how to determine protected species size exactly in RPA ?

Colin Dolphin c.t.dolphin at qmw.ac.uk
Tue Oct 7 10:35:04 EST 1997


In the past, I have used RPA to determine the transcriptional start
point(s) (TSP) of a gene by running the resulting protected species on a
sequencing gel alongside a DNA ladder. However, as the ssDNA fragments do
not migrate equally with their ssRNA countertparts this makes accurate
sizing of the protcted species difficult. Having perused some papers on
gene structures it appears that most workers are likely to use RPA in
conjunction with another technique, such as primer extension or S1
nuclease, to determine the TSP. I now want to determine another TSP and
would like to use RPA alone. 

So, how can I determine exactly the size of the protected species in RPA
? Is there a way of generating an RNA sequence ladder - say from
pBluscript using T7 pol and deoxyNTPs ? (I think there are a few old refs
to such a technique and I believe Pharmacia may once have provided some
sort of kit).


Colin



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