PCR and Dideoxy Sequencing Video Lessons

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Problem: A 13-nucleotide long DNA template was sequenced by the chain-terminator method (the Sanger method) using a 5'-TAG primer. The resulting fragments were analyzed by gel electrophoresis as shown below. In each of the four different gel lanes (or wells), a different ddNTP was used (i.e. ddATP for the 1st well, ddGTP for the 2nd, ddCTP for the 3rd, and ddTTP or the 4th).Determine the sequence of the DNA template strand.

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The Sanger method, or the chain-terminator method determines the sequence of the template strand by through the use of the DNA polymerase reaction while utilizing a small concentration of dideoxynucleoside triphosphates (ddNTPs). These ddNTPs attach to the elongating chain but cause a premature termination due to the lack of OH group in the 3' carbon. Subsequent electrophoresis would give the sequence provided by the differently sized fragments produced in the reactions.

By concept, the smallest fragment of the sequence should have traveled farthest in an electrophoretic profile. Thus, the first base added to the elongating chain is found at the bottom, which, in this case, is A. The sequence is then followed by two Gs, and so on.

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Problem Details

A 13-nucleotide long DNA template was sequenced by the chain-terminator method (the Sanger method) using a 5'-TAG primer. The resulting fragments were analyzed by gel electrophoresis as shown below. In each of the four different gel lanes (or wells), a different ddNTP was used (i.e. ddATP for the 1st well, ddGTP for the 2nd, ddCTP for the 3rd, and ddTTP or the 4th).

Determine the sequence of the DNA template strand.

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