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Maxum Gilbert Methods for DNA Sequencing
This method was developed by A. Maxum and W. Gilbert in 1977. This method uses different chemicals to break or alter the DNA at different points or bases in the DNA.

The process involves radiolabelling of the 5 ' -PO 4 of the DNA molecule to be sequenced by using alkaline phosphatase and A TP or using a chemical method. The DNA molecules are divided in four groups or transferred into four tubes. To the first tube, dimethyl sulphate is added, which methylates guanine at the N 7 position.

To the second tube, acid is added, which alters either adenine or guanine. Hydrazine is added to the third tube, which alters either thymine or cytosine. To the fourth tube, hydrazine along with NaCl is added to alter cytosine base pair. A fifth tube is kept as a reference containing only NaOH.

The fifth tube gives a redundant, but confirmatory inform.Piperidine is added to all the tubes to remove the altered base pairs and break the DNA at the sugar residue where the base is removed. Chemicals are added such that base specific reactions are carried out at one site per molecule.

This sequencing reaction is analysed by running the four or five samples side by side on a sequencing gel. A sequencing gel is a high-resolution gel designed to fractionate single stranded (denatured) DNA fragments based on their length. These gels contain 7M area and 6-20% polyacrylamide.

Electrophoresis is carried out at 70°C to prevent the formation of secondary structure. After electrophoresis, the gel is exposed to large sheets of X-ray film in dark for 24 hours. Radiolabelled phosphate present on the DNA strands or fragments emit energy, which reacts with the X-ray film to produce a band corresponding to the location or position on the gel.

This technique of generating bands by exposing X-ray film to radioactive material is called as autoradiography. The film is developed and the sequence is read directly from the sequencing ladder in adjacent base specific tracks.

 
 

 
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