Single-Molecule Sequencing
In this chapter, the use of single-molecule conductance for DNA sequencing, a principle target of the $1,000 Genome Project, will be discussed. Since starting the project, numerous universities and companies have attempted to develop single-molecule seque
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Single-Molecule Sequencing Masateru Taniguchi
Abstract In this chapter, the use of single-molecule conductance for DNA sequencing, a principle target of the $1,000 Genome Project, will be discussed. Since starting the project, numerous universities and companies have attempted to develop single-molecule sequencers but have not yet demonstrated a proof of concept. A major challenge has been the fabrication of nanoelectrodes with a 1 nm gap, equal to the diameter of single-stranded DNA molecules. The breakthrough discovery of the use of tunneling currents was required to perform single-molecule electrical sequencing. This discovery led to a proof of concept using a chemically modified scanning tunneling microscope (STM) and mechanically controllable break junction (MCBJ). These single-molecule measurement technologies are now being developed for application studies. Keywords Single molecule • Sequencing • Tunneling currents • DNA • RNA
9.1 Introduction Many people believed that the end of the Human Genome Project in 2003 meant the dawn of personalized medicine and therapeutics based on genomic information [1– 4]. However, the long time and exorbitant cost to read an entire human genome have been a significant barrier to realizing personalized medicine [5–7]. To overcome this barrier, the US National Institutes of Health (NIH) that led the Human Genome Project has founded the $10,000 and $1,000 genome projects with the goal of reading an entire human DNA sequence in 1 day for the cost of $10,000 and $1,000, respectively [8–13]. The final target of the $1,000 genome project is to develop single-molecule DNA sequencers that can identify the sequences of the four-base molecules in DNA by measuring single-molecule conductance. First- and second-generation DNA sequencers identify base molecules via light emission by laser excitation of dye molecules that are chemically bonded to base molecules [9–11]. They require polymerase chain reaction (PCR) to amplify
M. Taniguchi () The Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan e-mail: [email protected] © Springer Science+Business Media Singapore 2016 M. Kiguchi (ed.), Single-Molecule Electronics, DOI 10.1007/978-981-10-0724-8_9
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sequencing templates so that sufficient material is available for generating detectable signals. Furthermore, first- and second-generation DNA sequencing technologies require fluorescent labels. In contrast, third- and fourth-generation DNA sequencing technologies directly detect single-base molecules by changes in electric current such that neither PCR amplification nor fluorescent probes are necessary [14– 17]. Comparing the throughputs and total cost to determine a complete human genome shows that first-generation DNA sequencing technologies take 3 months and cost approximately $10 million, second-generation technologies take 2 months and cost approximately $0.1 million, and third- and fourth-generation technologies will take 1 day and cost approximately $1,000 [9–11]. The use
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