Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids. Part One: Chemiluminescent methods.
The growth of analytical methods for the detection of nucleic acid from 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 derived from Southern's blotting. However such assays, based on the use of 32P labelled DNA probes, bring with them the associated problems of handling radioactive materials. In order to overcome these difficulties, a number of chemiluminescent detection methods have recently been developed. These new, alternative probe labelling procedures and chemiluminescent detection methods are easy to use in routine assays performed in research laboratories as well as for medical applications, and can reach the level of sensitivity found in classical radiolabelling techniques. The techniques investigated include peroxydase, biotin 16-dUTP or digoxigenin 11-dUTP probe labelling. The target DNAs are transferred onto nitrocellulose or nylon membranes and further fixed by heat or UV crosslinking. Specific hybridization on the target DNA is finally revealed by the use of chemiluminescent substrates. For all these techniques the detection limit is 10 aM (attomol) of a 561 bp target DNA. However for the probes labelled with peroxydase and with digoxigenin the detection limit drops to 1.0 aM of the target DNA. In the present paper we shall compare several of these DNA labelling and detection procedures and show that the detection threshold can vary by as much as a factor of 20 from method to method. This is the first time that various chemiluminescent methods for label and detection of DNA are compared and evaluated in order to determine the best protocol.
D000218:Adamantane / Q000031:analogs & derivatives; D001483:Base Sequence; D001710:Biotin; D015139:Blotting, Southern; D004247:DNA / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D010544:Peroxidases; D012680:Sensitivity and Specificity
D003856:Deoxyuracil Nucleotides; D007202:Indicators and Reagents; C061523:3-(2'-spiroadamantane)-4-methoxy-4-(3''-phosphoryloxy)phenyl-1,2-dioxetane; D001710:Biotin; D004247:DNA; D010544:Peroxidases; D004076:Digoxigenin; D000218:Adamantane