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Molecular Pathology

DNA sequencing

Illumina Genome Analyzer


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 Illumina Genome Analyzer, similar to the 454 FLX Sequencer, begins by constructing a sequence mixed library of DNA fragments of up to several hundred bp
● Adaptor-sequences with forward and reverse PCR primers are attached to each end of these DNA fragments
● The adaptor-linked sequences are then amplified by bridge PCR, where both the forward and reverse PCR primers are tethered to a solid substrate by a flexible linker
● During PCR, the amplified sequences remain immobilized and clustered in one location on the array; eventually there are 1,000 to 1 million clustered amplicons, sufficient for reporting incorporated bases as the required signal intensity
● Several million different clusters can be amplified and distinguished at different locations within eight independent lanes that are on a single flow cell; thus, eight different libraries can be sequenced simultaneously
● Once the amplification step is completed, the amplicons are made single stranded and a universal sequencing primer is hybridized to a universal sequence flanking the DNA to be sequenced
● Sequencing occurs with each single-base addition, using a modified DNA polymerase and a mix of four modified dNTPs, each with two different modifications: (a) the dNTPs have a chemically cleavable moiety (a “reversible terminator”) at their 3’ hydroxyl position that allows only one nucleotide incorporation; and (b) each dNTP has a distinct fluorescent label that can also be chemically cleaved
● Thus, after one round of dNTP incorporation has occurred, it can be photometically measured
● The reversible terminator and fluorescent label are then chemically cleaved, washed away, and the DNA sequencing can proceed by adding another labeled dNTP series
● The read-length with this technique is about 36 bp; longer reads are possible but have higher error rates
● The main limiting factors are incomplete cleavage of the fluorescent labels and terminating moieties, all of which result in signal decay and dephasing
● Homopolyer stretches are far less of a problem with this sequencing platform that with the 454 FLX Sequencer
● Combining different sequences allows this platform to sequence several hundred nucleotides per run

End of Molecular Pathology > DNA sequencing > Illumina Genome Analyzer


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