Learning Goal: I’m working on a biology multi-part question and need an explanation and answer to help me learn.
- Consider the two proteins A and B. Protein A has a MW = 75kDa and protein B has a MW = 15kDa. Which protein will sediment faster during rate zonal centrifugation? Which protein will migrate faster during SDS polyacrilamide gel electrophoresis? 
- Describe the general features of restriction enzyme sites 
- Explain how bacterial restriction enzymes do not cut the bacteria’s own DNA 
- List and describe the 2 types of DNA ends generated after cutting DNA with restriction enzymes 
- Describe the reaction catalyzed by DNA ligase 
- Describe the features of both DNA topoisomerase I and DNA topoisomerase II, including how these enzymes work. 
- Define the term hybridization as applied to the screening of DNA sequences 
- How does equilibrium sedimentation provide direct evidence for the semiconservative replication of DNA? 
Use the University of Florida Health Science Center e-journals or any other applicable online resource to locate the following journal article: Journal of Bacteriology, July 2000, p. 3613-3618, Vol. 182, No. 13. Use this paper to answer the following questions.
- What are the advantages of cell free extracts and fractionation to biological research? 
- What was the rationale for conducting early DNA replication studies on prokaryotic systems? 
- Studies involving phage M13 and φ X174 led to some significant discoveries relating to DNA priming and replication – list them 
- What is the concept behind reverse genetics and genomics? 
- Describe the features that distinguish plasmid, phage, BAC and YAC cloning vectors. 
- Does lambda DNA packaging require any enzymes or ATP hydrolysis? Explain. 
- Compare the differences between cDNA clones and genomic DNA clones that originate from the same region of DNA. When is it more suitable to use one preparation over the other? 
- What is meant y an in situ hybridization experiment? Discuss and provide an example. 
- Once you had obtained a highly concentrated stock of purified phage, how would you separate the recombinant phage DNA away from the phage head and tail proteins? 
- Define and describe an application for suicide plasmids; include in your discussion a reference for a research journal article or website that discusses this. 
- Briefly explain how repetitive DNA is useful for identifying individuals by the techniques of DNA fingerprinting. 
- Locate the following article and briefly describe how the full-length cDNA sequence for the gene KCNV1 was determined 
Ebihara M, Ohba H, Kikuchi M, Yoshikawa T. (2004). Gene, 325: 89-96.
- Having just read the paper above, what exactly are deletion constructs? 
1. What types of information can be derived from performing a Western Blot? 
2. In choosing an animal model for performing genetic knockout experiments, what basic characteristics would be most beneficial? 
3. Aseptic technique is required in a cell culture lab or dedicated area, which would be required for many of the techniques described in this module. What does this entail? 
4. Discuss the differences between the Maxim-Gilbert and Sanger sequencing methods. 
5. Briefly explain what is meant by the term “chromosome painting”.
6. How might you explain an observation that the pattern of restriction fragment lengths from a region of the genome differs between homologous chromosomes and between two individuals? 
7. What exactly are single nucleotide polymorphisms (SNP’s) and what techniques can be used to identify them? 
8. Briefly discuss the pros and cons of integrating DNA microarray or chip technology into the forensic DNA laboratory 
9. Locate and read the following article: Roufosse CA, Direkze NC, Otto WR and Wright NA. (2004). The International Journal of Biochemistry & Cell Biology, 36, (4): 585-597.
a. What clinical implications would the identification of MPCs in adult circulating peripheral blood have? 
b. Exactly what was the FISH technique used to determine in these experiments? 
10. Locate and read the following article: Wang Z, Dooley T, Curty E, Davis R, VandeBerg J. (2004). Genomics,83(4): 588-599.
If we are able to spot thousands of human genes in a DNA microarray, what is the benefit of demonstrating cross-species application of the cDNA microarray technique described?