probe size: please help

[Martin Leach]

In article <CpoDKG.C3L at zoo.toronto.edu>, mes at zoo.toronto.edu (Mark Siddall)
wrote:

> 
> Aplogies for what may appear to be a naive query.  I am a molecular
> neophyte and hope to soon remedy that.
> 
> Here's my situation:  I am armed with sequences for ribosomal genes for
> two related parasites.  I desire a probe that will light-up parasite1 if
> it is there but not parasite2 and not the host.  Notably, the ssu rDNA
> gene, as a whole is highly conserved across taxa.  On the other hand I
> can find regions of hypervariability (across species but not within a
> species).
> Here's my question:  for a descent probe, how long should it be?  Will
> 20 bp do it?  How much sequence similarity with the non-target parasite2
> and host can I have without non-specific binding problems?  And is
> A-T : G-C composition going to be a problem for specificity?  Ideally
> I want to keep the prob(es) short as I may use them for priming
> PCR reactions at a later date.
> 
> Any help/pointers/redirections are most welcome!
> 
> Thanks in advance.
> 
> Mark


Dear mark

if hybridisation of the probe/washing is performed in alkylammonium salts
(see maniatis) then a single base-pair difference can be discriminated by a
5C difference in wash temp.

eg. 20bp sticking washes at 60C
    19 bp sticking washes at 55C

this is due to the alkylammonium salt concentration.

if u cant find it in the new maniatis - i have several ref.s on this.

In fact we used 20-25mer oligos to discriminate point mutations on pcr
products from cjd individuals.....

If you are interested in this - email me and i will post/mail some refs.

Martin


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.....          Martin Leach                Email:leach at mbcrr.harvard.edu 
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