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A family in which several individuals have arthritis and detached retina is diagnosed with Stickler syndrome. The locus for Stickler syndrome has been mapped near that for type II collagen on chromosome 12, and mutations in the COL2A1 gene have been described in Stickler syndrome. The family became interested in molecular diagnosis to distinguish normal from mildly affected individuals. Which of the following results would be expected in an individual with a promoter mutation at one COL2A1 gene locus?
A. Western blotting detects no type II collagen chains
B. Southern blotting using intronic restriction sites yields normal restriction fragment sizes
C. Reverse transcriptase“polymerase chain reaction (RT-PCR) detects one-half normal amounts of COL2A1 mRNA in affected individuals
D. Fluorescent in situ hybridization (FISH) analysis using a COL2A1 probe detects signals on only one chromosome 12
E. DNA sequencing reveals a single nucleotide difference between homologous COL2A1 exons
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Is C reasonable? I think so.
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I would go with the choice C.
This mutation is in the promoter region, so the transciption levels will be reduced.
Sequencing is a better stratgy, but the choice E) only metioned in exon region.
There is no changes in the intron so exclude Choice B.
Chioce A is not right, promoter mutation unlikely result in complete protein deficiency, I think.
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Correct answer is C
After the locus responsible for a genetic disease is mapped to a particular chromosome region, "candidate" genes can be examined for molecular abnormalities in affected individuals.
The connective tissue abnormalities in Stickler syndrome make the COL2A1 collagen locus an attractive candidate for disease mutations, prompting analysis of COL2A1 gene structure and expression.
Western blotting detects gene alterations that interfere with protein expression, while use of the reverse transcriptase“polymerase chain reaction (RT-PCR) detects alterations in mRNA levels. Each analysis should detect one-half the respective amounts of COL2A1 protein or mRNA in the case of a promoter mutation that abolishes transcription of one COL2A1 allele.
Southern blotting detects nucleotide changes that alter DNA restriction sites, but this is relatively insensitive unless large portions of the gene are deleted.
Fluorescent in situ hybridization (FISH) analysis using DNA probes from the COL2A1 locus is a sensitive method for detecting deletions of the entire locus, and DNA sequencing of the entire gene provides the gold standard for detecting any alteration in the regulatory or coding sequences.
Nucleotide sequence changes are still subject to interpretation, since they may represent polymorphisms that do not alter gene function. Population studies and/or in vitro studies of gene expression are often needed to discriminate DNA polymorphisms from mutations that disrupt gene function.
For any autosomal locus, the interpretation of molecular analyses is complicated by the presence of two homologous copies of the gene.
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Best assay is real time RT-PCR or called quantitative RT-PCR. Regular RT-PCR is hard to tell the difference.
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This is a good question with clear explanation. saton gets the best source. Doing PCR is a boring task that I did 20,000 reactions in the past at least! Do not mean anything!
