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Recombinant DNA Practice Problems

Recombinant DNA Practice Problems


1) Using the Southern Blot used in the Solving Problems section... You are studying a gene responsible for pigment biosynthesis in plants. RR individuals are red; Rr are pink; and rr are white. The restriction map of the R allele is:



You extract genomic DNA from a red, a pink, and a white plant; digest with EcoRI, HindIII and EcoRI+HindIII; run on a gel, and transfer to nitrocellulose. You have a cDNA for the R gene which you use as a probe. The results of the autoradiogram are as follows:



What would the resulting autoradiogram look like if you performed this blot, but skipped:

a) digesting the DNA with restriction enzymes.

b) gel electrophoresis.

c) separating the DNA strands with alkali.

d) capillary transfer.

e) blocking the nitrocellulose.

f) adding the probe.

g) washing the blot.

Solution

2) You are studying gene Q in yeast, which is an eukaryote but normally grows as a haploid. Protein Q is 50kDa. The restriction map of the wild-type allele of this gene is shown below:



You are studying 4 mutant strains with mutations in this gene. You analyze wild-type and each of the mutants in the following ways:

i) Southern blot: cut genomic DNA isolated from each strain with EcoRI, run on gel, blot, and probe with radioactive 4.8kbp BamHI fragment of gene Q.

ii) Northern blot: run RNA isolated from each strain on gel, blot, and probe with radioactive 4.8kb p BamHI fragment of gene Q.

iii) Western blot: run protein isolated from each strain on gel, blot, and probe with radioactive anti-Q antibodies.

Draw and explain the resulting three blots (i, ii, iii) from the following strains:

a) wild-type.

b) a mutant with a deletion of the region from 0.8kbp to 1.0kbp which partially inactivates the promoter, resulting in 5-fold lower levels of transcription. Assume that the translation rate of Q mRNA molecules (protein molecules per mRNA molecule) is normal.

c) a mutant with a point nonsense mutation at 2.5kbp.

d) a point missense mutant at 2.6kbp that prevents EcoRI from cutting at that site.

e) a deletion of the region from 2.0kbp to 2.5kbp.

Solution

3) In the presence of lactose, E. coli cells produce large quantities of the enzyme b-galactosidase (b-gal.) from the lac Z gene. In the absence of lactose, no b-gal. is produced. Describe an experiment or experiments to determine if the increase in b-gal. synthesis upon addition of lactose is due to:

i) an increase in transcription of lac Z mRNA, which is then translated - leading to more b-gal. production. (This is the mechanism used in nature.)

or: ii) an increase in translation of lac Z mRNA that is always-present at high levels. (This occurs with some genes)

You are given the following:

- DNA, RNA, and protein extracted from cells grown without lactose

- DNA, RNA, and protein extracted from cells grown with lactose

- radioactive DNA probe made from lac Z sequences

- radioactive antibody that binds to b-galactosidase

Describe the methods and the results of your experiment(s) (draw blots where appropriate) given:

a) model i) is true

b) model ii) is true

Note: this is a difficult question. We would not ask it on an exam, but it is important to try out this mode of thinking to test your understanding of how to apply these techniques.

Solution

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