Sequential Chemiluminescent Detection of Target DNAs without Stripping and Reprobing

We present a simple method for sequential chemiluminescent detections of two different DNA loci on a single Southern blot. First, an enzyme-linked DNA probe for a unique sequence is detected with a horseradish peroxidase (HRP) substrate followed by the detection of another enzyme-linked DNA probe for a different unique sequence with an alkaline phosphatase (AP) substrate that simultaneously inhibits the chemiluminescence generated by HRP. Such sequential detection steps eliminate the need to strip and reprobe blots and can be performed with no intervening steps.