Nucleic acid sequencing Introduction, Saccharomyces cerevisiae, Escherichia coli, Homo sapiens, DNA polymerase

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Nucleic Acid Sequencing Nucleic Acid Sequencing Introduction Chain Termination Sequencing Preparation of Single Stranded DNA Template Strand Synthesis Reaction for Chain Termination Sequencing Primer DNA Polymerase Deoxynucleoside Triphosphates Chain Terminating Nucleotide Sequencing Reactions Primer Annealing Strand Synthesis Running the Gel and Reading the Sequences

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	Introduction The ultimate description of a gene is the determination of the sequence of nucleotide bases of which it is comprised and which, in turn, determines the nature of the encoded protein product. From the first development of an accessible and routine technique to determine the nucleotide sequence of only small stretches of DNA, we have now advanced to the stage where nucleotide sequenced are determined at a prodigious rate. This has resulted in the sequencing of the complete genomes of a number of bacteria and the eukaryote yeast Saccharomyces cerevisiae.
Such is the pace of progress in the application of nucleotide sequencing technology that the sequence of the complete human genome is available now. This remarkable progress has been made possibly by the application of relatively simple technologies. Such was the importance of these techniques that their proponents, Walter Gilbert and Fred Sanger, were jointly awarded the Noble Prize for chemistry in 1980
Methods of DNA sequencing were developed in the late 1970s and have revolutionized the science of molecular genetics. The DNA sequences of many different genes from diverse sources have been determined. Projects are already underway to map the sequence of the entire genome of organisms such as Escherichia coli, Saccharomyces cerevisiae and Homo sapiens. Large-scale sequencing projects such as the Human Genome Project, product the DNA sequence of many unknown genes. DNA sequencing can act as a catalyst to stimulate future research into many diverse areas of science.
The two original methods of DNA sequencing described in 1977 differ considerably in principle. The enzymatic (or dideoxy chain termination) method of Sanger (Sanger et al., 1977) involves the synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The Maxam and Gilbert (or chemical degradation) method (Maxam and Gilbert, 1977) involves chemical degradation of the original DNA. Both methods produce populations of radioactivity labelled polynucleotides that begin from a fixed point and terminate at points dependent on the location of a particular base in the original DNA strand. The polynucleotides are separated by polyacrylamide gel electrophoresis, and the order of nucleotides in the original DNA can be read directly from an autoradiography of the gelal

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Direct Cycle Sequencing Chemical Degradation Sequencing Preparation of End Labelled DNA Conversion of End Labelled Molecule into a Form Suitable for Sequencing Chemical Degradation Reactions Automated Nucleic Acid Sequencing Applications of Gene Sequencing

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