Question

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Review of Concepts: Gene Identification and Gene Knockout In humans, Xeroderma Pigmentosis (XP) is characterized by hypersensitive to UV light. This light creates a very high amount DNA damage leading to increased cancer rates. You are very interested in identifying a gene that might be resulting in this UV induced DNA damage in people suffering from XP. Propose a research design that will identify, isolate, and characterize a gene involved in this disease. Include the methodology used and outcome/result you expect at each step along this research design. To begin the research design, the cells must be radiated by UV light which will affect DNA damage. After this is done, the tumor suppressor gene and protooncogene expression should be measured via Western blot analysis. And before this state, the genetic chaining pattern by restriction mapping or many other mappings can be viewed which will result in different bands because of mutation. The gene expression viewed will be lessened through RT PCR and western blot which will result in characterization of the protein as protooncogene or tumor suppressor gene depending on if their expression increases or decreases, respectively.

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Answer 1

Xeroderma Pigmentosum is an inherited disorder which is characterized by the hypersensitivity of your skin and eyes towards UV radiation as a consequence of the defect in the seven genes XPA involved in Nuclear Excision Repair (NER, responsible for the removing the strands of damaged DNA and directing for its correction) and more significantly the eighth gene XPV (DNA polymerase Ƞ. The application of UV radiation causes the formation of Thymidine dimer formation at random locations of the gene sequence disrupting the DNA double helical structure. The result is the continued replication of damaged DNA. The patient must not be exposed directly to sunlight.

In an experimentation to determine of the expression of XPV gene we can have following protocol:

Cells being irradiated with UV radiation. This will result in the mutation with the DNA of the cells.

Restriction mapping of the genes by cutting the gene into various fragments using restriction enzymes for both normal as well as UV irradiated cells. This will produce variation in the lengths of the fragments obtained for both due to the mutation in the UV irradiated cell.

The fragments are viewed as bands in the gel electrophoresis using a standard marker to determine the fragment lengths.

The RT-PCR can be carried out the gene but it may show little or no band since the mRNA is unstable. The PCR has to be carried immediately after the gene isolation.

Hence a more appropriate method is the use of Western Blot Analysis that will facilitate the characterization of proteins (that are stable) produced from both normal and UV irradiated cells depending upon their expressions.