DNA sequencing is the process of determining the precise order of the nucleotide bases in particular DNA molecules. DNA sequencing is one of the key technique used in nearly all molecular biology and genetics labs. Currently most labs use automated sanger sequencing techniques (see below), however, some labs (especially high throughput laboratories) are moving to new sequencing platforms like the 454 pyrosequencing based platform or the massively parallel sequencing-by-synthesis approach being developed by a number of companies like Solexa, Agencourt, and a number of other companies. While these new approach offer great potential for genome scale sequencing projects, they are not really suited to small gene level sequencing projects so sanger sequencing based approaches will be used for some time to come.
The most common approach used to sequencing DNA is the dideoxy or Sanger method. This techniques was developed by Fred Sanger in Cambridge in the mid 1970s and for which he was awarded his second nobel prize. This method involves creating DNA fragments terminated with nucleotide analogues (dideoxy) that are able to be incorporated by enzymes known as DNA polymerases, but are not able to be extended. Traditional Sanger sequencing (also known as Manual Sequencing) involved performing four separate reactions (one each for the four bases found in DNA: guanine, cytosine, adenine and thymidine) and labeling the DNA fragments with radioactive phosphorus or sulphur (typically 32P, 33P or 35S). The DNA fragments generated from each dideoxy reaction were separated by electrophoresis under conditions that allow DNA molecules that differed in length by only one nucleotide to be resolved. Each separate dideoxynucleotide reaction was run in separate gel lanes (normally next to one another) so that for each DNA fragment sequenced there were four lanes.
Once the fragments had been electrophoresis separated ("run") over several hours to the desired level of resolution (typically 300-400 nucleotides) the gel plates were separated and the gel wrapped in cling film, or dried, then exposed to X-ray film for anywhere from a few hours to several weeks. The film was developed and the nucleotide order of the DNA fragment sequenced determined by a process of examining the X-ray film for the radioactive DNA fragments which appear as thin lines or "bands". This process involved placing the developed X-ray film on a light box and recording the order of bands (from bottom to top). The nucleotide order was determined by observing which dideoxy reaction lane the band appeared in. For example, if the first band was in the ddATP lane, the second in the ddCTP lane, and the third in the dGTP lane, then the nucleotide order of the original sequence is ACG.
This processes of "counting bands" is fairly simple when the bands are well separated (ie at the bottom of the gel), but it becomes increasing difficult the higher up the gel you try to count. It was very common to find multiple consecutive bands in the one lane which were not separated and had formed into a single fat band or "blob". It is be extremely difficult to determine how many bands are in a blob. For example, a large blob made up of 6 consecutive bands (eg a sequence of TTTTTT) looks much the same a blob made up of 7 consecutive bands (eg TTTTTTT). In addition, faults in the gel electrophoresis or gel construction process were very common resulting in band distortions that made it hard to determine the correct band order. These two effects made the process of DNA sequencing increasing error prone the longer it was attempted to read a sequence from the gel.
The entire process of Manual DNA sequencing was very time consuming. A highly experienced and skilled scientist was able to determine the sequence of only 1000-2000 nucleotides per week. For this reason it used to be possible to obtain a scientific publication consisting of nothing more than the DNA sequence of a fragment of DNA. For example, pre-1990 Nucleic Acid Research was filled with such publications which were known as "For the record" papers - these papers made for quite scintillating reading but I guess they reflected the amount of work required to obtain the data!
DNA can also be manually sequenced using the method of Maxam and Gilbert and was developed around the same time as Sanger sequencing. This approach used a complex chemical based cleavage approach to generate the required DNA ladder. While Maxam and Gilbert sequencing is chemically much more clever than sanger sequencing, it requires the use of a lot of very nasty chemicals - for this reason it has fallen out of favor except for very specialized applications like oligonucleotide sequencing (even here mass spectroscopy is now a better approach).
The enormous amount of manual labor required for Manual sequencing led to the development of a range of sequencing techniques which are now known as Automated DNA Sequencing.
Automated DNA sequencing refers to a number of sanger-based DNA sequencing methods which use DNA fragments labeled with fluorescent dyes rather than a radioactivity isotope. The fluorescent dyes can be attached to the 5' ends of the sequencing oligonuceotide (primer), or to the dideoxynucleotides. The later is more commonly used as each sequencing reaction only requires on reaction rather than four separate reactions when using labeled primers. The use of the labeled dideoxynucleotides also avoids the need to have synthesized specific labeled oligonucleotides when using custom primers (primer walking).
It is an exciting time in the DNA sequencing world with many new sequencing platforms being released (or to be released later) in 2007. The pyrosequencing based chip platform was developed by 454 and is currently marketed by Roche. Other systems include the SOLiD system developed by Agencourt and now owned by Applied Biosystems and the Illumina Genome Sequencer. All these sequencers use approaches different to traditional Sanger sequencing.
Applied Biosystems SOLiD DNA sequencer
Roche/454 Genome Sequencer 20
Illumina Genome Sequencer
For a very general introduction see DNA sequencing - your questions answered. This site provides a very basic introduction to some of the concepts behind DNA sequencing.