We have been developing a genetic field-effect transistor (FET) based on the potentiometric detection of hybridization and intercalation on the Si3N4 gate insulator. In this study, we demonstrated the detection of charge density change as a result of hybridization and intercalation using genetic FETs. Since the electrical output signal is obtained with the genetic FET without any labeling reagent, as compared with the conventional fluorescence-based DNA chips, the genetic FET platform is suitable for a simple and inexpensive system for genetic analysis in clinical diagnostics.
A new method for SNP analysis based on the detection of pyrophosphate (PPi) is demonstrated, which is capable of detecting small allele frequency differences between two DNA pools for genetic association studies other than SNP typing. The method is based on specific primer extension reactions coupled with PPi detection. As the specificity of the primer-directed extension is not enough for quantitative SNP analysis, artificial mismatched bases are introduced into the 3′-terminal regions of the specific primers as a way of improving the switching characteristics of the primer extension reactions. The best position in the primer for such artificial mismatched bases is the third position from the primer 3′-terminus. Contamination with endogenous PPi, which produces a large background signal level in SNP analysis, was removed using PPase to degrade the PPi during the sample preparation process. It is possible to accurately and quantitatively analyze SNPs using a set of primers that correspond to the wild-type and mutant DNA segments. The termini of these primers are at the mutation positions. Various types of SNPs were successfully analyzed. It was possible to very accurately determine SNPs with frequencies as low 0.02. It is very reproducible and the allele frequency difference can be determined. It is accurate enough to detect meaningful genetic differences among pooled DNA samples. The method is sensitive enough to detect 14 amol ssM13 DNA. The proposed method seems very promising in terms of realizing a cost-effective, large-scale human genetic testing system.
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