Biology Reference
In-Depth Information
genomic or cloned DNA, eliminating the need to prepare large amounts of DNA
from tissues for sequencing and subcloning steps.
Conventional PCR requires primers of known sequence that flank the region
to be amplified. Several techniques have been developed for sequencing ds DNA
produced by the PCR. Cycle sequencing is advantageous because it requires only
very small amounts of template DNA, and it can work with ds templates as well
as ss templates, eliminating the need to subclone the DNA (
Murray 1989, Bevan
et al. 1992
). However, problems that need to be considered include confirming
that you have amplified the correct fragment, removing all residual PCR primers
and unincorporated nucleotides before sequencing, and providing good quality
primers for sequencing. Recombinant DNA sequences can be generated by the
PCR, so care must be taken especially when studying multigene families (
Bradley
and Hillis 1997
).
7.7 Automated Sanger Sequencers
The invention of automated, fluorescent Sanger sequencers made large-
scale genome projects feasible. Instruments can automate nearly every step
of the large-scale sequencing process. Integrated machines can isolate DNA,
clone or amplify DNA, prepare sequencing reactions, purify DNA, and sepa-
rate and detect DNA fragments containing fluorescent labels to obtain the
DNA sequence (
Meldrum 2000a
). Automated sequencers first used horizontal
or vertical slab gels, but more recent commercial systems use capillary sequenc-
ers (
Meldrum 2000b
). A commonly used sequencer, the ABI PRISM 377 DNA
Sequencer, uses multicolor fluorescence labeling and a four-dye, one-lane detec-
tion system. Two hundred bases per sample per hour can be analyzed, and 18,
36, 64 or 96 samples can be analyzed simultaneously.
Large-scale sequencing facilities used a random shotgun phase combined
with a directed finishing phase to complete analysis of the difficult regions of
the early genomes sequenced. Others used a whole-genome shotgun approach
in which random fragments of total genomic DNA were subcloned, and high-
throughput sequencing was used to generate sequences that provide at least
10-fold coverage of the genome. These sequences were ordered and put into a
linear sequence with the aid of high-speed computers (
Meldrum 2000a
).
7.7.1 Decreasing Costs of Sanger Sequencing
Sanger sequencing is now automated (
Men et al. 2008
). DNA is isolated, frag-
mented, and cloned into a vector for DNA amplification in bacterial cells.
