Data Analysis Task- Molecular (Southern Blotting).

BI3006: Data Analysis Task- Molecular (Southern Blotting).

Southern Blots are used to diagnose a range of genetic disorders, including Fragile X Syndrome and Friedreich’s Ataxia.  Ability to interpret a Southern Blot successfully not only demonstrates understanding of the technique but also of gene structure and function.
Basic introductory information on Southern blotting can be found in most Molecular Biology or Cell Biology textbooks, for example

McLennan, A., Bates, A., Turner, P. and White, M. (2012).  Instant Notes in Molecular Biology (4th edition), pages 294-296

Turner, P., McLennan, A. and White, M. (2005).  Instant Notes in Molecular Biology (3rd edition), pages 158-162.  NB This is available from the library as an e-book”
Interpretation of a Southern Blot
You are studying a gene associated with a genetic disorder. RR individuals (homozygotes for the wildtype allele) and Rr (carriers) are healthy and rr are patients who show symptoms of disease. The restriction map of the R allele is shown in Figure 1.

Figure 1: Restriction map of genomic DNA of a wildtype allele (R).

You extract genomic DNA from an RR individual, a carrier, and a patient; digest with the following restriction enzymes; Eco RI, Hind III and Eco RI + Hind III; run on a gel and after alkali treatment, transfer to a nitrocellulose or nylon filter. You have a full-length cDNA for the R gene, which you use as a probe on the Southern blot.  The resulting autoradiograph is shown in Figure 2.

Figure 2: Autoradiograph from Southern blot probed with the R gene cDNA.
From the restriction map (Figure 1) and resulting autoradiograph (Figure 2) answer the following questions.
1) Using the restriction map list the size of fragment(s) generated from digestion of the entire wildtype allele shown in Figure 1 with
a) Eco RI
b) Hind III
c) Eco RI + Hind III
Marks (12)
2) Explain why there are only two bands on the Southern blot of Eco RI digested genomic DNA from the RR individual?
Marks (6)
3) Using the information given for each restriction enzyme digest (Figure 1 and 2), explain the alteration in the R-gene DNA of the mutant r allele?
Marks (25)
4) What are the possible outcomes to the primary sequence of the translated protein?
Marks (8)
5) Why are there more bands in DNA from the carrier (Rr) than in the homozygous wildtype (RR) or patient – what do the fainter bands represent?
Marks (8)
6) Describe and explain what the resulting autoradiograph would look like if you performed this blot, but:
a) Samples were run without digesting the DNA with restriction enzymes.
b) The DNA separated by gel electrophoresis was transferred to nitrocellulose or nylon without prior treatment with alkali.
c) The nitrocellulose membrane was not blocked during pre-hybridisation.
d) The blot was processed without washing at high stringency.
Marks (8)
7) When the blot was probed at high stringency with a probe made with DNA that flanks the R gene (as seen in Figure 1) a smear typical of thousands of hybridising bands was seen.  How could this be explained?
Marks (8)
8) The turnaround time for Southern Blot analysis is typically around 10 days. Now that you have identified the region in which the mutation lies explain with detail how a faster test could be designed?
Marks (25)