Gene Cloning

184 Gene Cloning
A large DNA fragment or chromosome
(a)
Cloned random overlapping
fragments (vector not shown)
(b)
(c)
Figure 7.6 Shotgun sequencing. a) A large DNA fragment, chromosome or
complete genome is fragmented into a population of overlapping fragments and
cloned into a plasmid vector. b) Clones are chosen at random and the DNA
sequenced; sequence overlap between clones is detected. c) The sequence
information is assembled into a contiguous sequence (contig).
with each other to find regions of overlap and the sequence assembled into
one contiguous sequence. The advantage of this strategy is that the same
primer is used for all sequencing reactions. However, more sequence data
must be obtained to ensure that overlapping fragments covering the whole
region are sequenced. This will also lead to redundancy, i.e. where the
same region is sequenced several times. It is also possible that one region
is not sequenced, i.e. there are gaps in the sequence, and therefore the
sequence has to be “finished” by screening for specific clones and then
sequencing to fill the gaps. Redundancy has an advantage in that it should
eliminate errors generated by poor sequencing reactions or errors in reading
the autoradiograph. Shotgun sequencing, even of relatively short DNA
fragments, is heavily reliant on computer analysis to identify the overlapping
regions. Although shotgun sequencing is no longer required to
sequence a few kilobases, it is now the method of choice for whole genome
sequencing.
7.7 High-throughput Sequencing Protocols
Before scientists started sequencing whole genomes, most sequencing projects
involved the determination of just a few kilobases of DNA. Scientists
would therefore use a “manual” approach to sequencing. They would set up