ABSTRACT
In a recent survey [53] it was reported that 1 of 30 Jews of Eastern European descent in the United States and Canada is a carrier of the respective defective gene. Although several mutations in the respective gene were reported [53], the most frequent mutation includes the four-base (ATAG) insertion into the G-G base of the normal gene (P), to yield the mutant (Q). The primer (T) that is complementary to the normal gene as well as to the mutant till one base prior to the mutation site was immobilized on Au-electrodes. The human DNA was isolated from 0.5 mL blood samples of individuals that carry the heterocygotic gene (genetic disorder carrier), the homocygotic gene (carrier of the disease), and the normal gene. The DNA was denaturized and hydrolyzed to yield smaller fragments. The mixtures of the respective DNAs were interacted with the Tfunctionalized electrodes, with no pre-PCR amplification. The resulting electrodes were then interacted with polymerase (Klenow Fragment) in the presence of biotinylateddUTP. As dUTP complements the A base, attachment of the U-base by polymerase would occur only on the electrodes carrying the double-stranded assembly with the heterocygotic gene and homocygotic gene, whereas no polymerization occurs in the presence of the normal gene. Table 2 summarizes the changes in the interfacial electron transfer resistances, ∆Ret, ([Fe(CN)6]3−/4− is used as a redox-probe) as a result of the association of avidin-alkaline phosphatase to the biotinylated label and the precipitation of the insoluble product (12) on the electrode support. Only the samples carrying the heterocygotic or homocygotic mutants lead to the insulation of the electrode, ∆Ret=1.8 and 2.3 kΩ, respectively. Thus, a single measurement enables us to trace the individuals carrying the disease (homocygotic gene) or the carriers of the genetic disorders (heterocygotic gene). By a second measurement, the individuals carrying the homocygotic or heterocygotic genes could be differentiated by the reaction of the sensing electrodes, which includes the primer (T) hybridized with the respective genes, with polymerase in the presence of biotinylated-dCTP. In the heterocygotic gene sample, the normal gene, (P), and the mutant, (Q), are included in alleles and the biotinylated C-base is attached to the double-stranded assembly with the normal gene, Q. This enables the
binding of the avidin-alkaline phosphatase conjugate and the subsequent biocatalyzed precipitation of (12) on the electrode
Gene sample Reaction with biotinylated-dUTP followed by the binding of avidinalkaline and precipitation of (12).
