You are a scientist in a clinical lab studying the impact of mutations in BMPR2,
ID: 98132 • Letter: Y
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You are a scientist in a clinical lab studying the impact of mutations in BMPR2, the gene encoding the type II bone morphogenetic protein receptor on patients with pulmonary arterial hypertention (PAH). Roughly 25% of PAH patients possess heritable BMPR2 mutations. You genotype a DNA sample from one of the patients in your clinic using Sanger sequencing. A segment of the sequence from the electropherogram is shown below (control). You then collect skin fibroblasts from this patient and use them to isolate RNA, which you convert to cDNA using reverse transcriptase reaction. Upon PCR amplyfying and seqencing the region of cDNA corresponding to the genomic muation you produce the following electropherogram (patient mRNA sequence).
a) how do you explain the difference between this result and the electropherogram shown in question 1?
b) You treat the fibroblasts with the antibiotic puromycin and repeat RNA isolation, RT reaction, PCR and sequencing described above. What do you expect the electropherogram to look like now? Why?
You are a scientist in a clinical lab studying the impact of mutations in BMPR2, the gene encoding the type II bone morphogenetic protein receptor, on patients with pulmonary arterial hypertension (PAH). Roughly 25% of PAH patients possess heritable BMPR2 mutations. You genotype a DNA sample from one of the patients in your clinic using Sanger sequencing. A segment of the sequence from the electropherogram is shown below: Control Genome Sequence & Rearing Frame: G T G | G C C | G A A | C A You collect skin fibroblasts from this patient and use them to isolate RNA, which you convert to cDNA using a reverse transcriptase reaction. Upon PCR amplifying and sequencing the region of the cDNA corresponding to the genomic mutation, you produce the following electropherogram: a) How do you explain the difference between this result (mRNA sequence) to the (control genome sequence/patient genome)? b) You treat the fibroblasts with the antibiotic puromycin and repeat the RNA isolation, RT reaction, PCR and sequencing described above. What do you expect the electropherogram to look like now? Why?Explanation / Answer
According to fig.1 the electropherogram shows overlapped peaks i.e heterozygous (one mutant copy and normal copy ) in patient BMPR2 (bone morphogenic protein receptor type 2 (BMPR2) .Most of the mutations in BMPR2 are germ line mutations ,In second figure cDNA was isolated from skin fibroblats and mutations in skin fibroblast shows absence of heterozygous ,so mosaicisam was present in patient .so patient expresses 2 types of banding pattern .
b) puromycin is a translation inhibitor it effects only translation and it is acts as selecting agent in cell culture , when we treat fibroblast with puromycin it does not effect transcription so electropherogram look like same after treatment also .
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