Chapter 9, Problem 22P

You use the primer 5′ GCCTCGAATCGGGTACC 3′ to sequence part of the human DNA insert of a recombinant DNA molecule made with a plasmid vector. The result of the automated DNA sequence analysis is shown here. The height of the peaks is unimportant. (A = green; C = purple; G = black; T = red)

a.	Write the sequence of all the nucleotides of human DNA that you can determine. Indicate the 5′-to-3′ orientation of this sequence.
b.	Is the sequence you wrote in (a) part of the new DNA strand that was synthesized in the sequencing reaction or part of the template strand used in the sequencing reaction?
c.	How did you know how to design the primer you would need for the sequencing reaction? Diagram the recombinant DNA molecule to be sequenced, indicating the human and vector sequences, the position and orientation of the primer, and the position and orientation of the new DNA that would be synthesized during the sequencing reaction, using Fig. 9.7 as a guide.
d.	Show the full sequence of the smallest DNA molecule that would be synthesized in the sequencing reaction and that would contain dideoxyG (ddG). Indicate the 5′-to-3′ orientation of this molecule and the location of the ddG.
e.	How would the data differ from that shown if you accidentally left the dATP out of the reaction?