Lecture 28: Libraries, Probes, Restriction Maps, Southern and Northern Blots
1. 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?
2. What is a genomic library and what is its value?
3. 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.
4. 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?
5. 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.
6. 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.
7. You have a cloned cDNA for a protein. You want to find a restriction endonuclease that can be used to isolate the genomic coding sequence in a single DNA fragment. Describe the procedures you would use to identify an appropriate restriction endonuclease. (Assume that you are dealing with an eukaryotic gene that contains introns of unknown sequence).
8. You have a full length cDNA that codes for an eukaryotic protein. The cDNA was cloned using Pst I. When you do a genomic Southern blot with Eco RI, you detect three bands of distinctly different sizes that hybridize with the cDNA probe.
a. What are two very different possible interpretations of the data?
b. Describe the additional experiments that would be needed to distinguish clearly between the two possibilities. (Be aware that there are multiple possible ways of approaching this problem, and list as many as you can).
9. Do you expect cDNA preparations to contain cut sites for restiction endonucleases? Explain your answer.
10. Describe the process of chromosome walking and explain what it is used for. Include in your answer an explanation of how restriction mapping is employed in the process of chromosome walking.
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. Distinguish between Southern and Northern blots in a manner that makes it clear you know what each is and how they differ.
13. How does a Western blot differ from both of the above? When is a Western blot used in perference to a Northern or Southern?
14. Why is it generally desirable to use a relatively small probe when doing a Southern blot. What situations may make the use of a larger probe desirable?
15. Summarize the major steps that are involved in Southern blotting.
16. Describe a procedure that could be used to determine which tissues in a rat express the highest levels of a particular gene. What is the procedure called? What is the origin of the name?
17. 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.
18. 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.
19. 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.
20. Why is it sometimes desirable to reduce the stringency of probe hybridization reactions?
21. 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 overlappoing 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.
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