A novel method of identifying genetic mutations using an electrochemical DNA array

Abstract

We describe the development of a new type of DNA array chip that utilizes electrochemical reactions and a novel method of simultaneously identifying multiple genetic mutations on an array chip. The electrochemical array (ECA) uses a threading intercalator specific to double-stranded nucleotides, ferrocenylnaphthalene diimide (FND), as the indicator. ECA does not require target labeling, and the equipment is simple, durable and less expensive. The simultaneous multiple mutation detection (SMMD) system using an ECA chip and FND utilizes an enzyme to simultaneously distinguish several genetic mutations such as single nucleotide polymorphism (SNP), insertion, deletion, translocation and short tandem repeat. We examined this SMMD system using an ECA chip, by detecting seven different mutations on the lipoprotein lipase (LPL) gene for 50 patients in a blind test. It turned out that all the results obtained were concordant with the sequencing results, demonstrating that this system is a powerful tool for clinical applications.

Figure 1

LPL gene and properties of the seven LPL mutations used in this study. The substituted or deleted nucleotides are underlined.

Figure 2

An ECA chip and an ECA chip reader. (A) The ECA chip has 25 gold electrodes with a ceramic base. The left electrode in the front is the reference electrode, and the one to the right is the counter electrode. (B) The ECA chip reader is compact and durable. It weighs only 7 kg.

Figure 3

The process for the determination of mutations by the SMMD system using an ECA chip and FND. As the first step, asymmetric PCR (A-PCR) was performed with counter primer and special primer in the ratio of 1:4 and the first PCR product, as a template, from genomic DNA (A). The special primer contains the Tag sequence (cyan) at its 5′ end, which is a complement to the genomic region [yellow-colored region in (B)] between the mutation point (red) and the special primer. Since the A-PCR product contains self-complemental sequences [cyan- and yellow-colored sequences in (C)], it forms a self-loop (D). Before the hybridization and ligation reactions of the self-looped A-PCR product and a probe immobilized on the ECA chip, electric current (I₀, base line current response) was measured in a buffer containing FND (E). After the measurement of I₀, the self-looped A-PCR product was hybridized with two types of probes, a wild type and a mutant type, fixed on individual electrodes of the ECA chip. If the 3′ end nucleotide of the probe is completely matched with the self-looped A-PCR product, indicated as a bar, denoting hydrogen bond between red (c, cytosine) and pink (g, guanine) nucleotides, the enzyme ligase glues the 3′ end nucleotide on the probe with the 5′ end of the self-looped A-PCR product [left panel of (F)]. However, if the 3′ end nucleotide of the probe is mismatched with the 5′ end of the self-looped A-PCR product, the enzyme recognizes the gap, and it does not glue the probe and the A-PCR product [right panel of (F)]. After the ligation reaction, the ECA chip was denatured. When the A-PCR product is glued to the probe fixed on the electrode, the A-PCR product is not released from the probe [left panel of (G)]. When the A-PCR product is not glued, it is released from the probe [right panel of (G)]. After washing out excess nucleotides, individual electrode was measured for its electrochemical current of FND molecule by an ECA chip reader. The electrode containing double-stranded DNA gives higher current [left panels of (H) and (I)] than the one containing only a single-stranded short DNA [(right panels of (H) and (I)].

Figure 4

Establishment of criteria for the determination of zygosis by the SMMD system using an ECA chip. (A) Samples of wild homozygote (solid black circles), heterozygote (white squares) and mutant homozygote (solid black diamonds) were prepared from the plasmid DNA containing the seven mutations of the LPL gene or the corresponding wild DNA as described in Materials and Methods. Three experiments were carried out for individual samples, and the results have been plotted as the score values calculated from the equation α/δ − β/δ, where α is ΔI [(I₁ − I₀)/I₀ × 100] of W probe; β is ΔI of M probe; δ is average of α and β. (B) Samples of wild homozygote (solid black circles), heterozygote (white squares) and mutant homozygote (solid black diamonds) were prepared from genomic DNA samples of patients. The samples which are homozygotes of Y61X, V200A, A261T and A334T could not be obtained. Three experiments were performed for individual samples, and the results have been plotted as in (A).

Figure 5

Blind test for determination of LPL mutations by the SMMD system using an ECA chip. Samples from 50 patients, including homozygotes of A221-del and R243C, and heterozygotes of Y61X, V200A, A221-del, A261T, A334T and W382X were examined for the determination of genotype for the above seven mutations. The electrical currents measured were converted to the score (−2.0∼2.0) by using the equation described in Figure 4. The results of the 50 patients have been plotted for the seven mutations. The sample in which the score is between 1.0 and 2.0 is a wild homozygote, the sample in which the score is between −0.5 and 0.5 is a heterozygote, and the sample in which the score is between −2.0 and −1.0 is a mutant homozygote.