Scanning for mutations in the human prion protein open reading frame by temporal temperature gradient gel electrophoresis

Abstract

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.

Bacterial diversity and community composition in the chemocline of the meromictic alpine Lake Cadagno as revealed by 16S rDNA analysis

“…The TTGE technique is based on the same principle as DGGE, but does not require a chemical denaturing gradient [21,22]. The TTGE patterns obtained were highly reproducible.…”

Detection of point mutations by capillary electrophoresis with temporal temperature gradients

“…Linearized plasmids carrying the wild type (wt) [18] or the Pro102Leu mutation (mt) [19] sequence were amplified by PCR as described before [8] (Table 1). The PCR products were purified using the QIAquick spin PCR purification kit (Qiagen, Hilden, Germany) to remove nucleotides and primers and eluted with 100 mL 1´TBE (89 mM Tris, 89 mM borate, 2.5 mM EDTA, pH 8.3).…”

“…Best fragments were chosen by thermodynamic calculations based on the algorithm of Poland [9], which was improved by Steger [10] particularly for application to gradient gel electrophoresis. To ensure that any unknown mutation will be detected in a PCR fragment TGGE, the program MISMATCH [8] calculates the relative migration difference for a possible mismatch in any position of a PCR fragment. Optimizing the DNA fragments according to these calculations for experimental analysis by TGGE, it was possible to detect all mutations in the examples analyzed [8].…”

“…To ensure that any unknown mutation will be detected in a PCR fragment TGGE, the program MISMATCH [8] calculates the relative migration difference for a possible mismatch in any position of a PCR fragment. Optimizing the DNA fragments according to these calculations for experimental analysis by TGGE, it was possible to detect all mutations in the examples analyzed [8].…”

“…The most reliable methods, particularly to analyze large numbers of samples, are chemical mismatch cleavage [2] and gradient gel electrophoresis, either using a denaturing gradient (DGGE) [3] or a temperature gradient (TGGE). The temperature gradients were either spatial, perpendicular or parallel [4±6], or temporal [7,8]. Because the retardation of double-stranded DNA is greatest at the temperature of partial denaturation, DNA fragments of the same size can be separated according to different thermodynamic stabilities.…”