Analyzing Gene Mutations Using Southern, Northern, and Western Blotting

24. A series of people are found to have difficulty eliminating certain types of drily from their bloodstreams The problem has been linked to a gene. X, which encodes an enzyme Y. Six people were tested with the use of various techniques of molecular biology. Person A is a normal contra, person B is asymptomatic but some of his children have the metabolic problem, and persons C through F display the trait. Tissue samples from each person were obtained. Southern analysis was performed on the DNA after digestion with the restriction enzyme Hind lll. Northern analysis of mRNA also was done. In both Ives of analysis, the gels were probed with labelled X cDNA. Finally, a Western blot with an enzyme-linked monoclonal antibody was used to test for the presence of protein Y. The results are shown here Following concussion can be drawn 1. A mutation in person El hay altered -one if the of allele of gene X, leaving the other intact. The one functional allele is transcribed and translated enough protein to render person asymptomatic 2. Person C has only the smaller version (having deletion) of gene X. This gene transcribed but not translated 3. Person D has a normal copy of m-RNA but no RNA or protein. There may he mutation is promoter region of the gene which prevents transcription 4. Person E has normal copy of gene that is transcribed but no protein is made, which suggest mutation prevents translation, which may be due to incorporation of premature stop codon in m-RNA 5. Person F has a normal copy of gene that is transcribed and translated but still posses metabolic problem, It is unlikely and probably error has been made in experimentation Which of the following conclusion are not valid?
(A) 1, 2 and 3
(B) 2 and 5
(C) 3 and 4
(D) 1 and 4

Interpreting the Results of Molecular Blotting Analysis

The experiment used Southern blotting to analyze the presence of the gene X, Northern blotting to check mRNA transcription, and Western blotting to detect the enzyme Y protein. Based on these results, different mutations affecting transcription or translation were identified in individuals C to F.

Re-evaluating the Molecular Blotting Experiment and Conclusions

The study used three techniques to analyze gene X:

• Southern blot: Detects DNA changes (mutations, deletions, or rearrangements).
• Northern blot: Measures mRNA levels (gene transcription).
• Western blot: Identifies protein presence (gene translation).

Based on the blot results and the given conclusions, let’s check which ones are invalid.

Checking Each Conclusion

✅ Conclusion 1: “A mutation in person B has altered one allele of gene X, leaving the other intact. The functional allele produces enough protein to make the person asymptomatic.”

• Southern blot: Person B has both normal and mutant alleles.
• Northern blot: mRNA is produced.
• Western blot: Protein Y is detected.
• Valid? ❌ INVALID – This does not explain why some of person B’s children are affected. If only one allele were mutated, person B would be a recessive carrier, but the inheritance pattern suggests a dominant mutation or incomplete penetrance.

✅ Conclusion 2: “Person C has only the smaller version (with a deletion) of gene X. The gene is transcribed but not translated.”

• Southern blot: Shows a smaller DNA fragment, indicating a deletion.
• Northern blot: mRNA is present → transcription occurs.
• Western blot: No protein is produced → translation failure.
• Valid? ❌ INVALID – Even with a deletion, some truncated protein might be produced. A complete absence of protein suggests an issue beyond translation (such as nonsense-mediated mRNA decay).

✅ Conclusion 3: “Person D has a normal copy of mRNA but no RNA or protein. There may be a mutation in the promoter region of the gene preventing transcription.”

• Southern blot: Gene X is intact.
• Northern blot: No mRNA detected → no transcription.
• Western blot: No protein detected.
• Valid? ❌ INVALID – If no mRNA is produced, then mRNA presence should be zero. The conclusion incorrectly states that normal mRNA is present while also claiming a promoter mutation.

✅ Conclusion 4: “Person E has a normal gene that is transcribed, but no protein is made. This suggests a mutation prevents translation, possibly a premature stop codon.”

• Southern blot: Normal gene X.
• Northern blot: mRNA is present.
• Western blot: No protein detected → translation failure.
• Valid? ✅ VALID – A premature stop codon could lead to nonsense-mediated decay, preventing translation.

✅ Conclusion 5: “Person F has a normal gene that is transcribed and translated but still has a metabolic problem. This suggests an experimental error.”

• Southern blot: Normal gene X.
• Northern blot: mRNA is transcribed.
• Western blot: Protein Y is present.
• Valid? ✅ VALID – The metabolic problem may be due to a non-functional enzyme rather than an error in the experiment.

Final Answer: (A) 1, 2, and 3 are NOT valid.

• 1 is invalid (does not explain inheritance correctly).
• 2 is invalid (should show some truncated protein).
• 3 is invalid (contradicts the mRNA results).

Thus, the correct answer is A: 1, 2, and 3.