Dimethylsulphate Formic acid Hydrazine Hydrazine + salt
Methylates G Protonates purines Splits pyrimidine rings Splits only C rings migrate through the gel strictly according to their size. The migration speed is important because single-base resolution is required to interpret the sequence properly.
After electrophoresis, the gel apparatus is disassembled; the gel is removed to a sheet of filter paper, and it is dried on a gel dryer. The dried gel is exposed to lightsensitive film. Alternatively, wet gels can be exposed directly. An example of Maxam-Gilbert sequencing results is shown in Figure 10-2. The sequence is inferred from the bands on the film. The smallest (fastest-migrating) band represents the base closest to the labeled end of the fragment. The lane in which that band appears identifies the nucleotide. Bands in the purine (G + A) or pyrimidine (C + T) lane are called based on whether they are also present in the G- or C-only lanes. Note how the sequence is read from the bottom (5' end of the DNA molecule) to the top (3' end of the molecule) of the gel.
Although Maxam-Gilbert sequencing is a relatively efficient way to determine short runs of sequence data, the method is not practical for high throughput sequencing of long fragments. In addition, the hazardous chemicals hydrazine and piperidine require more elaborate precautions for use and storage. This method has therefore been replaced by the dideoxy chain termination sequencing method for most sequencing applications.
Polyacrylamide gels from 6% to 20% are used for sequencing. Bromophenol blue and xylene cyanol loading dyes are used to monitor the migration of the fragments. Run times range 1-2 hours for short fragments (up to 50 bp) to 7-8 hours for longer fragments (more than 150 bp).
Dideoxy (Sanger) Sequencing
The original dideoxy chain termination sequencing methods required a single-stranded template. Templates up to a few thousand bases long could be produced using M13 bacteriophage, a bacterial virus with a single-stranded DNA genome. This virus replicates by infecting Escherichia coli, in which the viral single-stranded circular genome is converted to a double-stranded plasmid, called the replication factor (RF). The plasmid codes for
■ Figure 10-2 Products of a Maxam-Gilbert sequencing reaction. The gel is read from the bottom to the top. The size of the fragments gives the order of the nucleotides. The nucleotides are inferred from the lane in which each band appears. A or T is indicated by bands that appear in the G + A lane or C + T lane, respectively, but not in the G lane or the C lane. G is present in the G + A lane and the G lane. C is present in the C + T lane and the C lane.
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