You wish to clone the gene for polyhydroxychickenwire hydroxylase from the Mexic

Question

You wish to clone the gene for polyhydroxychickenwire hydroxylase from the Mexican axolotl. Fortunately, you have already cloned the gene for this enzyme from the Uruguyan wild llama. You digest axolotl DNA with restriction enzymes, separate the DNA fragments by gel electrophoresis, make a Southern blot, probe the blot with labeled DNA from the complete llama clone and make an autoradiogram. You see the pattern on the gel at right: Explain how you would go about cloning the axolotl gene, including the following: a) How would you cut the axolotl DNA for cloning? Why? b) How would you detect the plasmid containing axolotl DNA? Explain. Propose an alternative approach to cloning the axolotl gene, using PCR. There are a wide variety of experimental approaches that can suggest or confirm that two proteins (products of separate genes interact physically in vivo. For each of the following kinds of observations, state clearly what you do and do not learn about protein-protein interaction: a) Co-immunoprecipitation b) Yeast two-hybrid interaction c) Genetic interaction between mutations (e.g., extragenic suppression) d) Non-allelic non-complementation

Explanation / Answer

3: we would clone the axolotl gene by first isolating the gene from the genome of axolotl , for this purpose we would first need to cut the DNA,

a. we cut the DNA by making use of any one of the restriction enzymes that are shwon here, because all of them have the ability to cut this gene and therefore we can isolate it using any one of them.

then we can make copies of this DNA to be inserted into the vector which is the plasmid, we can make the copies using PCR.

after this we insert our gene in the plasmid and then take the recombinant plasmid and insert it into the bacteria to make copies of it, after that we select the bacterial colonies that have the recombinant plasmid, by looking at the markers like the showing of a color, and these bacteria would express the gene and we can make proteins from it .

b. we can idenitfy a plasmid that contains the axolotl gene by using a combination of all 3 of these restriction enzymes, because when we digest it using these 3 enzymes, we would get the pattern that is shown in the autoradiogram above.

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