Temperature

The most critical parameter in denaturing DNA-PAGE is gel temperature. Highly concentrated urea, 6-7M, is the most commonly used denaturant, but to be fully effective, the temperature must be maintained above 40°C. Denaturing PAGE gels are generally run with a temperature in the range of 45 - 60°C, which is maintained by running the gel at constant power (wattage), as opposed to constant voltage or current. Since power measures the energy transferred through the gel maintaining constant power provides constant heat output and thus a stable temperature. It is crucial that denaturing gels be pre-run for at least 30 minutes prior to loading, to bring the gel up to operating temperature.

Denaturants

Some nucleic acid molecules are particularly difficult to denature. Sequences with a high guanine and cytosine content have more hydrogen bonds than adenine and thymine-rich sequences and require more vigorous denaturation. Sequences which can fold back on themselves are also difficult to denature fully, because the two complementary sequences are "tethered," increasing the likelihood of their re-annealing after denaturing. Difficult samples such as these may require gel temperatures in the 60-80°C range, which can give fuzzy bands, and distorted gels and may lead to cracked plates. Alternatively, formamide, a more active denaturant, may be included in the gel at concentrations up to 40%. This strategy is often used in DNA sequencing to alleviate GC compressions; regions of poor resolution caused by high GC content regions in the sample.

Buffer

Another important consideration in running denaturing PAGE is buffer selection. Some buffers used in molecular biology, most notably tris acetate EDTA (TAE), are easily exhausted. This means that they lose buffering capacity during the run, resulting in pH shifts at the ends of the gel. tris borate EDTA (TBE) is the buffer of choice for denaturing PAGE of nucleic acids. It has a high buffering capacity and can be run at high voltage for hours without exhaustion. Its only drawbacks are that it can interfere with some DNA recovery protocols and that it forms complexes with glycerol which can distort gel patterns. If glycerol is required as part of sample preparation, tris-taurine EDTA (TTE) buffer is recommended. TTE has buffering capabilities similar to TBE but shows no artifacts in the presence of glycerol.

Buffer Gradients

Gradients of buffer concentration can be employed to compress regions of the gel, thus providing more information per run. Fragment mobility is proportional to the logarithm of the molecular weight, so for 10 base fragments that differ by 1 base in length, the difference in mobility is 4%. For a 1 base difference at 100 bases, it is 0.3%, and at 500 bases, 1 base difference gives only a 0.03% difference. Thus, at the bottom of a 40cm gel, a 1 base difference in length will give band separations of 1-2cm, while 100 base fragments—having migrated 20cm—will separate from 101 base fragments by only 0.1cm. Clearly, compressing the pattern at the bottom of the gel—so as to allow longer runs without losing the smaller bands—will allow greater resolution of the larger fragments in the sample. This is accomplished by lowering the resistance across the lower portion of the gel which leads to a lessening of the voltage drop across this region. In the lower voltage gradient, bands will migrate more slowly. Small bands will thus migrate rapidly through the upper portion of the gel, and then slow down as they approach the end. This allows for longer runs, which facilitates the resolution of the larger bands on the gel.

The drop in resistance at the bottom of the gel can be accomplished by the use of wedge spacers, which are wider at the bottom, increasing the cross-sectional area of the gel. More often, however, a buffer gradient is used (protocol), with a higher conductivity buffer in the lower buffer chamber.

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