Chapter 8 Rapid DNA Sequencing by Direct Nanoscale Reading of Nucleotide Bases on Individual DNA chains

Abstract We present and discuss two concepts for rapid DNA sequencing by direct nanoscale reading of nucleotide bases on individual DNA molecules. Although these two concepts are based on substantially different principles, they both rely on the fabrication of nanoscale devices using state of the art technologies. The first method, which has been recently invented at Oak Ridge National Laboratory (ORNL), is based on a systematic nanoelectrode-gated tip-to-tip electron-tunneling molecular-detection concept. According to this concept, it should be possible to obtain genetic sequence information by probing through a DNA molecule base by base at a nanometer scale, as if looking at a strip of movie film. The nanoscale reading of DNA sequences is envisioned to take place at a nanogap (gate) defined by a pair of nanoelectrode tips as a DNA molecule moves through the gate base by base. The rationale is that sample molecules, such as the four different nucleotide bases, each with a distinct chemical composition and structure, should produce a specific perturbation effect on the tunneling electron beam across the two nanoelectrode tips. A sample molecular structure (base) could thus be detected when it enters the gate. This approach could lead to a single-molecule DNA sequencing technology that does not require the PCR amplification process and could, at least, be thousands of times faster than the current technology (Sanger's “dideoxy” protocol-based capillary electrophoresis systems). Theoretically, this new approach has the potential to perform DNA sequencing at a maximal rate of about 1,000,000 bases (∼1 Mb) per second per detection gate. This method can be paralleled using parallel arrays of multiple nanoelectrode-detection gates, thus magnifying the readout throughput by additional orders of magnitudes, achieving estimated maximal rates of possibly hundred millions bases (100 Mb) per second per device. The second method, developed at Harvard in collaboration with LingVitae AS, is based on the conversion of the natural DNA to another form of DNA in which each nucleotide is substituted with a group of 16 nucleotides (each base type is substituted with a unique sequence of 16 bases). Then fluorescently tagged oligonucleotides 16-mers, matching that of the converted DNA, are hybridized to the DNA and the molecule is electrophoretically fed through a nanoscale pore fabricated in thin solid-state film. The pore is used to sequentially remove the hybridized oligonucleotides, one by one, while the flashes of light in different colors arising from the attached fluorophores are detected. Moreover, according to the invention made by the Harvard group, the fluorophores attached to the 16-mer oligonucleotides are designed to be quenched until they reach the face of the nanopore, thus eliminating the undesired background fluorescence. This method is highly suitable for massive paralleled readout using high-density arrays of nanopores, which are simultaneously probed using a single imaging device. The readout throughput thus can be magnified by several orders of magnitudes, achieving estimated readout rates of ∼1–10 Mb/s.