Page 45 - Genetics_From_Genes_to_Genomes_6th_FULL_Part3

Solved Problems 339

removes phosphate groups that may be located at the a. Write the sequence of all the nucleotides of human
5′ ends of DNA strands. You will then add the frag- DNA that you can determine. Indicate the 5′-to-3′
ment of frog DNA to the vector and join the two orientation of this sequence.
together with the enzyme DNA ligase. b. Is the sequence you wrote in (a) part of the new
You don’t quite follow your advisor’s reasoning, so DNA strand that was synthesized in the sequencing
you set up two ligations, one with plasmid that was reaction or part of the template strand used in the
treated with alkaline phosphatase and the other with- sequencing reaction?
out such treatment. Otherwise, the ligation mixtures c. How did you know how to design the primer you
are identical. After the ligation reactions are com- would need for the sequencing reaction? Diagram
pleted, you transform E. coli with a small aliquot the recombinant DNA molecule to be sequenced,
(portion) of each ligation and spread the cells on pe- indicating the human and vector sequences, the po-
tri plates containing both ampicillin and X-gal. The sition and orientation of the primer, and the posi-
next day, you observe 100 white colonies and one tion and orientation of the new DNA that would be
blue colony on the plate transformed with alkaline- synthesized during the sequencing reaction, using
phosphatase-treated plasmids, and 100 blue colonies Fig. 9.7 as a guide.
and one white colony on the plate transformed with d. Show the full sequence of the smallest DNA mol-
plasmids that had not been treated with alkaline ecule that would be synthesized in the sequencing
phosphatase. reaction and that would contain dideoxyG (ddG).
a. Explain the results seen on the two plates. Indicate the 5′-to-3′ orientation of this molecule
b. Why was your research advisor’s suggestion a and the location of the ddG.
good one? e. How would the data differ from that shown if you
c. Why would you normally treat plasmid vectors accidentally left the dATP out of the reaction?
with alkaline phosphatase, but not the DNA
fragments you want to add to the vector? Section 9.4
23. a To make a genomic library useful for sequencing
Section 9.3 an entire genome, why would you ordinarily frag-
21. Which of the enzymes from the following list would ment the genomic DNA by mechanical shearing
you need to sequence DNA? What is the function of forces like sonication rather than by cutting the
those enzyme(s) in the process? DNA with a restriction enzyme?
a. DNA polymerase b. Suppose that you wanted to make a genomic li-
b. RNA polymerase brary to determine the complete sequence of a
newly discovered organism’s genome, but you did
c. A restriction enzyme not have a sonicator readily available. Explain how
d. DNA ligase you could nonetheless use two or more restriction
e. An aminoacyl-tRNA synthetase enzymes to make libraries whose clones could be
f. Peptidyl transferase sequenced so that a computer could assemble the
genomic sequence.
g. Reverse transcriptase c. Suppose you only had a single restriction enzyme
22. You use the primer 5′ GCCTCGAATCGGGTACC 3′ available, and you want to make a single genomic
to sequence part of the human DNA insert of a recom- library from which you could assemble the ge-
binant DNA molecule made with a plasmid vector. The nomic sequence. How might you be able to achieve
result of the automated DNA sequence analysis is this goal? (Hint: See Problem 9.) To make this li-
shown here. The height of the peaks is unimportant. brary, would it be preferable to use a restriction en-
(A = green; C = purple; G = black; T = red) zyme that recognizes a 4-base, 6-base, or 8-base
sequence of DNA?
24. Problem 15 showed part of the sequence of the plas-
mid vector pMBG36. Suppose you make a genomic
library by inserting EcoRI-digested fragments of a
genome into EcoRI-digested pMBG36. Write out the
sequences of two different primers you could use to
sequence (in separate reactions) the two ends of all
the clones in the library. These primers should be as
Smaller Larger long as possible based on the information given.