Electrochemical detection of DNA triplet repeat expansion.
Hereditary neurodegenerative diseases are connected with the expansion of trinucleotide repetitive sequences in genomic DNA. Molecular diagnosis of these diseases is based on the determination of the triplet repeat length. Currently used methods involve PCR amplification followed by electrophoretic determination of the amplicon size. We propose a novel electrochemical technique based on hybridization of target DNA (tDNA) immobilized at magnetic beads with a reporter probe (RP) complementary to the triplet repeats (12 units per RP). The biotin-labeled RP is detected via an enzyme-linked electrochemical assay involving binding of streptavidin-alkaline phosphatase conjugate and transformation of electroinactive 1-naphthyl phosphate to electroactive 1-naphthol. Pyrimidine residues within sequences flanking the homopurine (GAA)n repeat in tDNA are premodified with osmium tetroxide, 2,2'-bipyridine (Os,bipy), introducing electroactive labels in tDNA. The length of the triplet expansion is calculated from the ratio of the intensities of electrochemical signals of hybridized RP/tDNA-Os,bipy. The normalized signal increases linearly with the repeat length between 0 and about 200 triplet units, allowing for discrimination between normal, premutated, and mutated alleles. Application of this method for the detection of the asymptomatic heterozygous carrier of expanded alleles is demonstrated.
Fojta
Miroslav
M
Institute of Biophysics, Academy of Sciences of the Czech Republic, Kralovopolska 135, 612 65 Brno, Czech Republic. fojta@ibp.cz
Havran
Ludek
L
Vojtiskova
Marie
M
Palecek
Emil
E
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives* / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000378:metabolism; D000483:Alleles; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000235:genetics*; D004563:Electrochemistry / Q000379:methods*; D005621:Friedreich Ataxia / Q000235:genetics; D006147:Guanine; D006801:Humans; D015336:Molecular Probe Techniques; D009281:Naphthalenes / Q000032:analysis / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods; D009942:Organometallic Compounds / Q000737:chemistry; D019680:Trinucleotide Repeat Expansion
D009281:Naphthalenes; D009942:Organometallic Compounds; D015082:2,2'-Dipyridyl; D006147:Guanine; C076491:osmium tetroxide-2,2'-bipyridine; D001710:Biotin; D004247:DNA; D000469:Alkaline Phosphatase