title: 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.
authors.affiliation: Research Center for Genetic Medicine, Children's National Medical Center, Washington, District of Columbia 20010, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Thistlethwaite
authors.forename: Linda M
authors.initials: LM
authors.lastname: Moses
authors.forename: Kristen C
authors.initials: KC
authors.lastname: Hoffbuhr
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Devaney
authors.forename: Eric P
authors.initials: EP
authors.lastname: Hoffman
mesh_terms: D001331:Automation; D002851:Chromatography, High Pressure Liquid; D002868:Chromosomal Proteins, Non-Histone; D041321:Chromosomes, Human, X; D004268:DNA-Binding Proteins / Q000235:genetics*; D008040:Genetic Linkage; D005838:Genotype; D006801:Humans; D051783:Methyl-CpG-Binding Protein 2; D009154:Mutation; D036103:Nanotechnology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction; D012097:Repressor Proteins; D015518:Rett Syndrome / Q000235:genetics
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C497392:MECP2 protein, human; D051783:Methyl-CpG-Binding Protein 2; D012097:Repressor Proteins