Bioelectronic detection of point mutations using discrimination of the H63D polymorphism of the Hfe gene as a model.
BACKGROUND
A bioelectronic detection platform has recently been developed that facilitates the detection and characterization of nucleic acids. The DNA chip platform is compatible with homogeneous assays because separate labeling and wash steps are not required. A one-step, bioelectronic detection assay was developed to genotype patient samples with respect to the H63D polymorphism of the Hfe gene, associated with hereditary hemochromatosis.


RESULTS
Electrode arrays were modified with DNA capture probes that were perfectly matched to the wild-type or mutant allele of H63D. Amplicons containing the polymorphic site were hybridized with the capture probes on the electrode arrays in the presence of electronically labeled reporter (signaling) probes. Voltammetric analysis of the electrode arrays was conducted first at ambient temperature and then at elevated temperature. The electronic signal was preferentially diminished at elevated temperature from electrodes that hybridized with mismatched target amplicons.


CONCLUSIONS
An assay for bioelectronic genotyping of the H63D polymorphism was developed and used with six patient specimens to show the feasibility of this system as a model for point mutation detection.
Umek
R M
RM
Clinical Micro Sensors Division of Motorola, Inc, 757 South Raymond Ave., Pasadena, CA 91105, USA.
Lin
S S
SS
Chen Yp
Y
Y
Irvine
B
B
Paulluconi
G
G
Chan
V
V
Chong
Y
Y
Cheung
L
L
Vielmetter
J
J
Farkas
D H
DH
D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics*; D006432:Hemochromatosis / Q000235:genetics*; D000071020:Hemochromatosis Protein; D006639:Histidine / Q000235:genetics; D015395:Histocompatibility Antigens Class I / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010865:Pilot Projects; D017354:Point Mutation / Q000235:genetics*; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*
C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D001224:Aspartic Acid; D006639:Histidine