Vehicle Detector Technologies for Traffic Management Applications
Sunday, 8 April 2012Posted by
Crystal
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Single molecule sequencing (cyclic-array related; nanopore). Each of the methods discussed above requires either an in vitro or in vivo amplification step, such that the DNA to be sequenced is present at sufficient copy number to achieve the required signal. A method for directly sequencing single molecules of DNA would eliminate the need for costly and often problematic procedures such as cloning and PCR amplification.
A number of groups, including Solexa, Genovoxx, the Webb group at Cornell, and the Quake group at Caltech, are developing cyclic-array methods that are related to those discussed above, but attempt to dispense with the amplification step. Each method relies on extension of a primed DNA template by a polymerase with fluorescently-labeled nucleotides, but they differ in the specifics of biochemistry and signal detection. Additionally, both Solexa and Genovoxx have invested heavily in developing reversibly-terminating nucleotides, which would solve the problem (for single-molecule methods as well as amplified cyclic-array methods) of deciphering homopolymeric sequences, by limiting each extension step to a single incorporation. In so far as their research has been revealed at public conferences, Solexa has data on reversible terminators and has shown single molecule detection with an impressive signal-to-noise ratio. The Genovoxx team has shown the possibility of using standard optics for single-molecule detection and has given details on one class of reversible terminators52. In the academic sector, the Quake group has recently demonstrated that sequence information can be obtained from single DNA molecules using serial single base extensions and the clever use of fluorescence resonance energy transfer (FRET) to improve their signal-to-noise ratio53. The Webb group has recently shown the real-time detection of nucleotide-incorporation events via a nanofabricated zero-mode wave-guide. By performing the reaction in a zero-mode waveguide, only a zeptoliter volume of the reaction is excited by the laser so that in principle, one is only detecting fluorescent triphosphates that reside in the DNA polymerases active site54.
With respect to ease and reliability of detecting extension events, cyclic-array methods that sequence amplified molecules have an obvious advantage over single-molecule methods. However, there are several advantages of the single-molecule approach. Although all polymerase-based methods still require the introduction of some flanking “common” sequence (such that a single sequencing primer can be hybridized), single-molecule methods avoid a PCR amplification step, thereby reducing costs and avoiding potential biases. All polymerase-synthesis-driven methods will likely experience both a low frequency of nucleotide misincorporation events and non-incorporation events. For amplified-molecule methods, these manifest as eventual signal decay via “dephasing” of the identical individual templates within a single feature. For single molecule methods, in contrast, there is no risk of dephasing. A misincorporation event will manifest as a “dead” template that will not extend further, while non-incorporation events will simply appear as a “pause” in the sequence.
Another advantage of single-molecule methods is that they might require less starting material than other ULCS contenders and conventional sequencing52. Relevant to all technologies, we should take note that methods for amplification of large DNAs by multiple displacement amplification (MDA) or whole genome amplification (WGA) is improving rapidly96,97. This will enhance our ability to get complete sequence from single cells even when they are dead or hard to grow in culture55,56.
Cyclic array platforms operate via spatial separation of single molecules or amplified single molecules. As a consequence of this focus on single molecules, they also allow us to determine combinations of structures which are hard to disentangle in pools of molecules. For example, alternative RNA splicing contributes extensively to protein diversity and regulation but is poorly assayed by pooled RNAs on microarrays, while amplified single molecules allow accurate measures of over 1000 alternative spliceforms in RNAs like CD4457. Similarly haplotype (or diploid genotype) combinations of SNPs can be determined accurately from DNA molecules (or single cells)48.
A creative single-molecule approach that is quite unlike all of the above methods is nanopore sequencing, currently being developed by Agilent, and the Branton and Deamer groups. As DNA passes through a 1.5 nm nanopore, different base-pairs obstruct the pore to varying degrees, resulting in fluctuations in the pore’s electrical conductance. The pore conductance can be measured and used to infer the DNA sequence. The accuracies of base-calling range from 60% for single events to 99.9% for 15 events58. However, the method has thus far been limited to the terminal base-pairs of a specific type of hairpin.
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