Chapter 14 Practice Problems PDF

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Chapter 14 Practice Problems...

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Chapter 14 Practice Problems 2. Explain how gel electrophoresis is used to separate DNA fragments of different lengths. Gel electrophoresis uses an electric field to drive negatively charged DNA molecules through a gel that acts as a molecular sieve. Shorter DNA molecules are less hindered by the agarose or polyacrylamide matrix and migrate faster than do longer DNA molecules. 3. Give three important characteristics of cloning vectors. Cloning vectors should have: (1) An origin of DNA replication so they can be maintained in a cell (2) A gene, such as an antibiotic-resistance gene, to select for cells that carry the vector (3) A unique restriction site or series of sites to where a foreign DNA molecule may be inserted 4. Briefly explain how an antibiotic-resistance gene and the lacZ gene can be used as markers to determine which cells contain a particular plasmid. Foreign DNAs are inserted into one of the unique restriction sites in the lacZ gene and transformed into E. coli cells. Transformed cells are plated on a medium containing the appropriate antibiotic to select for cells that carry the plasmid, an inducer of the lac operon, and X-gal, a substrate for β -galactosidase that turns blue when cleaved. Colonies that carry the plasmid without foreign DNA inserts will have intact lacZ genes, make functional βgalactosidase, cleave X-gal, and turn blue. Colonies that carry plasmid with foreign DNA inserts will not make functional β-galactosidase and will remain white. 5. Briefly explain how the polymerase chain reaction is used to amplify a specific DNA sequence. First, the double-stranded template DNA is denatured by high temperature. Then, synthetic oligonucleotide primers corresponding to the ends of the DNA sequence to be amplified are annealed to the single-stranded DNA template strands. These primers are extended by a thermostable DNA polymerase so that the target DNA sequence is duplicated. These steps are repeated 30 times or more. Each cycle of denaturation, primer annealing, and extension doubles the number of copies of the target sequence between the primers. 12. How is RNA interference used in the analysis of gene function? (Not covered in this chapter, but covered in Chapter 12). RNA interference is one potential reverse-genetics approach to analyze gene function, by specifically repressing expression of that gene. Double-stranded RNA may be injected directly into a cell or organism, or the cell or organism may be genetically modified to express a doublestranded RNA molecule corresponding to the target gene. 16. How often, on average, would you expect a restriction endonuclease to cut a DNA molecule if the recognition sequence for the enzyme had 5 bp? (Assume that the four types of bases are 1

equally likely to be found in the DNA and that the bases in a recognition sequence are independent.) How often would the endonuclease cut the DNA if the recognition sequence had 8 bp? Because DNA has four different bases, the frequency of any sequence of n bases is equal to 1/ (4n). A 5-bp recognition sequence will appear with a frequency of 1/(45),or once every 1024 bp. An 8-bp recognition sequence will appear with a frequencyof 1/(48), or 65,536 bp. 20. A linear piece of DNA has the following EcoRI restriction sites.

a. This piece of DNA is cut by EcoRI, the resulting fragments are separated by gel electrophoresis, and the gel is stained with ethidium bromide. Draw a picture of the bands that will appear on the gel. b. If a mutation that alters EcoRI site 1 occurs in this piece of DNA, how will the banding pattern on the gel differ from the one that you drew in part a? c. If mutations that alter EcoRI sites 1 and 2 occur in this piece of DNA, how will the banding pattern on the gel differ from the one that you drew in part a? d. If 1000 bp of DNA were inserted between the two restriction sites, how would the banding pattern on the gel differ from the one you drew in part a? e. If 500 bp of DNA between the two restriction sites were deleted, how would the banding pattern on the gel differ from the one that you drew in part a?

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