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 program 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.