title: A highly specific microarray method for point mutation detection.
abstract: Improvements of microarray techniques for genotyping purposes have focused on increasing the reliability of this method. Here we report the development of a genotyping method where a microarray was spotted with stemloop probes, especially designed to optimize the hybridization specificity of complementary DNA sequences. This accurate method was used to screen for four common disease-causing mutations involved in a neurological disorder called Charcot-Marie-Tooth disease (CMT). Healthy individuals' and patients' DNA were amplified and labeled by PCR and hybridized on microarray. The spot signal intensities were 81 to 408 times greater for perfect compared with mismatched target sequences, differing by only one nucleotide (discrimination ratio) for healthy individual "homozygous" DNA. On the other hand, "heterozygous" mutant DNA samples gave rise to signal intensity ratios close to 1, as expected. The genotypes obtained by this method were perfectly consistent with those determined by direct PCR sequencing. Cross-hybridization rates were very low, resulting in further multiplexing improvements. In this study, we also demonstrated the feasibility of real-time hybridization detection of labeled synthetic oligonucleotides with concentrations as low as 2.5 nM.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Limoges, Paris.
authors.forename: Yasser
authors.initials: Y
authors.lastname: Baaj
authors.forename: Corinne
authors.initials: C
authors.lastname: Magdelaine
authors.forename: Virginie
authors.initials: V
authors.lastname: Ubertelli
authors.forename: Christophe
authors.initials: C
authors.lastname: Valat
authors.forename: Luc
authors.initials: L
authors.lastname: Talini
authors.forename: Françoise
authors.initials: F
authors.lastname: Soussaline
authors.forename: Elena
authors.initials: E
authors.lastname: Khomyakova
authors.forename: Benoît
authors.initials: B
authors.lastname: Funalot
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Vallat
authors.forename: Franck G
authors.initials: FG
authors.lastname: Sturtz
mesh_terms: D001483:Base Sequence; D016022:Case-Control Studies; D002607:Charcot-Marie-Tooth Disease / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D009841:Oligonucleotides / Q000737:chemistry / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D009841:Oligonucleotides