I am hypothetically looking at a genetic disease arising from a trinucleotide re

Question

I am hypothetically looking at a genetic disease arising from a trinucleotide repeat expansion (30 base pairs in total) after the 150'th nucleotide of a 450 nucleotide gene.

The question asks you to design a PCR-based method to detect this disorder. We learned about qPCR and gel electrophoresis.

GE: I was thinking that after PCR you could use gel electrophoresis to distinguish the size difference between the amplified product using the same primers. But at the same time I don't know if a 30 base pair difference is enough to be shown on a gel.

qPCR: But then we also talked about how qPCR utilizes the fact that each cycle doubles the # of copies of the target DNA and allows you to quantify the amount of transcript in treated vs untreated cells (done by looking at SYBR green fluorescent emission at the end of each extension stage)

Which method makes sense?

Explanation / Answer

30 bp difference is not enough for gel eletrophoresis . Do qPCR seems to be a better option.

QPCR doubles the number of copies of the targeted DNA. So the amount of 30bps will be amplified - in turn the amount of DNA will be increased. After doing the qPcR one can run a GE to get the purified DNA and then go for sequencing.

At the situation given qPCR makes more sense.

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