A genetic disease causing poor resistance to bacterial infection is linked to an unknown mutation in a gene carried by the X chromosome. The cDNA corresponding to this gene is used as a radioactive probe for Southern blot and Northern blot analyses. The results of these 3 experiments are illustrated below. (Please see attachment for the figure of all the gels)
Figure: Diagram representing the position (and size) of the DNA fragments (Southern blot, left panel) and RNA transcripts (Northern blot, middle panel) recognized by the radioactive probe. The range of size listed on the left side of the left panel applies only to the Southern blot. The size of the proteins coded by the wild type and mutant type gene is shown on the Western blot (right panel). You should assume that no fragments lower that 0.1 kb was detected by the radioactive probe.
The Southern blot is used to compare genomic DNA of a healthy individual (H) and a patient carrying the mutation (P). In this experiment, genomic DNA is digested with the restriction BamHI and the restriction fragments are separated by gel electrophoresis and transferred onto a membrane. The DNA attached to the membrane is denatured and the membrane is incubated with a radioactive cDNA probe. The position of the fragments annealing with the probe are visualized by placing an X-ray film in contact with the probed membrane. We assume that the experimental conditions are such that the cDNA probe detects every restriction fragment containing a portion of the studied gene (with or without mutation).
The Northern blot analysis is used to compare RNA transcripts isolated from samples isolated from the same healthy individual and patient. The purified RNA are not digested by BamHI but directly submitted to gel electrophoresis. The rest of the procedure (transfer, probing and detection) is similar to that described for the Southern blot.
Finally, protein extracts purified from the healthy donor and from the patient are compared by western blot. In this case, proteins extracts are submitted to gel electrophoresis, the proteins transferred onto a membrane and the membrane is probed with antibodies that can specifically detect the protein coded by the studied gene.
1) Could the Southern blot data suggest that in patients the studied gene contains a mutation that eliminates a BamHI site? Briefly explain
2) Using the combination of data from the Southern and Northern blots, could you predict the likely type of mutation (deletion, insertion, nucleotide's substitution) affecting the studied gene in patients? Briefly explain.
3) Explain the result of the Western blot in light of your answers to the previous questions.