Chapter Questions
Use a drawing to illustrate the principle of DNA gel electrophoresis. Indicate roughly the comparative electrophoretic mobilities of DNAs with 150,600 , and $1200 \mathrm{bp}$.
You have electrophoresed some DNA fragments on an agarose gel and obtain the results shown in Figure 5.2.(a) What is the size of a fragment that migrated $25 \mathrm{~mm}$ ?(b) How far did the $200 \mathrm{bp}$ fragment migrate?
What is SDS? What are its functions in SDS-PAGE?
Design a Southern blot experiment to check a chimeric mouse's DNA for insertion of the neomycin-resistance gene. You may assume any array of restriction sites you wish in the target gene and in the neo ${ }^r$ gene. Show sample results for a successful and an unsuccessful insertion.
Compare and contrast SDS-PAGE and modern twodimensional gel electrophoresis of proteins.
In a DNase footprinting experiment, either the template or nontemplate strand can be end-labeled. In Figure 5.37a, the template strand is labeled. Which strand is labeled in Figure $5.37 b$ ? How do you know?
Invent a pyrogram with 12 peaks and write the corresponding DNA sequence.
Describe the principle of ion-exchange chromatography. Use a graph to illustrate the separation of three different proteins by this method.
Describe the principle of gel filtration chromatography. Use a graph to illustrate the separation of three different proteins by this method. Indicate on the graph the largest and smallest of these proteins.
Compare and contrast the principles of autoradiography and phosphorimaging. Which method provides more quantitative information?
Describe a nonradioactive method for detecting a particular nucleic acid fragment in an electrophoretic gel.
Diagram the process of Southern blotting and probing to detect a DNA of interest. Compare and contrast this procedure with Northern blotting.
Describe a DNA fingerprinting method using a minisatellite probe. Compare this method with a modern forensic DNA typing method using probes to detect single variable DNA loci.
What kinds of information can we obtain from a Northern blot?
Describe fluorescence in situ hybridization (FISH). When would you use this method, rather than Southern blotting?
Draw a diagram of an imaginary Sanger sequencing autoradiograph, and provide the corresponding DNA sequence.
Show how a manual DNA sequencing method can be automated.
Show how to use restriction mapping to determine the orientation of a restriction fragment ligated into a restriction site in a vector. Use fragment sizes different from those in the text.
Explain the principle of site-directed mutagenesis, then describe a method to carry out this process.
Compare and contrast the $\mathrm{S} 1$ mapping and primer extension methods for mapping the $5^{\prime}$-end of an mRNA. Which of these methods can be used to map the $3^{\prime}$-end of an mRNA. Why would the other method not work?
Describe the run-off transcription method. Why does this method not work with in vivo transcripts, as $$\$ 1$$ mapping and primer extension do?
How would you label the $5^{\prime}$-ends of a double-stranded DNA? The $3^{\prime}$-ends?
Describe a nuclear run-on assay, and show how it differs from a run-off assay.
How does a dot blot differ from a Southern blot?
Describe the use of a reporter gene to measure the strength of a promoter.
Describe a filter-binding assay to measure binding between a DNA and a protein.
Compare and contrast the gel mobility shift and DNase footprinting methods of assaying specific DNA-protein interactions. What information does DNase footprinting provide that gel mobility shift does not?
Compare and contrast DMS and DNase footprinting. Why is the former method more precise than the latter?
Describe a ChIP assay to detect binding between protein $X$ and gene Y. Show sample positive results.
Describe a yeast two-hybrid assay for interaction between two known proteins.
Describe a yeast two-hybrid screen for finding an unknown protein that interacts with a known protein.
Describe a method for creating a knockout mouse. Explain the importance of the thymidine kinase and neomycinresistance genes in this procedure. What information can a knockout mouse provide?
Describe a procedure to produce a transgenic mouse.