Theoretical Melting Temperature of PCR primers?

Charlie Wright Genetics cw117 at mole.bio.cam.ac.uk
Fri Nov 18 07:35:28 EST 1994


I've bet putting together a little spreadsheet to keep track of my 
primers, their names, locations, GC content, temperature of melting, and 
the like.  It takes out the drudgery of mathematics, but I thought I 
would use a more complex formula for Tm now that the machine is doing the 
number crunching.  

I have been using the (simple but practical) formula:

        Tm = 4x[total no. of G & C] + 2x[total no. of A & T]

this works most of the time and is accurate up to about 18mers.

Sambrook et. al. list the formula from Bolton and McCarthy 1962:

        Tm = 81.5 - 16.6(log[Na]) + 0.41(%G & %C) - (600/N)

        where N=chain length and [Na] is Sodium ion concentration.

This is odd for two main reasons.  It implies to me that as Na 
concentration approaches zero (pure water) the log[Na] approaches 
negative-infinity... making the Tm approach an INFINITE temperature in 
water? Surely there must be a better substitute for [Na]... would any 
positive ion (potassium or even pH/hydrogen ion concentration) do? This 
is a very limited case if it only accounts for Sodium buffers.

It also seems backwards. In my experience, lowering salt concentration
lowers melting temperatures (high salt buffers are good for low stringincy
washes = high melting temperature of primer). Practical application:
stripping hybridized filters in boiling water with SDS is quicker and more
thorough than in 1x SSC buffer... though you can boil away your blotted
DNA too. 

Any mathematicians/theorists have any light to shed on this.  Anyone have 
a better formula that they like?

I'm baffled.

Charlie


-- 
C. R. Wright                                    Dept. of Genetics
+44 (0)223 333970 telephone                     Univ. of Cambridge
+44 (0)223 333992 telefax                       Downing Street, Cambs.
cw117 at mole.bio.cam.ac.uk                        CB2 3EH, England



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