Posted October 20, 1999
Lecture 16: Vectors for large inserts, cDNA, libraries, probes, expression vectors
1. 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. Bacterial artificial chromosome.
2. How can site-directed mutagenesis be achieved in a cloned cDNA sequence? Include in your answer a description of the type of vector that you would have to use and the way in which you would transfer the cDNA clone from its original vector to the vector used for site-directed mutagenesis.
3. Site-directed mutagenesis has become a valuable tool in molecular biology.
a. Use the table of codons in figure 4.4 or inside the front 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.
4. Expression vectors are widely used in recombinant DNA technology.
a. What is an expression vector? Include in your answer the special properties that a vector must have to function as an expression vector.
b. Describe as many different reasons as you can why it may be useful to clone a gene or a cDNA into an expression vector.
c. What are the potential advantages of using an expression vector with a strong constituitive promoter?
d. What are the potential disadvantages of using an expression vector with a strong constituitive promoter?
e. How can the disadvantages that you described in question 2 be avoided?
5. What is a genomic library and what is its value?
6. cDNA clones are widely used in recombinant DNA studies.
a. What does the term "cDNA" stand for?
b. How is cDNA prepared?
c. What features of cDNA make it a useful research tool?
d. What are the limitations that restrict the usefulness of cDNA clones?
e. When used as a hybridization probe in a Southern blot, cDNA may identify several separate bands. Explain as many different ways as you can why this might happen (this requires information from lecture 17).
7. You have a polyclonal antibody to a protein. You would like to clone the coding sequence for the protein. How would you go about it?
8. Explain the rationale for using IPTG rather than lactose or allo-lactose to induce expression of genes cloned downstream from the lac promoter/operator sequence.
9. You have the cloned cDNA for a protein. Describe the procedures you would use to isolate a clone that contains the genomic sequence from a genomic library contained in lambda phage vectors.
10. Do you expect cDNA preparations to contain cut sites for restiction endonucleases? Explain your answer.
11. An unaltered vector and the same vector containing a cloned cDNA are denatured in the same solution, allowed to anneal slowly, and prepared for electron microscopy. What would you expect to see with the electron microscope?
12. What is a degenerate probe and what is it used for? In designing such a probe, what problems are likely to be encountered and how can they best be overcome.
13. You know the amino acid sequence of a protein. Starting with a preparation of messenger RNA from a tissue that makes large amounts of the protein, how would you go about isolating a cloned cDNA that contains the nucleotide coding sequence for that protein? You should be able to think of two quite different approaches to this question.
14. Why is it sometimes desirable to reduce the stringency of probe hybridization reactions?
15. What is meant by the term "restriction map"? What is the value of using partial digests in such mapping?
16. What is the advantage of preparing a restriction map with more than one restriction endonuclease?
17. You have used a CDNA to isolate a series of clones of various sizes from a genomic library that was prepared by incomplete digestion with Eco RI. Explain how you might use restriction mapping to determine patterns of overlapping among these clones and to assemble a set that collectively contain the complete genomic sequence including the introns that were spliced out of the mRNA used to prepare the cDNA. Also explain how you could identify presumptive promoter sequences located immediately upstream from the start of transcription. This question will require you to do some projection beyond specific details that we have covered in class, but should be fully answerable with the informaiton that has been presented to you at various times during the semester.
18. Using the table of codons in the textbook, identify all possible amino acid sequences whose genomic coding sequences could generate a cut site for EcoRI (G|AATTC). (Be sure to examine all possible reading frames and exclude all nucleotide sequences that could not be found in the coding sequences for proteins.)
19. In an organism whose DNA is 50% AT, how long would a protein have to be to have a 50% chance of containing a cut site for EcoRI within its coding sequence?
20. Vectors derived from bacteriophage lambda have become quite popular.
a. Describe the modifications that are made in bacteriophage lambda to generate a typical vector (such as the Charon vectors)
b. Explain how blue-white selection is used to identify lambda vectors that contain cloned inserts.
c. What determines the size range of inserts that can be cloned in a lambda phage vector?
d. What is done to prevent lambda phage vectors from entering into a lysogenic relationship with their host bacterial cells.
e. What special modification is likely to be needed in order to clone a typical cDNA in a lambda phage vector? (You have not been given an answer for this question, but you should be able to figure it out, based on what you are supposed to know about cDNAs and lambda phage vectors).Go to Review Questions for the Following Lecture
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