Detecting base pair substitutions in DNA fragments by temperature-gradient gel electrophoresis

Abstract

A vertical gel electrophoresis apparatus is described which can distinguish DNA fragments differing by single base pair substitutions. The system employs a homogenous polyacrylamide gel containing urea-formamide and a temperature gradient which runs either perpendicular or parallel to the direction of electrophoresis. The temperature-gradient system simplifies several features of the denaturant-gradient system (1) and is relatively inexpensive to construct. Eight homologous 373 bp DNAs differing by one, two, or nine base pair substitutions were examined. DNA electrophoretic mobility changed abruptly with the temperature induced unwinding of DNA domains. GC to AT substitutions at different locations within the first melting domain, as well as an AT to TA transversion were separated with temperature gradients parallel to the electrophoretic direction. The relative stabilities of the DNAs observed in the gels were compared to predictions of DNA melting theory. General agreement was observed however complete correspondence was not obtained.

Chip-based mtDNA mutation screening enables fast and reliable genetic diagnosis of OXPHOS patients

“…Other methods for mtDNA mutation analysis are conventional sequence analysis, single strand conformation polymorphism (SSCP) analysis, denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), denaturant capillary electrophoresis (DCE) and denaturing high-performance liquid chromatography (dHPLC). [13][14][15][16][17] These techniques all have their advantages and disadvantages with respect to costs time consumption, high-output possibilities, heteroplasmic detection limit, and type of mutations that can be detected (Table 3). At this moment, heteroduplex-based methods (dHPLC, DGGE, TGGE, and DCE) appear to be more sensitive than the MitoChip for heteroplasmy detection, with detection sensitivities as low as 0.5%.…”

The in-depth evaluation of suspected mitochondrial disease

“…Laboratory methods that can be used to screen for known point mutations include PCR with restriction fragment length polymorphism (RFLP) analysis to screen for specific mutations on a single basis or multiplex PCR with allele specific oligonucleotide (ASO) analyis to screen for multiple known mutations simultaneously. Methods that can be used to screen for unknown mtDNA point mutations include single-strand conformation polymorphism (SSCP) [119], heteroduplex screening assays (such as temporal temperature gradient gel electrophoresis (TTGE) [120][121][122], temperature gradient gel electrophoresis (TGGE) [123], denaturant gradient gel electrophoresis (DGGE) [124,125], denaturing high performance liquid chromatography (dHPLC) [126]), and sequencing. As direct DNA sequencing is generally considered to be the gold standard for mutation detection in nuclear genes, the advent of rapid sequencing tools has resulted in some laboratories including mtDNA sequencing in their clinical diagnostic approach by one of several different sequencing methodologies available.…”

Quantitative predictions for DNA two‐dimensional display according to size and nucleotide sequence composition

“…Furthermore, since all fragments correspond to different parts of the genome, the denaturing gradient gel electrophoresis (DGGE) in the whole genome display cannot bene-fit from the improved sensitivity of "standard" TGGE/DGGE aimed at analyzing the polymorphism across individuals of a gene or part of a gene. In such a case, a GC-clamp added to the DNA fragments prevents the complete strand dissociation and ensures that base substitutions in the highest temperature melting domain can be detected [3,[7][8][9][10]. Finally, although simulations have been used to a limited extent to plan experiments and interpret results [11,12], the method is mostly empirical at the present time, and the optimization of the experimental conditions often requires a long trial-anderror process.…”