Direct detection of single nucleotide polymorphism (SNP) with genomic DNA by the ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA).
A ferrocenylnaphthalene diimide-based electrochemical hybridization assay (FND-EHA) was applied to the direct detection of a C-to-G transition in a codon (TCA) for Ser-447 of the human lipoprotein lipase (LPL) gene, which resulted in the termination of the LPL protein there. Either one of two 13-meric oligonucleotide probes, S447 WT and S447X MT, representing sequences complementary to those of the wild type (WT) and mutated (MT) forms, was immobilized on a gold electrode, followed by hybridization with chromosomal DNA extracted from human leukocytes under the condition in which both WT- and MT-type sequences can form a duplex. These two electrodes were soaked in an electrolyte containing FND under a condition [0.1 M HOAc/KOAc (pH 5.6) containing 0.1 KCl and 0.05 mM FND at 40 degrees C], in which only the MT duplex could undergo dissociation. FND was concentrated in proportion to the amount of the duplex formed on the electrode to give rise to a current signal. The electrochemical signal ratios obtained for WT/WT, WT/MT and MT/MT were close to the theoretical 2:1:0 with the S447 WT-modified electrode, and was again close to 0:1:2 with the S447X MT-modified one.
Nojima
Takahiko
T
Department of Applied Chemistry, School of Engineering, Kyushu University, Fukuoka 812-8581, Japan.
Yamashita
Kenichi
K
Takagi
Atsuko
A
Takagi
Makoto
M
Ikeda
Yasuyuki
Y
Kondo
Hiroki
H
Takenaka
Shigeori
S
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D004563:Electrochemistry / Q000379:methods*; D005296:Ferrous Compounds / Q000737:chemistry*; D006801:Humans; D007094:Imides / Q000737:chemistry*; D008071:Lipoprotein Lipase / Q000235:genetics; D009693:Nucleic Acid Hybridization; D020641:Polymorphism, Single Nucleotide
D017931:DNA Primers; D005296:Ferrous Compounds; D007094:Imides; C406363:ferrocenylnaphthalene diimide; D004247:DNA; D008071:Lipoprotein Lipase