not as sensitive as chemiluminescent detection and can also result in a higher background, especially with probes labeled by random priming.

The key to a successful blotting method is a high signal-to-noise ratio. Ideally, the probe and detection systems should yield a specific and robust signal. High specific signal, however, may be accompanied by high background (noise). Therefore, sensitivity of detection is sometimes sacrificed to generate a more specific signal.

Interpretation of Results

When a specific probe binds to its target immobilized on a membrane, the binding is detected as described in the previous section, with the end result being the visual ization of a "band" on the membrane or film. A band is simply seen as a line running across the width of the lane. Analysis of bands, i.e., presence or absence or location in the lane, produced by Southern blot can be straightforward or complex, depending on the sample and the design of the procedure. Figure 6-19 is a depiction of a Southern blot result. The bands shown can be visualized either on a membrane or on an autoradiographic film. If a gene locus has a known restriction pattern, for instance in lane 1, then samples can be tested to compare their restriction patterns. In the figure, the sample in lane 3 has the identical pattern, i.e., both lanes have the same number of bands, and the bands are all in the same location on the gel and are likely to be very similar if not identical in sequence to the sample in lane 1. Southern blot

0 0

Post a comment