The use of biotinylated DNA probes in parentage testing: non-isotopic labeling and non-toxic extraction.
With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time-consuming. Most isotopic labeling techniques use the isotope 32P and require 3-10 days to visualize bands after hybridization. An alternative approach is based on the use of non-isotopic detection methods. The available non-isotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non-toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non-isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmid and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3-5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.
Dykes
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Memorial Blood Center of Minneapolis, MN 55404.
D001710:Biotin; D015139:Blotting, Southern; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes; D005787:Gene Frequency; D006801:Humans; D008297:Male; D010334:Paternity; D012150:Polymorphism, Restriction Fragment Length; D013194:Staining and Labeling
D015342:DNA Probes; D001710:Biotin; D004247:DNA