EVALUATION OF NONRADIOACTIVE LABELING AND DETECTION OF DEOXYRIBONUCLEIC ACIDS .1. CHEMILUMINESCENT METHODS

The growth of analytical methods for the detection of nucleic acid fro m various biological samples reflects recent advances in biotechnology development especially in the areas of genetic, infections and cancer diagnosis. The target DNA is detected by hybridization techniques der ived from Southern's blotting. However such assays, based on the use o f P-32 labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulti es, a number of chemiluminescent detection methods have recently been developed. These new, alternative probe labelling procedures and chemi luminescent detection methods are easy to use in routine assays perfor med in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling t echniques. The techniques investigated include peroxydase, biotin 16-d UTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transf erred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is final ly revealed by the use of chemiluminescent substrates. For all these t echniques the detection limit is 10 aM (attomol) of a 561 bp target DN A. However for the probes labelled with peroxydase and with digoxigeni n the detection limit drops to 1.0 aM of the target DNA. In the presen t paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much a s a factor of 20 from method to method. This is the first time that va rious chemiluminescent methods for label and detection of DNA are comp ared and evaluated in order to determine the best protocol.