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Summary DNA sequencing and future of DNA sequencing
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BLGY1232 DNA sequencing: the Next GenerationSequencing whole genomes
If we can sequence the whole genome of an organism, then we can predict the
sequences of all the gene products (proteins) necessary for it’s programming
Simply knowing the sequence of a gene/polypeptide does not mean that we can
predict its biological function
Genome sequencing is only of value when combined with biochemical and
genetic evidence of gene functions/interactions/regulation
The first Genome sequencing projects involved all the techniques of cloning and
DNA sequencing we have discussed Two approaches were used. (i) The
development of a “minimum tiling path” – basically identifying the smallest
number of clones necessary to be sequenced to cover the entire genome. (ii)
High depth sequencing of very large numbers of clones - “random shotgun
sequence assembly”
Assembling the Human genome sequence used both approaches, by two very
different consortia The publicly funded Human Genome Consortium (HUGO)
used a miniumum tiling path method - Construct libraries of cloned genomic
DNA by “shotgun-cloning” total genomic DNA by restriction/ligation cut & paste
A commercially established company – Celera genomics – used random
shotgun assembly
The HUGO project was based on constructing large-insert (100kb+) clones in BAC
vectors These were then aligned by mapping “landmarks” within them like
restriction sites, and were assigned to individual chromosomes using molecular
genetic markers to first create chromosome-scale overlapping clusters of clones,
or “contigs” the smallest number of such clones that made up each contig
were subjected to sub-cloning fragments derived from them in plasmids, which
were then sequenced
Craig Venter created Celera Genomics – a private company that attracted private
finance to sequence the human genome This initiative stimulated the
development of automated sequencers and paved the way for the latest “Next
Generation” sequencing technologies
Celera attracted funding from pharmaceutical companies by offering them the
opportunity of inspect and patent sequences of potential use for therapy
development
Celera was a leader in developing computational approaches for sequence
assembly that HUGO did not believe were possible
The Celera sequence assembly benefited from access to the chromosome scale
assemblies already placed in the public domain by HUGO
The “First-draft” public release of the Human Genome sequence resulted from
the combination of these two approaches All subsequent genome sequencing
programmes use the random shotgun assembly methods pioneered by Celera
The Celera programme used the shotgun method:
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