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sequencing products. This is where liquid polymers in thin capillary tubes are used

obviating the need to pour sequencing gels and requiring little manual operation.

This substantially reduces the electrophoresis run times and allows high throughput to

be achieved. A number of large-scale sequence facilities are now fully automated

using 96-well microtitre-based formats. The derived sequences can be downloaded

automatically to databases and manipulated using a variety of bioinformatics

resources.

5.11.6 Alternative DNA sequencing methods

Developments in the technology of DNA sequencing have made whole-genome

sequencing projects a realistic proposition within achievable timescales; indeed the

first diploid genome sequence to be completed was of Craig Venter who pioneered

high-throughput sequencing. This makes studies on genome variation and evolution

viable, as evidenced by the 1000 Genomes Project which is providing high-resolution

sequence analysis of genomes. This has been made possible not only by refinements

in traditional automated sequencing but also by new developments such as sequen-

cing by synthesis and the development of sequencing byhybridisation arrays.These

methods are changing the way genome analysis is undertaken and makes individual

5  3 

3  5 

5  3 

3  5 

Cycle

sequencing

(one cycle)

Extension/termination reaction Primer annealing reaction

Label Primer

5  3 

3  5 

Labelled oligo anneals to target sequence

5  A

3  5 

5  A

3  5 

Ta q polymerase extends target sequences
until chain terminator is added (e.g. ddA)

Denaturation

ds DNA denatured by heating to > 94 °C

Fig. 5.39Simplified scheme of cycle sequencing. Linear amplification takes place with the use of labelled

primers. During the extension and termination reaction, the chain terminator dideoxynucleotides are

incorporated into the growing chain. This takes place in four separate reactions (A, C, G and T). The products

are then run on a polyacrylamide gel and the sequence analysed. The scheme indicates the events that take

place in the A reaction only. ds, double-stranded.

192 Molecular biology, bioinformatics and basic techniques