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Molecular Pathology

DNA sequencing

HeliScope Sequencer


Author: Rodney E. Shackelford, D.O., Ph.D., Tulane University (see Reviewers page)
Revised: 13 February 2011, last major update February 2011
Copyright: (c) 2008-2011, PathologyOutlines.com, Inc.

General
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● The Heliscope sequencing platform does not clonally amplify the DNA fragments to be sequenced
● Instead, DNA libraries are produced by random fragmentation and poly-A tails are added to the fragments
● The fragments are denatured and captured by surface-tethered poly-T oligomers, yielding a disordered array of primed, single molecule sequencing templates
● Each sequencing cycle consists of the addition of DNA polymerase and one uniquely fluorescently labeled dNTP, with the reaction moving forward by incorporating one dNTP (or more with homopolymeric nucleotide runs)
● Following a wash step to remove the unincorporated fluorescent dNTPs, the remaining florescent nucleotide type and position are interrogated and their positions are recorded
● The fluorescent group is then cleaved and another sequencing round is initiated with a different dNTP, until all four have been placed on the array
● Multiple four-phase sequencing cycles are performed, with up to 25-45 bp being sequenced, with billions of strands measured in a single run
● Not surprisingly, each run on this platform can require a large amount of computer storage space - about 14 terabytes
● As with the 454 FLX Sequencer, this technique is not efficient at sequencing longer homopolymer runs
● The resolution of the optical microscopy is a few hundred nM, so the captured molecules must be this distance apart for optimal image resolution
● This platform has been used to sequence the ~7,000 bp M13 viral genome (Science 2008;320:106)

Images
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Images captured on the HeliScope Sequencer

End of Molecular Pathology > DNA sequencing > HeliScope Sequencer


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