An improved protocol to increase sensitivity of Southern blot using dig- labelled DNA probes

The use of methods for nonradioactive labelling of nucleic acids has increased in recent years because they avoid disadvantages associated with radioisotopes. The most frequently used label is digoxigenin (DIG). The greatest problem of nonradioactive methods is their high nonspecific background mainly caused by the multistep detection. A diffuse background can mask the specific signal; furthermore nonspecific signals can make it difficult to interpret the result. In this study we have attempted to identify elements which could generate background. We have also determined the probe and antibody concentrations by which the higher sensitivity is obtained.