DNA sequencing gels have been a ‘workhorse’ technique for the molecular biologist, but have now been replaced by automated methods such as dideoxy sequencing for routine applications. However, for some particular applications, such as DNA footprinting, sequencing gels are still used.
Whereas agarose gel electrophoresis of DNA is highly suitable for relatively short DNA molecules, a different form of electrophoresis has to be used when DNA sequences are to be determined. Whichever DNA sequencing method is used, the final analysis usually involves separating single-stranded DNA molecules shorter than about 1000 nt and differing in size by only 1 nt. To achieve this, it is necessary to have a small-pored gel and so acrylamide gels are used instead of agarose. For example, 3.5% polyacrylamide gels are used to separate DNA in the range 80–1000 nt and 12% gels to resolve fragments of between 20 and 100 nt. If a wide range of sizes needs to be analysed, it is often convenient to run a gradient gel, for example from 3.5% to 7.5%. Sequencing gels are run in the presence of denaturing agents, urea and formamide. Since it is necessary to separate DNA molecules that are very similar in size, DNA sequencing gels tend to be very long (100 cm) to maximise the separation achieved. A typical DNA sequencing gel is shown in Figure 1.
Fig1. Autoradiograph of a DNA sequencing gel. Samples were prepared using the Sanger dideoxy method of DNA sequencing. Each set of four samples was loaded into adjacent tracks, indicated by A,C, G and T, depending on the identity of the dideoxyribonucleotide used for that sample. Two sets of samples were labelled with 35 S (1 and 3) and one was labelled with 32 P (2). It is evident that 32 P generates darker, but more diffuse bands than does 35 S, making the bands nearer the bottom of the autoradiograph easy to see. However, the broad bands produced by 32 P cannot be resolved near the top of the autoradiograph, making it impossible to read a sequence from this region. The much sharper bands produced by 35 S allow sequences to be read with confidence along most of the autoradiograph and so a longer sequence of DNA can be obtained from a single gel.
As mentioned above, electrophoresis in agarose can be used as a preparative method for DNA. The DNA bands of interest can be cut out of the gel and the DNA recovered by: (a) electroelution, (b) macerating the gel piece in buffer, centrifuging and collecting the supernatant or (c), if low melting point agarose is used, melting the gel piece and diluting with buffer. In each case, the DNA is finally recovered by precipitation of the supernatant with ethanol.
