DNA Sequencing Based on Intrinsic Molecular Charges

Abstract

Gene functional analyses have proceeded remarkably in the fields of molecular biology, pharmacogenomics, and clinical research, on the basis of completion of the decoding of the human genome. The analysis of nucleotide variation has become increasingly important for the assembly of a highresolution map of disease-related loci and for clinical diagnostics. The most common form of genomic variation is single-nucleotide polymorphism (SNP), which is an important marker in personalized medicine that affects disease susceptibility and resistance. Although a number of methods for SNP analysis have been developed, [1][2][3][4][5][6][7] DNA sequencing techniques still need to be improved in terms of cost, simplicity, and throughput to analyze not only SNPs but also genomic variations, such as insertion/deletion and short tandem repeats.We have been investigating a new approach to the direct, simple, and highly sensitive detection of nonlabeled molecular recognition events on a miniaturized and arrayed solidstate device.[8] Recently, several types of field-effect devices have been used for the electrochemical detection of hybridization events on a solid surface.[9] As DNA molecules are negatively charged in an aqueous solution, the number of negative charges at the gate surface of field-effect devices increases as a result of hybridization and extension reactions. The charge-density change is directly transduced into an electrical signal by the field effect. Based on this principle, point-mutation analysis was carried out by using the PCR products amplified with allele-specific primers.[9e] In this case, the overall specificity was determined from that of the allelespecific PCR. A single-base mismatch could also be distinguished by hybridization with complementary and mismatched DNA probes immobilized on the capacitor-type field-effect devices.[9b]We propose a new method for DNA sequencing of known as well as unknown sequence variants, which is based on detection of the intrinsic charges of DNA molecules by using the field effect. Herein, we report the direct transduction of single-base extension at the gate surface into an electrical signal, and the possibility of label-free DNA sequencing based on the intrinsic charges of DNA molecules.Oligonucleotide probes are immobilized on the Si 3 N 4 gate surface, and the complementary target DNA is hybridized with these probes. The hybridization events are followed by the introduction of DNA polymerase and one of each deoxynucleotide (dCTP, dATP, dGTP, or dTTP). DNA polymerase extends the immobilized oligonucleotide probes in a template-dependent manner ( Figure 1). As a result of the extension reaction, the number of negative charges increases at the gate surface of the field-effect transistor (FET) because of the intrinsic negative charges of the incorporated molecules. This change in charge density can be detected as a shift in the threshold voltage (V T ) of the FET. Thus, iterative addition of each deoxynucleotide and measurement of the threshold voltage allow direct, simple,...

Development of cell/transistor interface for real-time and noninvasive monitoring of potassium ion release based on apoptosis using biologically-coupled field effect transistor

“…Meanwhile, field effect transistors (FET) have been applied to biosensors; for example, enzyme-FET [1~3] and immuno-FET [4]. We have also reported some original manners for label-free and non-invasive bio-functional analyses using biologically coupled field effect transistors (bio-FET) [5,6]. The principle of bio-FET is based on the potentiometric detection of charge density changes induced at a gate insulator/solution interface accompanied by specific bio-molecular recognition events.…”

DNA Detection by an Extended-Gate FET Sensor with a High-Frequency Voltage Superimposed onto a Reference Electrode

“…Moreover, the charge-density change on a silicon-nitride (SiN) gate surface caused by singlebase extension could be measured as a shift in the gate voltage. 14 This result indicates that it is possible to realize labelfree DNA sequencing based on the intrinsic charges of DNA molecules. The DNA hybridization efficiency is affected by the immobilization procedure for the DNA probes on the sensing surface of the FET sensor.…”

“…Generally, FET sensors with a SiN gate insulator are used for DNA detection. 9,11,12,14 In this case, a silane-coupling method, which has many complicated steps, was applied to immobilize the DNA probes on the SiN surface; this method makes it difficult to control the DNA probe density. 9,11 In addition, the FET sensor with a SiN gate insulator must be operated in a dark environment, such as a lightshielding box, because of its light sensitivity.…”

“…[2][3][4][5][6][7][8][9][10][11][12][13][14] In particular, field-effect-transistor (FET) sensors have been used to electrochemically detect DNA hybridization events on solid surfaces. 3,[8][9][10][11][12][13][14] Since a DNA molecule contains phosphate ions, which are negatively charged in an aqueous solution, the number of negative charges on the gate surface of the FET sensor increases as the result of hybridization and extension reactions.The charge-density change can be directly transduced into an electrical signal by the field effect. The FET sensor can thus detect DNA without labeling.…”

Electronic Detection of DNA Adsorption and Hybridization

“…Comparative analysis of two different samples on the FET array provides reproducible mutation detection, the overall specificity is simply that of the allele-specific PCR. A combination of FET based DNA detection and sequence specific extension of surface bound oligonucleotides has been reported by Sakata and Miyahara [29].…”