We examined the WT αHL pore and the pore formed by E111N/K147N. 1 B), and here we quote the residual current ( I RES) as a percentage of the open pore current ( I O). The immobilized DNA molecules caused a sequence-dependent decrease in the current flow through the pore ( Fig. In this state, the DNAs were captured and immobilized by αHL pores in an applied potential, but they were not translocated into the trans compartment ( Fig. SsDNA oligonucleotides with biotin tags at the 3′ terminus were allowed to form complexes with streptavidin. In the present work, we return to the problem of base identification and show that all 4 DNA bases can be distinguished in both homopolymeric and heteropolymeric immobilized DNA strands. In a step toward the management of this problem, the Ghadiri group have shown that ssDNA can be ratcheted one base at a time through the αHL pore by the action of a DNA polymerase ( 20). Under the high applied potentials required for threading, freely moving DNA is translocated through the wild-type (WT) αHL pore too quickly for bases to be identified, unless the bases are modified with bulky groups ( 19). ( 16), and recent studies have shown that the rate of capture of DNA by protein pores is enhanced when the interior surfaces bear a net positive charge ( 17, 18). Wang and colleagues first showed that the direction in which DNA enters the αHL pore (5′ or 3′ threading) affects the extent of current block ( 15), an observation supported by the data of Mathé et al. Further, with a view toward exonuclease sequencing, in which bases are sequentially cleaved from a DNA strand, all 4 DNA bases can be identified as deoxyribonucleoside 5′-monophosphates by using an engineered αHL pore equipped with a cyclodextrin molecular adapter ( 12, 14). Notably, Ghadiri and coworkers showed that when ssDNA is immobilized within the α-hemolysin (αHL) protein pore, it is possible to distinguish between 2 bases when one or the other is located at a specific position within the pore ( 13). Several advances suggest that nanopore sequencing with protein pores is feasible. However, single-molecule sequencing-by-synthesis still requires expensive imaging and data-handling equipment.
One such approach is single-molecule sequencing-by-synthesis, which was initiated in academic laboratories ( 6, 7) and is now under commercial development ( 8, 9). Therefore, most probably, the cost of DNA sequencing can only be driven down further by using single-molecule technologies, all of which would avoid expensive, error-prone enzymatic DNA amplification techniques ( 4, 5). However, despite impressive recent price reductions, today's sequencing technologies require the use of costly enzymes, fluorescently labeled molecules, state-of-the-art imaging equipment, and high-capacity data storage devices ( 3). At that price, many people could afford to have their genomes sequenced and personalized medicine would become a reality ( 1, 2). A price of $1,000 for a human genome sequence would be a critical development in medicine ( ).
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