Influence of nearest neighbor sequence on the stability of base pair mismatches in long DNA: determination by temperature-gradient gel electrophoresis

Abstract

Temperature-gradient gel electrophoresis (TGGE) was employed to determine the thermal stabilities of 48 DNA fragments that differ by single base pair mismatches. The approach provides a rapid way for studying how specific base mismatches effect the stability of a long DNA fragment. Homologous 373 bp DNA fragments differing by single base pair substitutions in their first melting domain were employed. Heteroduplexes were formed by melting and reannealing pairs of DNAs, one of which was 32P-labeled on its 5'-end. Product DNAs were separated based on their thermal stability by parallel and perpendicular temperature-gradient gel electrophoresis. The order of stability was determined for all common base pairs and mismatched bases in four different nearest neighbor environments; d(GXT).d(AYC), d(GXG).d(CYC), d(CXA).d(TYG), and d(TXT).d(AYA) with X,Y = A, T, C, or G. DNA fragments containing a single mismatch were destabilized by 1 to 5 degrees C with respect to homologous DNAs with complete Watson-Crick base pairing. Both the bases at the mismatch site and neighboring stacking interactions influence the destabilization caused by a mismatch. G.T, G.G and G.A mismatches were always among the most stable mismatches for all nearest neighbor environments examined. Purine.purine mismatches were generally more stable than pyrimidine.pyrimidine mispairs. Our results are in very good agreement with data where available from solution studies of short DNA oligomers.

Rapid screening of β-Globin gene mutations by Real-Time PCR in Egyptian thalassemic children

“…In two samples, we observed different melting curves which were close to but distinct from the mutant curves of the detection probes; after sequencing, these samples were found to have the IVS I-5 and the frame shift mutations, which are less common in the Egyptian population. All genotype results were confirmed by routine methodologies, including denaturing gradient gel electrophoresis in combination with ARMS PCR and direct sequencing [3,11,12] The IVS I-1 mutation gave a Tm mean ± standard deviation of 59.07 ± 0.45° C, the IVS I-6 mutation gave a Tm mean ± standard deviation of 61.34 ± 0.27° C while IVS I-110 mutation gave Tm mean ± standard deviation of 66.96 ± 0.58° C. Here the design of the sensor probes of the IVS 1-1/6 and IVS I-110 takes the advantage of the fact that different mutations placed along the sequence produce distinct effects on the Tm [12] Thus, the melting curves permitted the distinction of the normal allele from IVS 1-1 and IVS 1-6 mutations separated by five nucleotides apart. The melting curves obtained immediately after the amplification procedure permitted the identification of the Tm peaks corresponding to normal or mutated alleles in each sample (fig 2A) …”

“…The software provided with the equipment (LightCycler 2) gives the temperature of melting (Tm) of the sensor probe. A base pair mismatch between the sensor probe and template causes a decrease in Tm that can be easily detected by the LightCycler [12,13] …”

Evaluation and application of denaturing HPLC for mutation detection in Marfan syndrome: Identification of 20 novel mutations and two novel polymorphisms in theFBN1gene

“…These results suggest that the degree of DHPLC pattern abnormality is mainly influenced by the sequences flanking the mismatch rather than by the nature of the mismatch itself. The reason for this phenomenon may lie in neighboring stacking interactions [Ke and Wartell, 1993] and hydrogen bonding between non-Watson-Crick base oppositions such as G-T and G-A [Aboul-ela et al, 1985]. In our study, two pairs of mutations (encoded 35, 34; and 4, 64 in Table 1) provide evidence for a mismatch stabilization effect of flanking bases, both representing neighboring mutations (∆1 bp) which result in different single-base mismatches but in FIGURE 4.…”

Genetic Variability of Immunomodulatory Genes in Ectromelia Virus Isolates Detected by Denaturing High-Performance Liquid Chromatography

“…No correlation of DHPLC profiles with mutation type was observed. This is expected since the degree of denaturation depends on the nature of both the mismatched and flanking sequences (22,31).…”