Chapter 10, Problem 10P

This problem investigates issues encountered in sequencing the inserts in cDNA libraries.

a.

If you sequenced many clones individually, wouldn’t you spend many of your resources inefficiently sequencing cDNAs for the same type of mRNA molecule over and over again? Explain. Does this apparently inefficient process provide any useful information beyond the sequences of individual mRNAs?

b.

Suppose that you identified a clone with a cDNA insert that was 4 kb long. You could determine the entire sequence of the clone by shearing the DNA into small random fragments, cloning these fragments into a vector to make a mini-shotgun library, and then sequencing hundreds of these clones to allow the computer to assemble the full sequence of the 4 kb–long insert. However, this procedure would be inefficient. An alternative that requires many fewer sequencing reactions is called primer walking. This technique involves the synthesis of additional oligonucleotide primers corresponding to cDNA sequences you have just obtained. Diagram how you would sequence the entire 4 kb–long cloned cDNA using primer walking, indicating the vector and insert, all primers that you would use, and all the sequences you would obtain. Assume that each sequence read is 1 kb.