PCR on repeated sequences

ELader at my-dejanews.com ELader at my-dejanews.com
Thu Jul 23 09:05:19 EST 1998


In article <6p2h7j$qnq$1 at nnrp1.dejanews.com>,
  mbarnhart at my-dejanews.com wrote:
> Hi,
>
> We want to do quantitative PCR on polyubiquitin.  This gene contains ten
> repeats coding for 76 amino acids.  The repeats are identical or have 1 base
> change. Does anyone have any experience doing PCR under such circumstances
> and can we do accurate quantitation using PCR on this gene?
>
> We have about 160 nt from the poly A signal to the end of the repeated
> sequences, but I'm not sure this is a large enough segment.
>

Looking at the rat seuquence in Genbank, It looks like the end of the protein
VNTKKVKQEDTRTFLTTV SRKSPSCACSWV is unique.

 Anchor the upstream primer 10 bases into the unique sequence (the 5' end of
it 10 bases into the last repeated sequence), the downstream primer near the
polyA signal.
Something 160 bp is small for PCR unless you run a 4% agarose gel. A
polyacrylamide gel would be perfect. By the same token, doing an RPA on the
repeats would be difficult. You could use the same region of the gene for the
RPA.

Eric

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