched. This way, we can also pick up multiple mismatches in the same substrate. The CEL I signal peak is in transition from the full-length to the fragment of truncation at mismatch to degradation to short oligos. It is important to use as little CEL I as needed for a signal in your fragment analysis instrument.

Lastly, dilution of CEL I should be done in a buffer containing 0.1% Triton X-100. This finding is consistent in the hands of several lab members. This fact was neglected to be mentioned in our publications. We happened to use enzyme grade 10% Triton X-100, but not sure if that is really better than other grades of Triton X-100. For our purpose, we do not dilute the SURVEYOR nucleases because we use close to single turn over kinetics of more enzyme and shorter time for higher signal to noise ratio.

Updated May 7, 2008

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Created by: admin Last Modification: Tuesday 16 of December, 2008 22:14:26 EST by yeung