( the conditions under which all alterati...)A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis

the conditions under which all alterations within that fragment should theoretically be resolved. The juxtaposition of GC-rich and AT-rich regions commonly seen within the human genome often result in complex melting profiles with adjacent domains demonstrating widely divergent melting temperatures. Such multiple melting domain fragments do not permit the resolution of sequence alterations in the higher melting temperature domains due to the more rapid melting of the lower temperature domains. The addition of a GC rich sequence, (GC clamp), to the 3' or 5' end of an amplicon results in a single melting domain across the fragment, thus allowing the detection of sequence variation across the entirety of the sequence (Myers, 1985c). The prediction of melting domains within a DNA fragment of known sequence has been reduced to a computer algorithm by Lerman and co-workers (Lerman, 1987). The melting profiles determined for the current work were performed using an adaptation of Lermans MELT program created by Bio-Rad, called MacMelt software.

This study presents our efforts within a model system to determine the feasibility of using DGGE in a clinical environment for the detection of mutations in the mismatch repair genes relevant to the development of Hereditary Non-Polyposis Colon Cancer. We selected exon 4 of the hMSH2 gene as a model system for analysis. A series of mutations was created by site directed mutagenesis in an exon 4 containing clone. The individual mutant-containing clones were then analyzed via DGGE to determine the limits of detection of the system.

Materials and Methods
SITE-DIRECTED MUTAGENESIS OF hMSH2 EXON 4

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