Lecture 27: Restriction endonucleases, vectors, DNA cloning
(You may need to use the table of cut sites in the lecture 27 notes to answer some of these questions).
1. Restriction endonucleases are widely used in recombinant DNA research.
a. Distinguish between exonuclease and endonuclease in a manner that makes it clear you understand what each is and how they differ.
b. What are the characteristic features of a target site for cutting by a restriction endonuclease?
c. What range of frequencies of cutting is encountered with the various restriction nucleases that are currently in widespread use?
d. What is the size of the restriction endonuclease recognition site that is most useful for routine gene cloning operations? Explain the reasoning behind your answer.
e. What is a sticky end and why is it considered useful in gene cloning?
2. The frequency with which a particular restriction endonuclease can be expected to cut a random DNA is influenced by the overall base composition of the DNA.
a. What effect does the overall base composition of a DNA sample have on the average fragment length obtained with a restriction endonuclease?
b. Will the effect in part a be the same for every restriction endonuclease? Explain the reasoning behing your answer
c. The cut site for Eco RI is G|AATTC. What average fragment lengths do you expect to be produced from DNA with each of the following fractions of GC base pairs? 1/4, 1/3, 1/2, 2/3, 3/4
d. Describe a situation in which the shortest restriction fragments would be obtained with a DNA that was 50% A+T.
e. Would you expect Sma I (CCC|GGG) to be able to cut DNA that is 50% AT? Explain your answer.
f. Among the enzymes on the list in lecture 27 that have cut sites consisting of 6 nucleotide pairs, which would you expect to yield the largest DNA fragments from a GC-rich DNA and why?
3. Distinguish between 5'-overhangs and 3'-overhangs and describe a hypothetical (or real) example of each.
4. What role do restriction endonucleases play in nature? (What were they good for before molecular biologists discovered how useful they can be in recombinant DNA studies?)
5. Do different restriction endonucleases ever generate sticky ends that are the same? Explain your answer.
6. Eco RI cuts its GAATTC target site between G and A. A hypothetical enzyme Hyp I cuts the same sequence between T and C. Both enzymes yield sticky ends with the sequence 5'AATT3'.
a. What problems would be encountered trying to rejoin the ends of DNA fragments cut with these two enzymes? Use a diagram to explain your answer.
b. Can you think of any clever methods to overcome the problems described in your answer to part a? Using the same enzyme to cut both fragments is not an available option.
7. What prevents restriction endonucleases from destroying the genomic DNA of the bacteria that produce them?
8. A DNA fragment prepared with Bgl II was successfully cloned into a vector that had been cut with Bam H1.
a. What property of these two enzymes made it possible to perform this cloning without problems.
b. It was subsequently discovered that neither of the original enzymes would release the cloned DNA from the vector. Explain why this occurred.
c. Would it be possible to use Mbo I to release the DNA from the vector? Explain what problems might be encountered.
d. If the Bam HI site were located in the middle of a multicloning site (polylinker), would you expect to be able to release the cloned insert with a different restriction endonuclease than those used in cloning? Explain your answer?
e. Would you expect to be able to use site-directed mutagenesis to restore a cut site for one of the two original enzymes? Explain the reasoning behind your answer and be careful (this question contains hidden traps).
9. What is a vector? (Be careful not to define vectors too narrowly).
10. What is the value of using plasmids that carry antibiotic resistance genes as vectors?
11. What is the value of inserting a cloned DNA sequence into the middle of an antibiotic resistance gene?
12. Replica plating was an extremely valuable technique in early gene cloning studies, but is no longer as widely used.
a. Describe the processes involved in replica plating.
b. Explain the value of replica plating in the early studies.
c. What has been done to eliminate the need for replica plating?
13. What is the rationale for the inclusion of multiple cloning sites in modern cloning vectors. Include in your answer a description of what multiple cloning sites are.
14. Describe a cloning technique in which both the cloning vector and the bacterial strain that it is infected into have been engineered to work together to facilitate the process of screening.
15. What is the rationale for using two different restriction endonucleases to prepare a DNA fragment for cloning. (You should be able to think of at least two different answers.)
16. You are trying to clone a gene by ligating Eco RI fragments into a plasmid vector. You get some successful clones, but sequencing studies reveal that you have only half of the coding sequence because there is an Eco RI restriction site in the middle of the gene. What would you do to obtain a full length clone of the gene?
17. What are the advantages of using each of the following vectors as alternatives to plasmids?
a. Lambda-phage (Charon) vector.
b. Cosmid
c. M13 viral vector
d. Yeast artificial chromosome.
e. Shuittle vector
18. Site-directed mutagenesis has become a valuable tool in molecular biology.
a. Use the table of codons in figure 12.6 or inside the back cover of the textbook to generate a DNA code for a protein with the following partial sequence: Met-His-Leu-Val-Pro-Gly-Val-trp-Ile-.......
b. Describe in detail how you would use site directed mutagenesis to change the proline to serine.
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