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DNA sequencing using helicase-modified alpha-hemolysin nanopores (HELIPORE)
Start date: Apr 16, 2012, End date: Apr 15, 2014 PROJECT  FINISHED 

"Most of the current, second generation, methods for DNA sequencing are based on alternating cycles of enzyme manipulation and imaging the incorporation of fluorescently labeled nucleotides. These methods, however, hold several disadvantages, mainly the long duration of each sequencing experiment and the requirement for fluorescently labeled nucleotides and primer sequences. Tremendous efforts have been taken in order to develop cheap and ultra-fast sequencing systems. Specifically, the sequencing of single DNA molecules is of great importance, as no pre-amplification step and no labeled nucleotides are required. I intend to use biological nanometer-scale pores (alpha-hemolysin) modified with a DNA manipulating enzyme, a helicase, embedded in a membrane that separates two compartments, each contains an Ag/AgCl electrode, for the sequencing of single DNA molecules. Based on previous results, I expect that the ionic current, associated with DNA translocation through the nanopore, will exhibit a specific pattern for each nucleobase, allowing sequence determination. I intend to construct two different systems, in the first system the helicase will be soluble in the cis compartment, whereas in the second configuration the helicase will be attached to the nanopore via double stranded DNA linkers. The helicase enzyme will unwind double stranded DNA, form a ratchet-like movement of the DNA strand through the pore and control the pace of DNA translocation, leading to efficient sequencing."

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