GENOMIC SOUTHERN ANALYSIS WITH ALKALINE-PHOSPHATASE-CONJUGATED OLIGONUCLEOTIDE PROBES AND THE CHEMILUMINESCENT SUBSTRATE AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a P-32-labeled oligomer. After 16 h, the signal was 12 times greater than the P-32 signal. The detection of the single-copy tissue plasminogen activator gene in 0.25-mu-g of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.