High throughput and economical mutation detection and RFLP analysis using a minimethod for DNA preparation from whole blood and acrylamide gel electrophoresis
✍ Scribed by François Rousseau; Richard Réhel; Patricia Rouillard; Pierre DeGranpré; Edward W. Khandjian
- Publisher
- John Wiley and Sons
- Year
- 1994
- Tongue
- English
- Weight
- 416 KB
- Volume
- 4
- Category
- Article
- ISSN
- 1059-7794
No coin nor oath required. For personal study only.
✦ Synopsis
Cottoti
We report a simple, rapid, and high throughput method which allows the simultaneous processing of multiple whole blood samples for routine D N A purification and analysis. The method is based on a microscale D N A preparation and digestion using minimal amounts of reagents and handling. The amount of material necessary for a Southern blot analysis (5-7 pg) is obtained from 200 p1 of whole blood. All steps involved in D N A preparation and restriction digestion are processed in a single 1.5-ml Eppendorf T" tube. D N A 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 (Vicm 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 D N A preparation, and multiples of this number depend on the available equipment. This economical and rapid method allows routine D N A 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 D N A purified by the minimethod can be used as an economical source for PCR analysis. o