ISSN:
0173-0835
Keywords:
Scanning for mutations
;
Prions
;
Temperature gradient gel electrophoresis
;
Open reading frame
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Gerstmann-Sträussler-Scheinker syndrome (GSS), fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (CJD) are caused by point mutations or octarepeat insertions in the prion protein (PrP) gene. In the present work a method was established that is appropriate for a thorough screening for mutations in the PrP gene and is generally applicable to screenings of any given gene. Temperature gradient gel electrophoresis (TGGE) was modified at two critical steps by UV cross-linking of the DNA strands and by replacing the spatial gradient with a temporal one. The shift of a DNA band in temporal temperature gradient gel electrophoresis (tTGGE) due to a mutation can be calculated as a function of the position of the mutation in the sequence. Appropriate DNA fragments were selected for polymerase chain reaction (PCR) amplification and analysis by tTGGE on the basis that the predicted band shifts amount to more than 10% of the migration distance for all possible mutations. The accuracy of the prediction was tested experimentally with ten known mutations in the human PrP gene, and quantitative agreement between theory and experiment was achieved. Thus, this screening method is also a suitable means to verify the absence of mutations in a given gene segment.
Additional Material:
6 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.11501601304
