High throughput and economical mutation detection and RFLP analysis using a minimethod for DNA preparation from whole blood and acrylamide gel electrophoresis.
We report a simple, rapid, and high throughput method which allows the simultaneous processing of multiple whole blood samples for routine DNA purification and analysis. The method is based on a microscale DNA preparation and digestion using minimal amounts of reagents and handling. The amount of material necessary for a Southern blot analysis (5-7 micrograms) is obtained from 200 microliters of whole blood. All steps involved in DNA preparation and restriction digestion are processed in a single 1.5-ml Eppendorf tube. DNA preparation is performed using a salting out procedure with a proteinase K digestion step but no phenol/chloroform extraction. Restricted fragments are separated by electrophoresis through polyacrylamide slab gels followed by electrotransfer to nylon membranes. By varying the electrophoresis parameters (V/cm or duration), fragments of interest up to 12 kb length can be separated with high resolution. At least 80 samples can be processed at once per DNA preparation, and multiples of this number depend on the available equipment. This economical and rapid method allows routine DNA analysis for mutation or RFLP detection to be performed on a large scale which is a mandatory feature in any DNA-based population screening program. In addition, the DNA purified by the minimethod can be used as an economical source for PCR analysis.
Unité de Recherche en Génétique Humaine et Moléculaire, Centre de Recherche de l'Hôpital St-François d'Assise, Québec, Canada.
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Rousseau
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Réhel
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Rouillard
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DeGranpré
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Khandjian
D015139:Blotting, Southern; D003365:Costs and Cost Analysis; D004247:DNA / Q000097:blood* / Q000235:genetics*; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005600:Fragile X Syndrome / Q000175:diagnosis / Q000235:genetics; D005821:Genetic Techniques / Q000191:economics; D006801:Humans; D008297:Male; D012150:Polymorphism, Restriction Fragment Length
D004247:DNA