probe size: please help
leach at mbcrr.harvard.edu
Thu May 12 08:14:19 EST 1994
In article <CpoDKG.C3L at zoo.toronto.edu>, mes at zoo.toronto.edu (Mark Siddall)
> 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
> 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.
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 Leach Email:leach at mbcrr.harvard.edu
_|____ Dept. of Pharmacology Phone: (617) 638-5323
/ o / Boston Univ. School of Med. Fax: (617) 638-4329
_/ |-/__==/ 80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118 "Not the old underpants on your
USA head.....WIBBLE" -BLACKADDER
More information about the Methods