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 D NA purification and analysis. The method is based on a microscale DNA preparation and digestion using minimal amounts of reagents and handli ng. The amount of material necessary for a Southern blot analysis (5-7 mu g) is obtained from 200 mu l of whole blood. All steps involved in DNA preparation and restriction digestion are processed in a single 1 .5-ml Eppendorf(TM) tube. DNA preparation is performed using a salting out procedure with a proteinase K digestion step but no phenol/chloro form 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 duratio n), fragments of interest up to 12 kb length can be separated with hig h resolution. At least 80 samples can be processed at once per DNA pre paration, and multiples of this number depend on the available equipme nt. This economical and rapid method allows routine DNA analysis for m utation or RFLP detection to be performed on a large scale which is a mandatory feature in any DNA based population screening program. In ad dition, the DNA purified by the minimethod can be used as an economica l source for PCR analysis. (C) 1994 Wiley-Liss, Inc.