Hints for reading sequencing gels

The accurate derivation of a sequence from a sequencing gel is not always a straightforward procedure and the purpose of this section is to set out some general guidelines which should be followed when interpreting the results of a sequencing experiment. 

Two types of primer DNA (Sections 4.2.1. and 4.8.1.) may be employed for the chain-extension reaction. When the desired sequence, of chain length up to —400 nucleotides, is cloned into M13mp2, a flanking primer which hybridizes to a region of the 

In the dideoxy chain-termination procedure all the bands produced on the sequencing gel should ideally result from specific chain termination caused by the incorporation of a dideoxynucleo-tide. However, spurious chain termination can also occur and while the reasons for this are not altogether clear, it is supposed that this 

When an over-exposed autoradiograph of a sequencing gel is examined it is seen that the desired bands are in fact, superimposed on a much fainter background of bands corresponding to 

G band is an artifact. In the T reaction consecutive residues usually give a band pattern of equal intensities except where the run of T s follow a C. In this case the pattern is often reminiscent of a run of A s. A run of consecutive C residues also has a characteristic pattern in which the 5 -C is much weaker than the second band (Fig. 4.16.(d)), while the third and subsequent bands fall off in intensity towards the 3 -end. Thus for example, the sequence 5 XCCC3 (where X is G, A or T) is represented by four consecutive bands in the C channel and whatever the intensity of the first band it can be recognised as an artifact since it will be followed by a characteristic weak band and not the intense band expected if it was the second C in a run of consecutive C s (Fig. 4.16.(e)). Runs of G s, where they follow T residues, give a similar pattern of intensities to runs of C (Fig. 4.16.(f)). 

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