Dideoxy Sequencing of DNA

Abstract

The enzymic sequencing approach uses a DNA polymerase to synthesize transcripts that have been terminated at specific bases. A primer anneals to a complementary region on the template strand and acts as a starting point for DNA synthesis, which occurs in the presence of a mixture of deoxyribonucleotides (building blocks) and specific dideoxyribonucleotides (chain‐terminators). Originally four separate reactions were set up, each with a base‐specific chain‐terminator. Separation of these reaction products on a DNA sequencing gel will generate a ladder of bands indicating the position of each terminated base relative to the end of the sequencing primer. A homogeneous sequencing template should generate a single band in one of the four lanes where a band in the ‘A stop’ lane indicates an ‘A’, a band in the ‘C stop’ lane represents a ‘C’ and so on. In this manner it is possible to deduce the DNA sequence of the target template by identifying consecutive bands in one of the four lanes of the ladder. Since DNA synthesis occurs in the 5′ to 3′ direction, the sequence is read 5′ to 3′ from the bottom to the top (origin) of the gel. Although it was originally suited to the sequencing of single‐stranded DNA templates, there have been many advances in the chemistry of enzymatic sequencing, including the application of thermostable DNA polymerases and fluorescent dye‐terminators as used in automated DNA sequencers.

Keywords: DNA sequencing; manual; polyacrylamide gel; radioactive isotope; autoradiograph

Figure 1.

Deoxynucleoside triphosphate and its analogue the dideoxynucleoside triphosphate.

Figure 2.

Chain termination upon the incorporation of a dideoxynucleoside triphosphate.

Figure 3.

5′‐terminal labelling of DNA sequencing fragments via the primer.

Figure 4.

Incorporation of α‐radiolabel into the sugar phosphate backbone.

Figure 5.

Overview of manual sequencing steps.

Figure 6.

Comparison of well‐forming and sharktooth combs for sequencing gels.

Figure 7.

Inserting the gel into the gel apparatus.

Figure 8.

Pouring acrylamide mix into the gel cassette.

Figure 9.

Bubble formation during gel pouring.

Figure 10.

Dideoxy sequencing ladder.

Figure 11.

Autoradiograph.

Figure 12.

Thin layer chromatography to check 5′‐radiolabelling efficiency.

Figure 13.

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.

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References

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How to Cite close
Wilton, S(Oct 2002) Dideoxy Sequencing of DNA. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003768]