A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis ( A series of mutations was designed...)

A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis

A series of mutations was designed across an amplified region of hMSH2 exon 4. To accomplish the mutagenesis, a series of exon 4 specific PCR primers each with a single base change corresponding to the designed mutation was synthesized. The incorporation of the single base change within the primer resulted in the site-directed mutagenesis of the amplified product. As the mutagenic primers were internal to a larger exon 4 fragment, subsequent amplification of the larger product resulted in an amplified exon 4 containing a known mutation at a known site. The amplified, mutagenized fragment was then cloned into a pGEM-T vector and selected clones were sequenced to verify the presence of the mutation. All subsequent DGGE model experiments were then completed using the cloned material.

PRIMER DESIGN
The design of DGGE primers follows the general principles of PCR primer design with respect to sequence specificity, lack of internal homology and minimal primer dimer formation. In addition, a GC-rich sequence or clamp is often appended to one of the primers. Such a clamp can be of various lengths and base composition as determined by its ability to produce a single melting domain for the sequence under study. For clinical laboratory use additional criteria include: uniform PCR conditions, amplification of exonic regions inclusive of exon/intron boundaries and a minimum of different DGGE conditions.

The exon 4 model used in this study is an exception to some of these criteria; its purpose was to test the limits of the DGGE assay. To illustrate the general appearance of melt profiles, Figure 1a shows the profile of amplicons with a high divergence from the ideal profile. Figure 1b shows the i
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( A series of mutations was designed...)A Multiple Mutation Model System as a Test Development and Training Tool for Denaturing Gradient Gel Electrophoresis