You have been characterizing the gene responsible for chronic hereditary ear hai

Question

You have been characterizing the gene responsible for chronic hereditary ear hair disorder by mapping restriction enzyme sites in the gene. The coding region of the gene contains a single Hind III site, but no Eco RI sites (there will be sites for both outside the gene in the other genomic DNA). At this point, you want to clone the gene so you can study it further and use it as a probe. The plasmid you want to use contains the ampicillin resistance gene, which has a Hind Ill site within it, and the tetracycline resistance gene, which has an Eco RI site within it. Describe the steps you would take to clone the intact gene. Chronic hereditary ear hair disorder (CHEHD) is a recessive trait. The recessive allele differs from the wild-type allele by only a single point mutation, which conveniently falls within the Hind III site. The mutation changes the DNA sequence within the site such that Hind III does not recognize and cut the sequence Utilizing this information, you obtain blood samples from five newborn subjects, and isolate their DNA. You cut the DNA with Hind III, and perform a Southern blot on the DNA, using your cloned gene as a probe. The following bands show up on the Southern blot: CHEHD Southern Blot State the genotype of each of the newborns, and indicate which you expect to develop the heartbreak of CHEHD, and why.

Explanation / Answer

Hi,
The gel image is not visible. I can only answer the first part of the question. I will give the logic behind the second part as well.

The gene for the disorder has a HindIII site within it. The gene is flanked by EcoR1 restriction site on both the sides. That means if you use the EcoR1 enzyme, it will cleave at both the sites and the gene will be excised.
The plasmid has both the ecor1 and HindIII site. In order to clone the gene into the plasmid following steps can be taken.
1. Designing primers for the gene such that it includes the EcoR1 sites as well. These primers will amplify the gene from the patient DNA.
2. Restriction digestion of the PCR amplified product using EcoR1 enzyme. This will cut the ends of the gene and create sticky ends.
3. Restriction digestion of the plasmid using EcoR1.
4. Ligating the digested gene with the plasmid. The successful ligation reaction will result in gene being inserted into the plasmid in correct orientation in te tetracycline site.
5. The recombinant plasmid has lost its resistence to tetracycline and such clones can be identified using replica plating.

In the second question, the mutant gene has lost its ability to be cut by HindIII. So the homozygous mutant will show no cleavage to HindIII and thus only a single band. The wild type will be cut by this enzyme and hence will have 2 bands on the gel. If the person is homozygous to the disease, then he will have only single band, if he is heterozygous, then one with no cut and another with 2 bands. The homozygous mutants are more prone to heartbreak.

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