Gene Cloning and DNA Analysis by Brown T. A.;

Author:Brown, T. A.;
Language: eng
Format: epub
ISBN: 9781119072546
Publisher: Wiley
Published: 2015-10-01T00:00:00+00:00

Figure 10.7 Sequencing a cloned DNA fragment with a universal primer.

10.1.4 Limitations of chain-termination sequencing

With the most up-to-date versions of the chain-termination method it is possible to obtain over 750 bp of sequence per experiment. Although most genes – especially eukaryotic ones – are longer than this it is easy to carry out two or more chain-termination experiments, directed at different parts of a gene, in order to build up the complete sequence. Chain-termination sequencing is therefore the method of choice for sequencing genes and other DNA fragments obtained by cloning or PCR.

The chain-termination method was also used to obtain the first complete genome sequences. This was a much more challenging task, because even the smallest bacterial genomes are over 1 Mb in length, and the human genome, which initially was sequenced by the chain-termination method, is 3200 Mb (Table 10.1). Furthermore, as no sequencing method is entirely accurate it is necessary to sequence each region of a genome multiple times, in order to identify errors present in individual sequence reads (Figure 10.8). With the chain-termination method, to ensure that errors are identified, at least a fivefold sequence depth or coverage is required, which means that every nucleotide is present in five different reads. So, for the human genome, a total of 5 × 3200 = 16 000 Mb of sequence would be required. This is equivalent to over 21 million chain-termination sequences averaging 750 bp in length. When the human genome was sequenced during the 1990s and early 2000s, chain-termination sequencing was the only method available, so this challenge had to be met. In fact it was exceeded, as the Human Genome Project had generated 23 147 Mb of sequence by the time that the first draft of the genome was published in 2001. This capacity was achieved by automated sequencing machines, capable of generating 384 sequences in parallel in a single run, each run taking about one hour, corresponding to an output of almost 7 Mb per day under optimal working conditions.

Table 10.1 Sizes of representative genomes.

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