Molecular Biology of the Gene 7th Edition by Richard Losick, James Watson, Michael Levine, Tamara Baker, Alexander Gann

Exercise 5

You decide to perform dideoxy sequencing on a PCR product. You add the appropriate 32 P-labeled primer, DNA polymerase, DNA template (the PCR product), buffer, dNTP mix, and a small amount of one of the four ddNTPs to four reaction tubes. You run the reactions in the thermal cycler, load each reaction into a separate lane of a polyacrylamide gel, and separate the products by gel electrophoresis. In the figure below, the lanes are labeled according to the ddNTP added.

A. What is the sequence of the template strand? Be sure to label the 5? and 3? ends.
B. Suppose that you accidentally added 10-fold more ddGTP to the reaction in lane 4. What effect would that have on the banding pattern in lane 4?
C. In lane 5, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only dATP, dTTP, dCTP, and dGTP.
D. In lane 6, draw what you would expect to see if you prepared a reaction using a nucleotide mix containing only ddATP, dTTP, dCTP, dGTP.