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?

Answer 1

Using a modified Rrpeat Expansion Detection (RED) assay, that was optimized for individual oligonucleotides, unrelated individuals were systematically screened for maximal repeat sizes of each of the ten possible trinucleotides repeats. Cloned trinucleotides repeats were generated ans used as standards for the detectability of single copy trinucleotide repeat fragments.

when the size distributions of trinucleotides repeats were compared to previously reported data, significant differnces were found for the CTT repeats, which corresponds to the expanded GAA repeat in Friedreich ataxia, as well as for ATT, CCT and GTT repeats.