APPLICATION OF DHPLC FOR MOLECULAR ANALYSIS IN
GONADAL DISORDERS
Matsuo
N, Kosaki K
Keio
University School of Medicine, Tokyo, Japan
Objective: The more the geneticists unravel the genetic basis of endocrine
disorders, the more the practicing clinicians get frustrated because they
are confronted with a situation in which molecular diagnostic tests cannot
be offered to the patients simply because of the cost and labor associated
with such testing. A
cost-effective and automated strategy needs to be implemented.
Methods: Denaturing high-performance liquid chromatography (DHPLC), the
new technology, based on temperature-modulated liquid chromatography and a
high-resolution matrix was implemented in a way suitable for clinical
application. Primers were designed so that all of the primers would have
the same annealing temperature. In this way, all the exons can be amplified
simultaneously in a single PCR machine. Primer pairs were aliquoted on a 96-well
format PCR plate referred to as COPPER plate (Condition-Oriented-PCR
primer-Embedded-Reactor). The COPPER plate was
accompanied with a corresponding computer programs optimized for DHPLC
analysis of the particular PCR product.
Results: COPPER plate and accompanying computer programs were developed
for analysis of the androgen receptor gene, the 5 alpha reductase gene, and
the arylhydrocarbon receptor repressor gene. All the exons could be
analyzed sequentially by the optimized method and all the operation will be
totally automatic once the operator sets the plate and starts the “gene-specific”
analysis program. We have finished screening >100 patients with
micropenis in a few months.
Conclusions:
The DHPLC technology, when complemented with
the COPPER plate system, offers an ideal tool for mutation analysis in
clinical applications. The key aspects to setting the operating parameters
will be discussed.