Biomedical Engineering Reference
In-Depth Information
Fig. 1. Schematic presentation of heteroduplex formation through hybridization.
ing part of the heteroduplexes close to the mismatched base single-stranded
whereas the homoduplexes remain double-stranded. The percentage of organic
mobile phase that disrupts the interactions between DNA fragments and the
DNAsep cartridge matrix is lower for heteroduplex DNA strands than for
homoduplex DNA strands. Therefore, heteroduplex DNA fragments elute ear-
lier in the gradient, and this signals the presence of a mutation.
DHPLC is fully automated, eliminating time and labor in gel preparation,
loading, running, analyzing, and photographic documentation. The polymerase
chain reaction (PCR) products are directly loaded without any prior purifica-
tion as would be required in various enzymatic methods and capillary electro-
phoresis. The separation efficiency and the optimized time for DNA fragment
elution and column equilibrium allow the analysis of up to 200 samples per
day on an instrument with a single analytical column.
A number of clinical applications for mutational analysis have been pub-
lished
(
2-9
)
, and disease-causing genes that have been screened by DHPLC
can be found on the website of Dr. Oefner
(http://insertion.stanford.edu/
