Question

What step of DNA sequencing is skipped during ahotgun sequencing? (A) computer analysis (B) physical mapping (C) primer reactions (D) cloning of DNA fragment​

Answer 1

Sanger sequencing: The chain termination method

Regions of DNA up to about 900 base pairs in length are routinely sequenced using a method called Sanger sequencing or the chain termination method. Sanger sequencing was developed by the British biochemist Fred Sanger and his colleagues in 1977.

Answer 2

There are three basic strategies for DNA sequencing. These can be divided into two ordered strategies (primer walking and unidirectional deletions (Henikoff, 1987)) and one random strategy, shotgun sequencing (Messing et al., 1981). The ordered strategies have the advantage of being more efficient than a shotgun approach. However, they have significant drawbacks as strategies for large-scale projects. Primer walking, as it was carried out when we began our projects in early 1990, was not well suited to automated sequencers. Initial chemistry for carrying out sequencing using custom primers on ABI 370 DNA sequencers gave inadequate results in our experience. In addition, primer walking requires the synthesis of a large number of oligonucleotides, one for each sequencing reaction. This results in adding about $30 to the cost of each reaction. Moreover, the results of each reaction must be obtained and analyzed before the next primer can be chosen and synthesized. This requires that a large number of start points must be initiated to scale-up the process and this in turn can be a burden on project management. The combined requirements of choosing a primer, coupling it with the correct template and carrying out that reaction makes automation less straightforward than with a random approach where many templates are used, but each is used in exactly the same way.
Ordered deletions are also an efficient strategy for sequencing. However, the use of this approach in large projects has serious disadvantages since it requires substantial effort to subclone the cosmid DNA into pieces that can readily be subjected to exonuclease III (ExoIII) deletions. This requires extensive labor from individuals with a fairly high skill level. Moreover, it is not clear how such procedures could be highly automated. While very effective for small fragments, this is basically an approach that does not scale-up well for a large-scale sequencing project which requires parallel operations. As a result of these factors we chose to implement a shotgun sequencing strategy for our two large-scale sequencing projects (Martin-Gallardo et al., 1992; McCombie et al., 1992c).
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