Multiplex Detection and Genotyping of Point Mutations Involved in Charcot-Marie-Tooth Disease Using a Hairpin Microarray-Based Assay

Abstract EN

We previously developed a highly specific method for detecting SNPs with a microarray-based system using stem-loop probes.

In this paper we demonstrate that coupling a multiplexing procedure with our microarray method is possible for the simultaneous detection and genotyping of four point mutations, in three different genes, involved in Charcot-Marie-Tooth disease.

DNA from healthy individuals and patients was amplified, labeled with Cy3 by multiplex PCR; and hybridized to microarrays.

Spot signal intensities were 18 to 74 times greater for perfect matches than for mismatched target sequences differing by a single nucleotide (discrimination ratio) for “homozygous” DNA from healthy individuals.

“Heterozygous” mutant DNA samples gave signal intensity ratios close to 1 at the positions of the mutations as expected.

Genotyping by this method was therefore reliable.

This system now combines the principle of highly specific genotyping based on stem-loop structure probes with the advantages of multiplex analysis.