Rapid Genotyping of Common MeCP2 Mutations with an Electronic DNA Microchip Using Serial Differential Hybridization

Abstract

Rett syndrome is a neurodevelopmental disorder that affects females almost exclusively, and in which eight common point mutations on the X-linked MeCP2 gene are knows to cause over 70% of mutation-positive cases. We explored the use of a novel platform to detect the eight common mutations in Rett syndrome patients to expedite and simplify the process of identification of known genotypes. The Nanogen workstation consists of a two-color assay based on electric hybridization and thermal discrimination, all performed on an electronically active NanoChip. This genotyping platform was tested on 362 samples of a pre-determined genotype, which had been previously identified by a combination of DHPLC (denaturing high performance liquid chromatography) and direct sequencing. This genotyping technique proved to be rapid, facile, and displayed a specificity of 100% with 3% ambiguity. In addition, we present consecutive testing of seven mutations on a single pad of the NanoChip. This was accomplished by tagging down two amplimers together and serially hybridizing for seven different loci, allowing us to genotype samples for seven of the eight common Rett mutations on a single pad. This novel method displayed the same level of specificity and accuracy as the single amplimer reactions, and proved to be faster and more economical.

Nanogen Microelectronic Chip for Large-Scale Genotyping

“…This lowers the cost per SNP to almost one-third of the cost of using a new microelectronic chip and more lowers the cost compared with RFLP analysis by more than one-half. This is approximately the same cost reported by others for detecting eight SNPs on one test site simultaneously (€1.62 per SNP) (6 ). It should be noted, however, that purchase of the Nanogen NMW 1000 Nanochip Molecular Biology Workstation is not included in these calculations.…”

“…Other studies have also shown the multiplexing ability of the microelectronic chip by analyzing eight different SNPs on one test site simultaneously (6 ). Furthermore, compared with other microarray systems, the microelectronic chip allows simultaneous use of a wide variety of probes of different lengths and chemical compositions on the same chip.…”

An Overview of Current Microarray-Based Human Globin Gene Mutation Detection Methods

“…With well designed assays, including multiplex PCR, multiple addressing of uniplexed PCR-products to the same pad, or serial hybridization of reporters (involving intermediate stripping of previously hybridized reporters) ( Figure 1) (16,18,(23)(24)(25), costs per sample for nine β-globin mutations range between €5-8, including sample preparation and PCR amplification. A recently updated version of the platform (NanoChip 400; Nanogen Inc.) includes 400 pads per cartridge that can be scanned in a single day, further facilitating large-scale screening applications.…”

Microelectronic DNA chip for hereditary hyperferritinemia cataract syndrome, a model for large-scale analysis of disorders of iron metabolism

“…A serial hybridization format was performed by chemically stripping previously hybridized reporters (NaOH 0.1N) for 3 min [Frusconi et al, 2004;Thistlethwaite et al, 2003]. Amplicons were subjected to 24 subsequent hybridization steps, with all different sets of reporters specific for the 24 mutations.…”