( ssay. Since this work was completed a ...)A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis

ssay. Since this work was completed, a redesigned set of clamped primers is now used to analyze exon 4, reducing the total number of primer pairs from the three used in this work to two sets of GC-clamped primers pairs. Through the use of GC-clamps of various lengths and GC content it is possible to reduce the number of melting domains in virtually all fragments to a single uniform temperature. With careful planning and design of primers and gel conditions, it is possible to analyze large genes in a relatively high throughput manner while maintaining a high degree of sequence alteration detection. Sequencing to determine the precise character of a mutation is then reduced to a single easily analyzed region rather than an entire gene. In summary, DGGE is a robust, reliable technique applicable to both research and clinical endeavors.

References
1. Dianzani, I., Camaschella, C., Ponzone, A. and Cotton, R. G. H., Dilemmas and progress in mutation detection, Trends in Genetics, 9(12), 403405 (1993).

2. Lerman, L. S., Silverstein, K. Computational simulation of DNA melting and its application to denaturing gradient gel electrophoresis. Methods in Enzymology 155, 482501 (1987).

3. Myers, R. M., Lumelsky, N., Lerman, L. S. and Maniatis, T,. Detection of single base substitutions in total genomic DNA, Science, 313, 495498 (1985a).

4. Myers, R. M., Fischer, S. G., Maniatis, T and Lerman, L. S., Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis, Nucleic Acids Research, 13(9), 31113129 (1985b).

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