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Some groups of research workers have developed alternate methods to Sanger method for sequencing of DNA. Unfortunately, despite the initial excitement, most of these methods have disappeared from the scene. There are at least two methods with some promise for DNA sequencing pyrosequencing, and gene chips (microarrays).

Pyrosequencing

Pyrosequencing is a DNA sequencing method that is based on the principle of determining which one of the four bases (A, G, C, and T) is incorporated at each step while a DNA template is copied. In this technique, the template copies in a straight forward manner without added dideoxynucleotides. As the new strand is synthesized, the order in which the deoxynucleotides are incorporated is detected, and the sequence can be read as the reaction proceeds. The identification of base addition becomes possible since the addition of a nucleotide is accompanied by the release of a molecule of pyrophosphate. This can be detected by chemiluminescene technique.

In the procedure of pyrosequencing, each dNTP is added individually, along with a nucleotidase enzyme. This enzyme degrades the dNTP if it is not incorporated into the newly synthesized DNA strand. Once the appropriate nucleotide is incorporated into the new DNA strand, a molecule of pyrophosphate is released. This can be converted by the enzyme sulfurylase into a flash of light. By the addition of each dNTP separately, one after another, the order of the nucleotides added to the growing DNA strand can be followed, and the sequence can be identified.

Detection of the molecule pyrophosphate is the basis of DNA sequencing, hence the name pyrosequencing. Since pyrosequencing does not require electrophoresis or any other DNA fragment separation technique, it is more rapid than chain termination sequencing.

The major limitation of pyrosequencing is that it is suitable to detect the sequence of about 200 nucleotides. This is much less than the Sanger method. Many improvements are being made in pyrosequencing so that much longer DNA molecules can be sequenced. In fact, an automated system for pyrosequencing is also available now.