| The MIDAScan AT Mutation
Detection kit allows for the rapid scanning of A:G and G:G mutations by
heteroduplex analysis. DNA from wild-type sequences and sample sequences
are PCR amplified using labeled oligonucleotide primers. The amplified
DNA fragment is then heat-denatured, mixed together, and reannealed by
slow cooling in a hybridization-reaction buffer. The reannealed mixture
is incubated in the presence of the TDG enzyme from Methanobacterium
thermoautotrophicum which cleaves the DNA at sites of A:G and G:G
mismatches. Following the reaction, the DNA fragments are resolved by
gel or capillary electrophoresis. Results from the MIDAScan AT
reaction clearly indicate the presence or absence of a T mutation as
well as the relative position of that mutation with respect to the
labeled DNA end. Unlike other scanning methods, the MIDAScan AT provides
a simple and cost effective searching method without the need for
special equipment and results can be obtained in as little as one day.
The results are easily interpreted and do not require time consuming gel
scanning or sequence reading.
The MIDAScan kits provide a unique (patent pending) set of control
DNAs to produce every possible base mismatch upon heteroduplex
annealing. The DNA is provided as restriction fragments that are labeled
using the Klenow fragment of DNA polymerase. Following labeling, the two
DNA fragments are denatured, mixed, then allowed to reanneal. This
combination produces heteroduplexes with every mismatch represented at
20 base pair intervals. When cleaved by the mismatch repair enzyme, it
produces a defined pattern for each enzyme with mismatch activity. |
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