Lecture 16: PCR, Southern, Northern, Western blots
1. The polymerase chain reaction (PCR) is a widely used technique in molecular genetics.
a. What special property must be possessed by the DNA polymerase used in PCR, and why is this so important for doing PCR?
b. Explain the use of primers in PCR, including why two different primers must be used.
c. What relationship must exist between the two primers used in PCR?
d. Summarize all of the components that must be present in a PCR reaction mixture.
e. Summarize the overall process that makes possible extensive amplification of specific DNA sequences with minimal effort through the use of PCR.
2. One of the ways of determining whether a sequence has been successfully amplified by PCR is to do electrophoresis and look for a band of DNA of sharply defined size. Explain how such a band arises during PCR.
3. Why is it not necessary to add new DNA polymerase and primers for each new cycle of PCR?
4. You have a cDNA of unknown sequence in a plasmid vector. How could you do a PCR amplification of the cDNA without first determining its end sequences?
5. What types of precautions must be taken when doing PCR to be certain that the desired results are obtained?
6. What aspects of PCR make it particularly useful in forensic investigations? How do the same properties make PCR particularly susceptible to challenge by defense lawyers?
7. Distinguish between Southern and Northern blots in a manner that makes it clear you know what each is and how they differ.
8. How does a Western blot differ from both of the above? When is a Western blot used in perference to a Northern or Southern blot?
9. 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).
10. 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).
11. 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?
12. Summarize the major steps that are involved in Southern blotting.
13. 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?
14. Explain how it is possible to separate proteins by size alone, by isoelectric point alone, or by a combination of isoelectric point and size.
15. What result would you expect to see if you probed a Southern blot of a PCR product derived from genomic DNA separately with each of its primers? How might the results differ if different restriction endonucleases were used on the PCR product prior to running the Southern blot (this calls for some speculation -- explain any assumptions that you have made in arriving at your answer.
16. What is a dot blot and when would you be most likely to use it?
17. You have a full-length genomic clone for an eukaryotic gene. You isolate and purify the cloned insert and use it as the starting point for a Southern blot of the clone. Using a cDNA for the gene as a probe, you detect five bands on the Southern blot. However, when you label the cloned genomic sequence and use it as a probe, you see two additional bands.
a. Propose an explanation for the additional bands.
b. You isolate the restriction fragment that is responsible for one of the additional bands and use it as a probe for a Southern blot prepared the same way as the first one. How many bands do you expect to see? Explain the reasoning behind your answer.
c. You purify the cDNA for the gene and do a Southern blot of it using the same restriction endonuclease that was used for the genomic Southern blot. When you probe that Southern blot with the full length cDNA, you only find three bands. Propose an explanation for the reduced number of bands.
d. You purify one of the bands from part c and use it as a probe on the genomic Southern blot. How many bands do you expect to detect? Explain the reasoning behind your answer.
e. You use the probe from part d on a Southern blot of the cDNA prepared with a different restriction endonuclease. What possible range of results might you see?
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