DHPLC in clinical molecular diagnostic services

A high-capacity low-cost mutation scanning method based on denaturing high-performance liquid chromatography (DHPLC) has been recently introduced. We have implemented an automated and cost-effective strategy using DHPLC. To facilitate the semi-automated analysis of multiple exons, two steps were taken. The first step was the development of a PCR protocol for the amplification of multiple exons under the same conditions. Primer sets, which amplify each exon in the entire gene, were aliquoted to and air-dried on a 96-well format PCR plate. In this way, all the exons in a gene can be simultaneously amplified on a single PCR machine. The second step was the serial DHPLC analysis of multiple amplicons under conditions optimal for each amplicon. We named the 96-well plate containing the primer pairs and the corresponding computer file used to analyze each amplicon under the pre-determined optimal conditions as the "Condition-Oriented-PCR primer-Embedded-Reactor plate," or the COPPER plate. We have developed COPPER plate systems for more than 20 congenital disorders including classic congenital syndromes like Marfan syndrome (FBN1: 65 amplicons), CHARGE syndrome (CHD7: 39 amplicons), de Lange syndrome (NIPBL: 46 amplicons), Sotos syndrome (NSDI: 30 amplicons), and Rubinstein-Taybi syndrome (CREBBP: 41 amplicons). Using the COPPER plate system, we are functioning as a reference laboratory for the clinical molecular diagnosis of congenital malformation syndromes and are presently analyzing more than 200 samples annually from all over Japan. (c) 2005 Elsevier Inc. All rights reserved.