GC-clamp

Myers, R.M., Fischer, S.G., Lerman, L.S. and Maniatis, T. (1985a) Nearly all base substitutions in DNA fragments joined to a GC-clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3131-3145. 

Sheffield, V.C., Cox, D.R., Lerman, L.S. and Myers, R.M. (1989) Attachment of a 40-base pair G+C-rich sequence (GC-clamp) to genomic DNA fragments by polymerase chain reaction results in improved detection of single-base changes. Proceedings of the National Academy of Sciences USA 86, 232—236. 

Sheffield, V.C., Beck, J.S., Nichols, B., Cousineau, A, Lidral, A. and Stone, E.M. (1992) Detection of multiallele polymorphisms within gene sequences by GC-clamped denaturing gradient gel electrophoresis. American Journal of Human Genetics 50, 567—575. 

PCR primers for DGGE should be designed using dedicated software, which is commercially available. Of greatest importance is a uniform (flat) melting temperature for the whole PCR product with the exception of the GC tail introduced by means of a 40 - 60 nucleotide GC clamp. For many disorders, DGGE primer sequences are available in the literature. 

Prepare a 100-//1 reaction for double-stranded amplification of a single specimen for perpendicular DGGE. Use the same primer combination planned for subsequent comparisons of multiple samples. One of the primers should include a GC clamp. 

Obtain double-stranded amplifications of all samples with the same pair of primers used for perpendicular DGGE (one should include a GC clamp). Use total reaction volumes of 25 or 12.5 pi. Run on agarose minigels and take plugs by puncturing with clean, positive displacement pipettes. Save the plugs in 250 pi of LTE buffer. 

Use 25 pi of the melted products to prepare 50-//1 reactions for asymmetric amplifications by PCR. Use primer versions without GC clamps. 

Greiner TC, Raffeld M, Lutz C, Dick F, Jaffe ES. Analysis of T cell receptor-gamma gene rearrangements by denaturing gradient gel electrophoresis of GC-clamped polymerase chain reaction products. Correlation with tumor-specific sequences. Am J Pathol 1995 146 46-55. 

Name and Sequence of GC clamps (2). The following clamps can be added to... 

Denaturating gradient gel electrophoresis (DGGE) High sensitivity Choice of primers is critical GC-clamped primers e5q>ensive does not reveal position of change... 

Myers, R.M., V.C. Sheffield and D.R. Cox 1989 Mutation detection by PCR, GC-clamps, and denaturing gradient gel electrophoresis. In, PCR Technology H.A. Erlich, Editor. Stockton Press, N.Y. 

Table 1 List of selected primers used to amplify small subunit ribosomal RNA used for DGGE. The target sites, sequences and specificity of the primers are shown. R (reverse) and F (forward) indicate the orientation of the primers. Y indicates C/T, W indicates A/T, M indicates A/C, R indicates A/G, S indicates C/G. A GC clamp (GC-rich sequence) is attached to the 5 -end of the forward primers to prevent complete dissociation... 

For PCR amplification prior to DGGE, one of the primers should be ordered with a GC clamp on the 5 end. The following is a suitable GC clamp sequence and the modification of primer 968F with this clamp sequence ... 

Once the gel casting cassette is ready, the denaturant gradient gel can be prepared. This involves making two solutions, one for each limit of the denaturant range. The description in this case assumes a denaturant range of 30-60% and a 7% gel but can be modified as necessary to achieve optimum separation of GC-clamped PCR products. The solutions are prepared in 50-ml centrifuge tubes according to the recipes in Table 3.2 and then filtered with a 0.45-pm filter. To 20 ml of each filtered solution. 

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