A chemiluminescence-based detection system for human DNA quantitation and restriction fragment length polymorphism (RFLP) analysis.

A protocol for the chemiluminescent detection of restriction fragment length polymorphism (RFLP) profiles with the sensitivity of radioisotope-based detection systems is presented. RFLP profiles for the loci D2S44, D17S79, D1S7, D4S139, and D5S110 were obtained from ten nanograms of HaeIII-digested K562 DNA and 50 nanograms of human genomic DNA isolated from bloodstains. DNA is transferred to an amphoteric membrane using a neutral, high salt transfer procedure. Oligonucleotide probes directly conjugated to alkaline phosphatase are hybridized to the membranes, then the membranes are incubated with a substrate that emits light upon chemical cleavage by the alkaline phosphatase. Several variables were investigated to determine the optimum conditions for RFLP profile generation, among them transfer conditions, film selection, and exposure conditions. A chemiluminscent human DNA quantitation protocol, consistent with the RFLP detection protocol, is also presented.