[BioC] Development of GCRMA-like methods

Richard Finney rfinney5 at yahoo.com
Fri Feb 6 02:01:56 MET 2004

Hay.  Just a couple of notes on your questions ...

> > My understanding is that-
> > The first of these papers shows that MM intensity
> is related to GC content, and weights MM values
> towards the average distribution of the binding of
> MM with similar GC contents.

The signal intensity is more a function of the 
CT content.  The lights attach to the back of the
of Gs and As on the target cDNA. 
 Remarkeably, this is syergistic :
the more Cs and Ts you have lined up together, 
the more the signal.  The locaion of the Cs and Ts
are also important.  They are stronger in the middle.

> > 
> > The second proposes that most MM>PM occur because
> when the middle PM A/G is changed to MM T/C the
> smaller size of the substituted pyrimidine (C or T)
> allows room for the label on the target RNA (U or C)
> which would otherwise interfere with the binding to
> the PM.

Label gets put on the cDNA.  RNA is converted to
cDNA at the last step.

Cross hybridization comes from everywhere.
The Gs and As overhelm the the correct hybridization
at low levels of expressions.  At higher levels,
the PM goes above MM.  It's not just that middle
base, it's what's around it, too.  The more Cs and
T's surrounding the mismatch spot, the stronger
the signal.

> > 
> > Are there plans to combine these ideas and would
> there be any benefit from doing so? Would
> sub-setting the MM based on both GC content and the
> middle base provide more accurate distributions to
> weight the MM's to? The majority of the MM>PM would
> have C (or T) as their middle base and 'averaging'
> them must surely distort things for the MM's with A
> or G?
> > 
> > Finally Fig 3 in ref (2) shows nice fits of the
> positional effect due to having individual bases at
> different positions (1-25) in the PM probe. What
> would such fits look like for the MM probes, would
> it be similar or random/distorted due to
> non-specific binding? And would it help in answering
> why C has a smaller effect than G on intensity - or
> is this already known?

I don't have the Fig.; but MM should look the same
with a dip at the mismatch spot.

Nota Bene : The signal strenth is also a function
of the probablility that the target RNA will fold
and a function of the distance from the 3` end.


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