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Study Notes: Denaturing Gels - DNA and RNA

This section deals mainly with SDS-PAGE of proteins but you should be aware that a lot of DNA sequencing work is also carried out using polyacrylamide gels. RNA is easily broken down, or ‘unstable’. This is because ‘RNAses’, enzymes whose job is to chew up RNA, are found everywhere, even in our fingerprints. Special procedures must be employed to ensure the cleanliness of all apparatus and reagents used with RNA analysis. Analysis of DNA is performed on the single-stranded form, not the double-stranded form, but even so problems may arise due to the secondary structures assumed by these molecules.

The inclusion of denaturing agents such as urea or formamide is required to unfold the DNA/RNA strand and remove the influence of shape on the mobility of the molecule.

A diagram showing the DNA molecule tightly bound at one end but open at the other, just like a zip fastener partly undone.
Denaturation of DNA by urea. Notice that the urea in effect opens the DNA molecule like a zip fastener.

Some factors affecting DNA-PAGE

The gel
Sequencing gels are poured between two long glass plates with very thin spacers (0.4 mm or thinner). Bubbles may become a problem due to pouring and they will interfere with the polymerisation of the gel leading to unusable lanes. Scrupulously clean plates, siliconising one plate and extreme care during pouring are ways to minimise bubble formation.

A line diagram of a typical DNA sequencing gel (polyacrylamide), in a vertical tank, the negative electrode chamber at the top (with the sample wells) and the positive electrode chamber at the bottom.
This is an example of a typical DNA sequencing gel (polyacrylamide).

Temperature is a critical factor in DNA-PAGE and must be kept above 40°C to avoid renaturation of the DNA even in the presence of high concentrations of urea (typically 6 - 7 M). Usually 45 - 60°C is used. To achieve this, gels are run at constant power (wattage) rather than constant voltage or current.

Buffer selection is important for DNA-PAGE and Tris-Borate EDTA is the buffer of choice as it has a high buffering capacity and can run at high voltages for hours without exhaustion. Sometimes Tris-Taurine EDTA buffer is used, especially where glycerol is used as part of the sample preparation process.

Buffer gradients
Buffer gradients can be employed to compress regions of the gel leading to more information from the run. For example, compressing the pattern at the bottom of the gel will allow longer runs without losing smaller bands and is a useful approach. Without such compression the bands would run off the bottom of the gel and be lost. This compression effect can be achieved by using ‘wedge’ spacers at the bottom of the gel or by using a buffer gradient coupled with a higher conductivity buffer in the lower buffer chamber.

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