Tm and Hybridization temperature

[Andy Phillips]

CVRL wrote:
> 
> My question concerns calculation of  Tm value for determining
> hybridization temperature.
> 
> I am trying to screen a genomic library with DIG labelled (CA)15 probe.
> We order our oligos from MWG Biotech and the synthesis report states Tm
> as 68 degrees (I have no idea how they caluclated this). Can I use this
> as a starting value for choosing a suitable hybridization temperature
> i.e Tm-25 degrees?
> 
> When I tried to calculate Tm uising the simple method: multiplying G and
> C by 4 degrees and the number of A and T by 2 degrees the sum works out
> to 90 degrees. With the more elaborate equation involving [Na+] and
> length of the oligonucleotide, Tm works out to around 54 degrees. What
> would you recommend as a starting value?
> 
> Any help on this matter would be much appreciated! Please respond to me
> by e-mail.
> 
> Sincerely,
> Maxy
> E-mail: microbio at emirates.net.ae

The Wallace rule (GC*4+AT*2) is very quick & dirty and applies only to
short oligos. I use a program I wrote using the formula of Rychlik et al
(Rychlik, W., Spencer, W.J. & Rhoads, R.E. (1990) Nucleic Acids Research
18, 6409-6412), who use next-neighbour analysis to calculate Tm and also
T(opt) for PCR. There's also a Web-based version, written by Jon Boxall,
at http://www.res.bbsrc.ac.uk/biochem/oligos/input.html.

I would expect the oligosynthesis synthesis company to use a similar
method, but they are probably basing the Tm on a monovalent cation
concentration of 50mM, as it would be in a PCR reaction. The higher []
in a library screen would push the Tm up considerably.

Andy


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