Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
3 posts • Page 1 of 1
For thirty years a huge fraction of DNA sequencing has been achieved using the chain termination method , developed by Frederick Sanger and Howard Chadwell (Student) in 1975. This technique uses sequence-specific termination of an in vitro DNA synthesis reaction using modified nucleotide substrates. DNA Sequencing has revolutionized biological discovery. The speed of development in DNA sequencing technology was astonishing and had facilitated the first ever large-scale biological project. The Human Genome and related projects spanned many continents in an amazing co-operative effort to generate the DNA sequences of many genomes. The focus is now shifting to resequencing in an immense effort to establish a link between genotypic variation and phenotype. 'Next generation' sequencing technologies from 454 Sequencing, Solexa, and Applied Biosystems are beginning to deliver large amounts.
In 1976-7, Allan Maxam and Walter Gilbert developed a method of DNA sequencing based on chemical modification of DNA followed by its subsequent cleavage at specific bases. Although this is contemporaneous with the development of Sanger sequencing, Maxim-Gilbert sequencing was initially more popular since purified DNA could be used directly, while the initial Sanger method required that each read start be cloned for production of single-stranded DNA. However, as the chain termination method has been developed and improved, Maxam-Gilbert sequencing has fallen out of favour due to its technical complexity, the need for use of hazardous chemicals, and difficulties with scale-up.
In brief, the method requires purifying a particular DNA fragment, radioactively labeled at one end. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). Thus a series of labelled fragments is generated, from the radiolabelled end to the first 'cut' site in each molecule. The fragments are then size-separated in a gel, with the four reactions arranged side by side. The gel can then be exposed to photographic film, yielding an image of a series of 'bands', from which the sequence may be inferred.
Also sometimes known as 'chemical sequencing', this method originated in the study of DNA-protein interactions (footprinting), nucleic acid structure and epigenetic modifications to DNA, and within these it still has important applications.
3 posts • Page 1 of 1
Who is online
Users browsing this forum: claudepa and 4 guests