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sanger_sequencing

by Ted Wong (Submitted: 03/02/2009)

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THE SANGER METHOD OF DNA SEQUENCING

This model simulates a classic method of determining the primary nucleotide sequence of a single strand of DNA. The Sanger method is a low-toxicity, low-radioactivity, low-technology method of sequencing, and it is still a popular method where automated and high-throughput sequencing is unavailable.

HOW IT WORKS

The model defines two types of agents: nucleotides and strands. Nucleotides can be A, T, C, G -- for dATP, dTTP, dCTP, and dGTP -- or the polymerization-terminating dideoxy counterparts to these. Nucleotides float aroud the space randomly, occasionally occupying the one patch (colored sky-blue) designated as the nucleotide binding-site of the DNA polymerase.

The template strand is generated automatically on set up. During model runs, complementary strands are synthesized one base at a time as appropriate nucleotides float into the blue patch. Complementary strands are synthesized right-to-left. If a dideoxy nucleotide is incorporated, the complementary strand is terminated and allowed to float in the space.

HOW TO USE IT

Short instructions: Set up, run the model for a while for each target base, and inspect the resulting gel bands.

Longer instructions: Choose a template-length and click "set up." The model will generate a template out of random bases. The model will also start the DNA polymerase reaction with a three-base primer -- visible in the gray patch. Click "go," and the model will begin synthesizing strands that are complementary to the template. If the polymerase incorporates a dideoxy-nucleotide or reaches the end of the template, the complementary strand will detach from the olymerase and float freely. The model will output that strand's sequence and place a band in the gel section of the model space.

Run the model with all four target bases: A, T, C, and G. (Do not click "set up" between target runs. Click "reset" instead, or just "go." Setting up will erase all gel bands and generate an entirely new template.) When there is a gel band for each strand-length greater than three (since the primer is three bases long), determine the sequence from the gel and confirm that it is the sequence (from right to left) of the template's complement.

THINGS TO NOTICE

Note how the efficiency of the method changes slightly when some bases are grossly over- or under-represented in the template.

CREDITS AND REFERENCES

This model was created in March 2009 by Ted Wong, tgwong - at - gmail - dot - com.
For more information: http://en.wikipedia.org/wiki/DNA_sequencing#Chain-termination_methods
http://scienceblogs.com/geneticfuture/2009/01/sanger_sequencing_is_not_dead.php

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