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Dideoxy Sequencing of DNA

  1. S Wilton

Published Online: 24 OCT 2002

DOI: 10.1038/npg.els.0003768

eLS

eLS

How to Cite

Wilton, S. 2002. Dideoxy Sequencing of DNA. eLS. .

Author Information

  1. Centre for Neuromuscular and Neurological Disorders, University of Western Australia, Australia

Publication History

  1. Published Online: 24 OCT 2002
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Figure 1. Deoxynucleoside triphosphate and its analogue the dideoxynucleoside triphosphate.

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Figure 2. Chain termination upon the incorporation of a dideoxynucleoside triphosphate.

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Figure 3. 5′-terminal labelling of DNA sequencing fragments via the primer.

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Figure 4. Incorporation of α-radiolabel into the sugar phosphate backbone.

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Figure 5. Overview of manual sequencing steps.

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Figure 6. Comparison of well-forming and sharktooth combs for sequencing gels.

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Figure 7(a). Inserting the gel into the gel apparatus.

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Figure 7(b). Pouring acrylamide mix into the gel cassette.

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Figure 7(c). Bubble formation during gel pouring.

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Figure 8. Dideoxy sequencing ladder.

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Figure 9. Autoradiograph.

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Figure 10. Thin layer chromatography to check 5′-radiolabelling efficiency.

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Figure 11. Reading the sequence of either strand from a sequencing ladder. The layout ACGT for each set of reactions is used so that it is possible to turn the autoradiograph over and read the complementary strand. The T is now on the left side of the inverted set of four reactions and is read as A. The next lane was G, which is read as C, and so on. Note that the polarity of the complementary sequence is now 3′ at the bottom to 5′ at the gel origin.