Sanger's Dideoxynucleotide Synthetic Method,Frederic Sanger,Plus Minus Method,Dideoxynucleotides,Triphosphates,Autoradiograph

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Isolation, Sequencing and Synthesis of Genes Isolation, Sequencing and Synthesis of Genes - Introduction Isolation of Genes Isolation of Genes Coding for Specific Proteins Isolation of Genes with Known or Unknown Products having Tissue Specific Expression Isolation of Genes with Known or Unknown Products Using DNA or RNA Probes Use of Heterologus Probes Use of Synthetic Probes Isolation of Genes Coding Use of Transposable Elements Transposon Tagging T-DNA-Insertion Mutagenesis for Isolation of Plant Genes Promoter Enhancer and Gene Trap for Isolation of Genes Differential Screeing and Differential Display Technique for Isolation of Genes Subtractive Hybridizatioon for Gene Isolation Map Based Cloning for Gene Isolation Isolation of Novel Genes by Asis Data at JNU New Delhi

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Sanger's Dideoxynucleotide Synthetic Method Frederic Sanger (who won Nobel Prize twice) had initially developed a method for DNA sequencing, which utilized DNA polymerase to extend DNA chain length. This was termed plus-minus method. Subsequently, he developed a more powerful method, utilizing single stranded DNA as template for DNA synthesis, in which 2,3, dideoxynucleotides were incorporated leading to termination of DNA synthesis.
A dideoxy base analogue showing dideoxy sugar absence of oxygen at 2' and 3' positions
These dideoxynucleotides are used as triphosphates (ddNTP) and can be incorporated in a growing chain, but they terminate synthesis, since they cannot form a phosphodiester bond with next incoming deoxynucleotide triphosphate (dNTP). Following steps are involved in Sanger's dideoxy method for DNA sequencing. Strategy used in chain termination using dideoxy analogue of a base only reaction with ddATP
(i) Four reaction tubes are set up, each containing single stranded DNA sample (cloned in M13 phage) to be sequenced, all the four dNTPs, an oligonucleotide sequencing primer (radioactively labelled) and an enzyme for DNA synthesis (DNA polymerase I = sequenase). Each tube also contains a small amount (much smaller amount relative to four dNTPs) of one of the four ddNTP, bringing about termination at a specific base-adenine (A), cytosine (C), guanine (G) and thymine (T). (ii) The fragments, generated by random incorporation of ddNTP leading to termination of reaction, are then separated by electrophoresis on a high resolution polyacrylamide gel. This is done f r all the four reaction mixtures on adjoining lanes in the gel. (iii) The gel' is used for autoradiography so that the position of different bands in each lane can be visualized. (iv) The bands on autoradiogram can be used for getting the DNA sequence as shown in.

An Autiograph of a Sangers sequencing Gel

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