Abstract
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.
Highlights
The need for efficient large-scale genotyping methods has rapidly increased over the last ten years
We show here that the multiplex genotyping of multiple mutation sites is possible, based on multiplex polymerase chain reaction (PCR) DNA amplification and a stem-loop probe method
All DNA sequences for the three genes known to be involved in Charcot-Marie-Tooth disease (CMT) disease, for healthy individuals and patients, were confirmed by direct sequencing
Summary
The need for efficient large-scale genotyping methods has rapidly increased over the last ten years. Microarray-based methods offer a convenient solution to this need, but multiplexing is essential to increase the power of this approach for detection and discrimination This multiplexing can be achieved in several ways, including reducing the complexity of the genome before genotyping. This is achieved by cleaving genomic DNA into fragments with a restriction enzyme and introducing common adaptor sequences into the restriction products by ligation. These common sequences are used as binding sites for common PCR primers [1, 2]. Long-range PCR for amplifying long PCR fragments, up to 10 kb in length, in each individual PCR has been described [4, 5]
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