Dideoxynucleotide Chain Terminators

The reaction mixture is then divided into four aliquots, representing the four dNTPs, A, C, G and T. Using the adenine (A) tube as an example, in addition to all of the dNTPs being present in the mix, an analogue of dATP is added [2´,3´-dideoxyadenosine triphosphate (ddATP)], which is similar to A except that it has no 3´-hydroxyl group. Since a 5´ to 3´ phosphodiester linkage cannot be formed without a 3´-hydroxyl group,the prese nce of the ddATP will terminate the growing chain. The situation for tube C is identical except that ddCTP is added; similarly, the G and T tubes contain ddGTP and ddTTP, respectively. Since the incorporation of a ddNTP rather than a dNTP is a random event, the reaction will produce new molecules varying widely in length, but all terminating at the same type of base. Thus four sets of DNA sequence are generated, each terminating at a different type of base, but all having a common 5´ end (the primer). The four labelled and chainterminated samples are then denatured by heating and loaded next to each other on a polyacrylamide gel for electrophoresis. Electrophoresis is performed at approximately 70 °C in the presence of urea, to prevent renaturation of the DNA, since even partial renaturation alters the rates of migration of DNA fragments. Very thin, long electrophoresis gels are used for maximum resolution over a wide range of fragment lengths.
After electrophoresis, the positions of radioactive DNA bands on the gel are determined by autoradiography. Since every band in the lane from the ddATP sample must contain molecules which terminate at adenine and that those in the ddCTP terminate at cytosine, etc., it is possible to read the sequence of the newly synthesised strand from the autoradiogram. Under ideal conditions, sequences of approximately 300 DNA bases can be read from one gel.

مواضيع ذات صلة

A Common Mechanism for Splicing Reactions

آليات واهمية النقل transduction mechanisms

النقل transduction

Regulation of Transcription by Chromatin

RNA Polymerase Binding and Regulation by Transcription Factors

The Discovery of Self-Splicing RNAs

Splicing Reactions and Complexes

آلية واهمية التحول

التحول transformation

Involvement of the U1 snRNP Particle in Splicing

البادئ (Promoter)

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