Functionalized self-assembled monolayer on gold for detection of human mitochondrial tRNA gene mutations.
We developed a rapid and simple method to identify single-nucleotide polymorphisms (SNPs) in the human mitochondrial tRNA genes. This method is based on a universal, functionalized, self-assembled monolayer, XNA on Gold chip platform. A set of probes sharing a given allele-specific sequence with a single base substitution near the middle of the sequence was immobilized on chips and the chips were then hybridized with fluorescence-labeled reference targets produced by asymmetric polymerase chain reaction from patient DNA. The ratio of the hybridization signals from the reference and test targets with each probe was then calculated. A ratio of above 3 indicates the presence of a wild-type sequence and a ratio of below 0.3 indicates a mutant sequence. We tested the sensitivity of the chip for known mutations in tRNA(Leu(UUR)) and tRNA(Lys) genes and found that it can also be used to discriminate multiple mutations and heteroplasmy, two typical features of human mitochondrial DNA. The XNA on Gold biochip method is a simple and rapid microarray method that can be used to test rapidly and reliably any SNP in the mitochondrial genome or elsewhere. It will be particularly useful for detecting SNPs associated with human diseases.
Du
Weidong
W
ThermoHybaid, Interactiva Division, 89077 Ulm, Germany.
Marsac
Cécile
C
Kruschina
Margit
M
Ortigao
Flavio
F
Florentz
Catherine
C
D000483:Alleles; D006046:Gold; D006801:Humans; D017241:MELAS Syndrome / Q000235:genetics; D017243:MERRF Syndrome / Q000235:genetics; D009693:Nucleic Acid Hybridization / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012313:RNA / Q000235:genetics*; D000077278:RNA, Mitochondrial; D012343:RNA, Transfer / Q000235:genetics*
D000077278:RNA, Mitochondrial; D012313:RNA; D006046:Gold; D012343:RNA, Transfer