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Decoding the DNAj0283803.gif (5750 bytes)

Sequencing:

A sequencing method, developed by Sanger, uses dideoxynucleotides with the DNA sample and labeled phosphorus or sulfur.

The first step is:

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Separation of the two strands of DNA.

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Reanealing the DNA with a primer that complements a piece within the DNA.

Note: dNTP= deoxynucleotide triphosphate. There are 4 nucleotides that are made up of four bases: Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). A always pairs with T and G always pairs with C. In RNA molecule, Thymine is substituted for Uracil (U) which still pairs with A.

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Extension of this primer by the enzyme DNA polymerase and deoxynucleotides (dNTP).

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Termination reaction using different nucleotide for each reaction (ddNTP). [Four separate incubations, where each sample containing the same mix, but different ddNTP]. Since the ddNTP do not have the 3'hydroxyl group, it cannot form a bond with the next phosphodiester group. Thus, the reaction is terminated.

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Fragments of the DNA are separated on a polyacrylamide gel electrophoresis.

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Learn about sequencing through animation.  You need shockwave plug-in to view it in a web browser. Just click on

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DNALearningCenter

 

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