Future Technologies

Until recently, there were few serious contenders to challenge the supremacy of Sanger sequencing with respect to data quality, read-length and flexibility of application. However recent broadening of the applications of microarrays and the emergence of several ultra-throughput SBS platforms to market reality has changed the spectrum of future technologies to emerging technologies. Single-molecule technologies that promise large gains in sequencing performance remain in accelerating development.

4.1. Nanopore membranes

Recently, Nakane et al. (2004) described the detection of the ion current signal during single DNA molecule capture at an a-Hemolysin nanopore in a lipid bilayer membrane (Figure 6). The successful capture of an analyte molecule in the pore results in a reduction of the potential to +10 mV for a short period. The exit of the probe from the pore is then prevented by the bound analyte, and impedance remains at blocked channel values. When the potential is reversed to —60 mV this provides the force to withdraw the probe from the pore and after time the probe disassociates from the analyte and an open channel (reverse) current is restored. More speculative SMS technologies involving nanopore membranes (Deamer and Akeson, 2000; Rhee and Burns, 2006) are also under development (Lee and Meller, 2007 this volume). While most development of nanopore techniques involves DNA sequence transducers to detect an electrical or ionic signal from individual DNA molecules (Nakane et al, 2004), Lee intends to examine stretched DNA molecules made to pass between ultra-sharp electrodes spaced 2-5 nm apart to distinguish between the four types of nuc-leotides based on differences in a physical phenomenon called electron tunneling (Lee and Meller, 2007).

In contrast, the Meller group intends to use nanopores to simultaneously detect electrical and fluorescent signals. The method employs ultra-fast optical reading of Design DNA polymers (Meller et al., 2005; Lee and Meller, 2007). Here, each nucleotide in the original DNA is substituted with a group (composed of a unique binary sequence of 3-16 nucleotides). Fluorescently tagged oligonucleotides that are complementary to this sequence are hybridized to the converted DNA. A nanopore is then used to unwrap the hybrid, and different fluorescent signals are detected briefly (1 ms) as they emerge from process, and achieve read rates of ~1 Mb/s (Lee and Meller, 2007). Other evidence collected by Mathe et al. (2005) suggests that the translocation process through a narrow pore involves base tilting and stretching of ssDNA molecules inside the confining pore and is strand-orientation dependent. In other studies, Viasnoff et al. (2006) found that rapid cooling (>100 °C/ms) locks DNA molecules into a unique alternative conformation that is retained over weeks at room temperature, and that this state can be probed using fluorescent energy transfer. These observations suggest that non-equilibrium DNA switch states may be more amenable to nanotechnology applications (such as for SMS). Fologea et al. (2005) observed that voltage biased solid-state nanopores can detect and characterize individual single-stranded DNA molecules of fixed length by operating a nanopore detector at pH values greater than approximately 11.6. They found that at this pH a large component of the DNA molecules are unfolded and can access the pore in this state.

Others intend to amplify the nucleotide signals by producing conical gold-coated nanopores in a synthetic membrane to control DNA transport and then

Fig. 6. Cartoon representing ion current signal detection during single DNA molecule capture at an a-Hemolysin nanopore in a lipid bilayer membrane. Upper, experimental data from the unsuccessful capture of an analyte molecule. Lower, experimental data from the successful capture of an analyte molecule. After the probe is captured in the pore, the potential is reduced to +10 mV for a short period. The exit of the probe from the pore is then prevented by the bound analyte, and impedance remains at blocked channel values. The potential is then reversed to — 60 mV, this provides the force to withdraw the probe from the pore, and after time toff the probe disassociates from the analyte and an open channel (reverse) current is restored. Analysis of many different dissociation event lifetimes (toff) at different reverse potentials (Vrev) can identify the different analyte characteristics. Reprinted from Nakane et al. (2004). Copyright (2004), reprinted with permission from The BioPhysical Society and Highwire Press.

Fig. 6. Cartoon representing ion current signal detection during single DNA molecule capture at an a-Hemolysin nanopore in a lipid bilayer membrane. Upper, experimental data from the unsuccessful capture of an analyte molecule. Lower, experimental data from the successful capture of an analyte molecule. After the probe is captured in the pore, the potential is reduced to +10 mV for a short period. The exit of the probe from the pore is then prevented by the bound analyte, and impedance remains at blocked channel values. The potential is then reversed to — 60 mV, this provides the force to withdraw the probe from the pore, and after time toff the probe disassociates from the analyte and an open channel (reverse) current is restored. Analysis of many different dissociation event lifetimes (toff) at different reverse potentials (Vrev) can identify the different analyte characteristics. Reprinted from Nakane et al. (2004). Copyright (2004), reprinted with permission from The BioPhysical Society and Highwire Press.

detect different signals from the four types of chemically modified nucleotides introduced into the DNA. Karhanek et al. (2005) reported an alternative to nanopore membranes where single DNA molecules labeled with nanoparticles can be detected as they translocate through a finely pulled nanopipette tip by their blockades of ionic current. SMS promises to radically improve DNA sequencing as it is potentially 10,000 times faster than present production systems that rely on single lanes. It can potentially start directly with genomic DNA, reducing the need for sample preparation. SMS read lengths are also potentially significantly longer than those obtained from gel electrophoresis systems. Longer read lengths will simplify sequence reconstruction and reduce the total number of runs required to get full coverage of the genome, although at its present state of development only short reads are possible (Lee and Meller, 2007). Uniquely, because fragments from single alleles of chromosomes will be used, SMS sequencing potentially can directly detect haplotypes over several linked polymorphisms.

4.2. Direct electrical detection of DNA synthesis

Pourmand et al. (2006) reported the development of electrical biosensors for direct electrical detection of enzymatically catalyzed DNA synthesis by induced surface-charge perturbation. The incorporation of a complementary deoxynucleotide (dNTP) into a self-primed single-stranded DNA attached to the surface of a gold electrode evokes an electrode surface-charge perturbation, which can be detected as a transient current by a voltage-clamp amplifier. It is proposed that the electrode detects proton removal from the 30-hydroxyl group of the DNA molecule during phosphodiester bond formation and this phenomenon can be potentially exploited at polarizable interfaces for evoking surface-charge perturbations specific for the addition of individual nucleotide species. The use of electrical biosensors are of interest for genotyping and re-sequencing, because of their potential high speed and for the elimination of the need for DNA labeling and optical detection, as well as their potential for miniaturization and automation.

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