PCR on repeated sequences

Patrick F.H. Lai pfhlai at LOOKSMART.COM
Tue Jul 21 21:00:20 EST 1998


---- Begin Original Message ----
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.

Any ideas will be greatly appreciated.

Thanks,
Michael

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Dear Micheal,

I think you may be able to get away with part of the last repeat plus
the last 160 nt to make it about 200 something bases, and try RPA 
instead of RT-PCR.  THis way you don't have to worry about your primer
binding to many sites.  200-sth bases should be enough for RPA, I think=
......  ELADER at Ambion will probably be more helpful than me.











Hope this is useful info.   :-)


Patrick F.H. Lai  
Graduate Student, University of Toronto 
< PFHLai at looksmart.com >




      
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