CEL I enzymatic detection of the presence and location of a mutation in a PCR product is a high throughput reliable method that complements traditional methods of mutation detection. The benefits are: very low levels, if any, of false positive or false negative conclusions, the ability to detect all mismatches, minimal interference by flanking sequences, the ability to detect mutations in kilobase long fragments, the ability to detect two mutations in the same fragment, and the ability to detect some mutations in a mixture of 30 normal alleles.
Fluorescent labeled PCR primers are used to PCR segments of a gene of interest. Two alleles of the same segment can be denatured and renatured to form heteroduplexes at the site of mutations. CEL I is a mismatch endonuclease studied in Dr. Yeung's laboratory. CEL I nicks one strand of the DNA at the 3' side of the mismatch in the heteroduplex. The length of the truncated PCR fragments are measured with Perkin Elmer's GeneScan method, using the automated DNA sequencers Model 373 or 377. Truncated fragments of the two colors, from the upper strand and the lower strand of the PCR product heteroduplexes, respectively, independently pin-point the site of the mutation.
It can easily be adapted to other genes of interest by making appropriate sets of fluorescent PCR primers. Please contact Kate Oleykowski for further information.
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Oleykowski, C. A., Bronson Mullins, C. R., Godwin, A. K., and Yeung, A. T. (1998) Mutation detection using a novel plant endonuclease. Nucleic Acids Research, 26, 4597-4602.
CA_Oleykowski@FCCC.edu
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