The melting characteristics of PCR products screened for point mutations are crucial for DGGE. It is important that the fragment, when it reaches the critical point in the gel, denatures immediately, instead of slowly denaturing at one end and progressing with this process as the product runs deeper in the gel. Such a slow-melting process will result in fuzzy bands or smears, rendering mutation detection impossible.
Because the melting characteristics are vital for success, primers to amplify the target should be chosen with great care. With this aim, special software that analyzes the melting curves of possible PCR products is used for primer selection. A number of programs are available for various platforms, either commercially or for free. There are websites where a sequence can be analyzed online as well. The experimenter will usually see a rather irregular melting curve when analyzing a target sequence. Attachment of a GC clamp of 40–60 nucleotides most often flattens this curve dramatically.
The curve should be flat within a range of 1°C. Of course, the melting temperature around the GC clamp is very high. If attaching a GC clamp at one side does not flatten the curve, one should try attaching it to the other side, because for DGGE, it does not matter whether the GC clamp is attached to the forward or to the reverse primer.
The selection process involves trying various combinations of forward and reverse primers to find products with a good flat curve and primers that will work well together in a PCR. DGGE products typically range from 200 to 400 bp. It is difficult to find the correct melting curve for products longer than 400 bp.