Maxam-Gilbert sequencing

Maxam – Gilbert sequence is a method of DNA sequencing was developed by Walter Gilbert and Allan Maxam in 1977-1976. Chemical modifications partial DNA base-specific, this method is based on the cleavage of the subsequent DNA backbone at a position adjacent to the nucleotides and modified. Maxam – Gilbert sequence is a method widely accepted for the first DNA sequencing, of and along with the DNA methods Sanger dideoxy method, the first generation. Maxam – are no longer in use, they, Gilbert sequencing, has been replaced by the next generation sequencing method.

Walter Gilbert and Allan Maxam, announced the DNA sequencing method in 1977 on the basis of change of the chemical DNA, the cleavage of subsequent specific site. Also known as a chemical sequence, this method, samples of purified double-stranded DNA, could be used without further cloning. After done improved method of Sanger, and discouraged radiolabeled spread, its technical complexity, will use this method. Maxam – it requires a radioactive label at one end, Gilbert sequencing, were sequenced purified DNA 5 ‘and 3’ of the DNA fragment. The chemical treatment, and then generate an interrupt to the small part of one or two 1 (G, G, C, CT) of the nucleotide bases four each of the four reactions. The concentration of modifying chemicals is controlled to introduce a modified average of the DNA molecule. Thus, a series of fragments that have been labeled generated from radioactive end “cuts” the first spot of each molecule. Fragments in four reactions are electrophoresed side by side in denaturing acrylamide gel for size separation. The obtained corresponding to the DNA fragment was radioactively labeled in order to visualize the fragment, it is possible to infer the sequence of the gel but not included, anything black strip and subjected to autoradiography for X-ray film.

The DNA is purified in two years later published a work of plus or minus sequencing, it is possible to have the original method, to be used directly, Maxam Alan Coulson and Frederick Sanger Gilbert and Maxam – Gilbert sequencing rapidly , it becomes more and more popular, it exposes the chemical sequencing method of them is Sanger, you need to get a single-stranded DNA to start reading all that is cloned. However, the improvement chain termination method (see below), Maxam – widespread use molecular biology standard kit, of hazardous chemicals, and difficult to scale-up, prohibits the use, Gilbert sequencing, the I fell out of favor because of technical complexity.

Maxam – it requires a radiolabeled at one end (by kinase reaction using γ -32 P ATP usually) After sequencing, Gilbert sequencing, and purification of DNA, 5 ‘DNA fragment. Chemical treatment produces a disruption of a small part of one or two 1 (G, G, C, CT) of the nucleotide bases four each of the four reactions. For example, purine (G) was methylated using was methylated pyrimidine and dimethyl sulphate in (CT) (degree of adenine) guanine, hydrazine was depurinated with formic acid. Salt (sodium chloride), and the addition of the hydrazine reaction inhibits the reaction of thymine the response of only C. Then modified DNA may be cleaved by heat at the position of the piperidine modified bases. The concentration of modifying chemicals is controlled to introduce a modified average of the DNA molecule. Thus, a series of fragments that have been labeled generated from radioactive end “cuts” the first spot of each molecule. Fragments in four reactions are electrophoresed side by side in denaturing acrylamide gel for size separation. In order to visualize the fragment obtained indicates the position of the radioactively labeled DNA molecules of the same each gel, a series of dark strip and subjected to autoradiography for X-ray film. It is possible to estimate the presence or absence of a particular fragment of the sequence.