Temperature and pH effects on single-strand conformation polymorphism analysis by capillary electrophoresis

Abstract

We investigated the effects of temperature and pH on single strand-conformation polymorphism (SSCP) analyzed by capillary electrophoresis (CE) using short-chain linear polyacrylamide as the sieving medium. Nine different mutations (in factor V, cystathionine ~-synthase, and methylenetetrahydrofolate reductase genes), including both transitions and transversions, were investigated. We confirmed that low temperature in general increased the number of detectable single-strand conformations and thereby the sensitivity of the analysis. The pH effects of the separation matrix on the migration pattern, and thus the assay sensitivity, varied markedly between the different DNA fragments. Seven of nine single point mutations were detected at the ordinary pH of 8.3, whereas the CBS T833C mutation was discriminated at the extreme pH values of 9.0 and 6.4, and the CBS G 797A mutation could not be detected at any pH value within the range 6.4-9.0. These data emphasize the importance of the pH of the separation matrix in detecting certain mutations by SSCP ' Hum Mutat 13:458-463, 1999.

Rapid Detection of Deletion, Insertion, and Substitution Mutations via Heteroduplex Analysis Using Capillary- and Microchip-Based Electrophoresis

“…This differs dramatically from methods described for CE-based SSCP analysis where the separation temperature was critical (Ren at al. 1997;Ren and Ueland 1999;Tian et al 2000b). …”

Development of a simple and highly sensitive mutation screening system by enzyme mismatch cleavage with optimized conditions for standard laboratories

“…It is now possible that labeling PCR products with fluorescent and adapted SSCP and HA to automated DNA sequencing machines either slab gel electrophoresis [24][25][26][27][28] or capillary electrophoresis (CE) [29][30][31][32][33]. PCR products can be labeled by fluorescent primers [24][25][26] or fluorescent deoxynucleotides either during PCR [27] or after PCR [34,35].…”

Technical challenges in applying capillary electrophoresis-single strand conformation polymorphism for routine genetic analysis

“…Partial purification of crude PCR products by use of a Microcon-PCR (Millipore, Bedford, MA, USA) filtration device or QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) reduces the amount of primer-ssDNA constructs [6,13], while complete purification of crude PCR samples by preparative slab-gel electrophoresis can eliminate them entirely [14]. If additional PCR product purification is undesired or impossible, a similar but less pronounced beneficial reduction in primer-ssDNA annealing can be achieved by a mere decrease in initial primer concentration during PCR amplification [14].…”

“…While analysis temperatures between 20 and 307C typically are recommended as a starting point in temperature optimization for attempts to analyze novel DNA fragments by SSCP, one needs to keep in mind that lower or higher temperatures may in fact provide more sensitive analysis. Tris-borate-EDTA (TBE) running buffer that contains 10% glycerol remains the top choice in most CE-SSCP protocols, but other buffers such as Tris-HEPES and Tris-MES-EDTA have also been used successfully [13]. Gelfi et al [30] evaluated the combined effects of temperature, sieving matrix, applied voltage, capillary length, capillary inner diameter, and pH on CE-SSCP, and noted a significant improvement in the resolution and sensitivity of CE-SSCP when a low-pH buffer (Tris-MES-EDTA, pH 6.8) was used; at the same time, no beneficial changes were recorded when the temperature of analysis was lowered.…”