Recent advances in single-molecule sequencing
Introduction
The first milestone regarding DNA sequencing was set in 1977 with the publication of two articles. Maxam and Gilbert described a method in which terminally labelled DNA molecules were chemically cleaved at specific bases and the resulting fragments were separated using gel electrophoresis [1]. Sanger et al. proposed an alternative approach, which applied dideoxynucleotide analogues as specific chain-terminating inhibitors of DNA polymerase [2]. Through automation and refinements the Sanger method has long since been the method of choice and a gold standard for DNA sequencing. Rising interest in genomic research and whole-genome sequencing with reasonable costs and expenditure of time resulted in recent developments of novel sequencing technologies. Those second generation sequencing systems include a number of non-Sanger ultra-high-throughput systems from 454 [3, 4], Illumina [5, 6] and Applied Biosystems [7], for instance. Another approach shows in the application of single-molecule sequencing, which is also referred to as third generation sequencing technology. The development of these new techniques aims towards meeting the demand for sequence information in various fields of research, such as study of genomics and evolution, forensics, epidemiology and diagnostics and applied therapeutics. This article intends to provide an overview about current advances in single-molecule sequencing, with an emphasis on Raman-based methods. The methods that will be described are listed in Table 1.
Section snippets
Exonucleolytic degradation
Fluorescently labelled DNA strands are attached to carrier particles (beads) via a biotin–streptavidin linkage. One of those beads is placed in a microchannel and fixed with an ‘optical tweezer’. Sequential degradation is started by adding an exonuclease enzyme, which cleaves one base at a time from the DNA strand. These monomers are moved by electro-osmotic flow towards the detection area. There the fluorescent dyes are excited by a laser and the emitted photon burst is analysed (Figure 1) [8•
Nanopores
Nanopore sequencing involves electrophoretical threading of DNA molecules through a nano-scale pore, which can be a membrane protein such as α-haemolysin, sometimes modified with an adapter such as cyclodextrin [26] or cyclodextrin derivatives [27••], or a synthetic pore [28]. A current is applied across the nanopore [29] and alterations in conductivity are measured while the DNA traverses through this channel. The major challenge of this method is the stochastic motion of the DNA while it
Sequencing using surface-enhanced Raman spectroscopy (SERS)
Surface-enhanced Raman spectroscopy (SERS) is a technique to increase the intrinsically weak Raman signal by conducting measurements of sample molecules adsorbed on rough metal (e.g. silver, gold and copper) surfaces or colloids. The signal enhancement is mainly attained by the excitation of localized surface plasmons as an electromagnetic effect, but also by the so-called charge transfer enhancement generated by the formation of metal–ligand complexes [43, 44].
A first efficient approach
Conclusion
Over the last years there was a remarkable increase in the interest in single-molecule sequencing technologies, which have even been developed into commercially available sequencing platforms. All aim towards a massive reduction in cost and expenditure of time and dispose with sample amplification methods. Table 2 gives an overview of some of these sequencing platforms. Although the cost is extensive regarding the equipment (from approx. $600 000 for the Roche/454, Illumina, and Applied
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgement
We gratefully acknowledge the support from the Federal Ministry of Education and Research (BMBF) through Project No. 0312032B.
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