When a repetitive sequence is broken into fragments, many of the resulting pieces contain the same, or very similar, sequence motifs. A second problem with the shotgun method is that it can lead to errors when repetitive regions of a genome are analyzed.
This shotgun method is the standard approach for sequencing small prokaryotic genomes, but is much more difficult with larger genomes because the required data analysis becomes disproportionately more complex as the number of fragments increases (for n fragments the number of possible overlaps is given by 2 n 2 - 2 n). This is done by breaking the molecule into fragments, determining the sequence of each one, and using a computer to search for overlaps and build up the master sequence ( Figure 5.1). This means that the sequence of a long DNA molecule has to be constructed from a series of shorter sequences.
DNA sequencing is obviously paramount among these techniques, but sequencing has one major limitation: even with the most sophisticated technology it is rarely possible to obtain a sequence of more than about 750 bp in a single experiment. The next two chapters describe the techniques and strategies used to obtain genome sequences. Describe how radiation hybrids and clone libraries are used in STS mapping
