Nearly all single base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis

Abstract

Duplex DNA fragments differing by single base substitutions can be separated by electrophoresis in denaturing gradient polyacrylamide gels, but only substitutions in a restricted part of the molecule lead to a separation (1). In an effort to circumvent this problem, we demonstrated that the melting properties and electrophoretic behavior of a 135 base pair DNA fragment containing a beta-globin promoter are changed by attaching a GC-rich sequence, called a 'GC-clamp' (2). We predicted that these changes should make it possible to resolve most, if not all, single base substitutions within fragments attached to the clamp. To test this possibility we examined the effect of several different single base substitutions on the electrophoretic behavior of the beta-globin promoter fragment in denaturing gradient gels. We find that the GC-clamp allows the separation of fragments containing substitutions throughout the promoter fragment. Many of these substitutions do not lead to a separation when the fragment is not attached to the clamp. Theoretical calculations and analysis of a large number of different mutations indicate that approximately 95% of all possible single base substitutions should be separable when attached to a GC-clamp.

Microbial contamination of laboratory constructed removable orthodontic appliances

“…The use of molecular techniques has shown far superior results in identification of the bacterial species present in a sample in comparison to bacterial cultures on enriched media [18]. The use of PCR and DGGE combined with GC-rich primers at the 5′ end has been shown to identify almost all of the sequence variations in a sample [20] .This method allows direct identification and relative abundance of the bacterial species present in a sample, without the risks of inability to directly culture by conventional methods only [18]. Some deficiencies of DGGE are that multiple species may be present in one band when examining complex microbial communities [21], but PCR amplification and identification were undertaken of the samples taken at each stage in the construction process to enable identification of the species present.…”

The link between mitochondrial DNA hypervariable segment I heteroplasmy and ageing among genetically unrelated Latvians

“…A GC clamp added to the 5′ end of one primer in each pair (GC) made the resulting PCR products suitable for DGGE analysis (Myers et al 1985;Tully et al 2000). PCR amplification was performed by combining 10-50 ng DNA templates, 10xPfu buffer with MgSO 4 , 10 mM primers, 10 mM dNTPs, deionized water, and 2,5 U/μl Pfu DNA polymerase (Fermentas, Lithuania).…”

Analysis of mutational spectra by denaturing capillary electrophoresis

“…The goal of melting profile analysis of DNA fragments with computer programs prior to DCE is to select and manipulate the target sequence, attach GC-clamp if necessary, so that the region of interest is in a low melting domain adjacent to a sequence with high temperature melting properties 28,53,82,87,[89][90][91] . The introduction of the thermally stable clamp increases the percentage of detectable DNA variants in the low melting domain by DGGE to close to 100% 92,93 . Even if this statement must be further evaluated, it suggests that most of the genome can be analyzed with this methodology.…”

“…DNA amplification does not only increase number of copies of DNA exponentially, but also restricts the amplification to specific target sequences determined by the primers. Furthermore DNA amplification allows for incorporation of fluorescent dyes and high temperature melting domain, also known as GC-clamps 92,93 . DNA amplification protocols amplifying DNA with GC-clamped primers has been published elsewhere 13,14,35,36,78,79,84,[105][106][107] .…”