Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence

D Pollard-Knight, A C Simmonds, A P Schaap, H Akhavan, M A Brady

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)


A chemiluminescent reaction based on the deprotection of a phosphorylated phenyl dioxetane by alkaline phosphatase has recently been described (Schaap, A.P., 1988, J. Biolumin. Chemilumin. 2, 253). Light output is enhanced by intermolecular energy transfer to a micelle-solubilized fluorophore. This system is applied here to the detection of DNA probes on Southern blots. Enzyme solution assays which give an indication of sensitivity show that using this substrate 100 fg (0.7 amol) alkaline phosphatase can be detected on a luminescence plate reader (200 ms reading time). In a model Southern blotting system 180 fg HindIII digested lambda DNA was detected on film with homologous biotinylated DNA and a streptavidin-alkaline phosphatase complex. The single copy genes mos and raf-1, representing targets of 4.2 and 2.4 pg target DNA respectively, have also been detected in Southern-blotted human genomic DNA. A delay in reaching a plateau level of light output which is dependent on pH is observed but signal continues for at least 7 days. Typically, 12-h exposures to X-ray film were performed but once a steady-state light output had been achieved this time could be reduced to 2 h by preflashing film. This detection system represents a sensitive nonradioactive method, which is applicable not only to Southern blots but also to Northern and Western blots and any assay in which alkaline phosphatase is the label.
Original languageEnglish
Pages (from-to)353-8
Number of pages6
JournalAnalytical Biochemistry
Issue number2
Publication statusPublished - 1990


Dive into the research topics of 'Nonradioactive DNA detection on Southern blots by enzymatically triggered chemiluminescence'. Together they form a unique fingerprint.

Cite this