Melting Temperature (Tm) Calculationfrom biophp.org, adapted for Gscope Clonage by Raymond RippSource code is freely downloable at biophp.org |
For sequences less than 14 nucleotides the formula is:
Tm= (wA+xT) * 2 + (yG+zC) * 4
where w,x,y,z are the number of the bases A,T,G,C in the sequence, respectively.
For sequences longer than 13 nucleotides, the equation used is
Tm= 64.9 +41*(yG+zC-16.4)/(wA+xT+yG+zC)
When degenerated nucleotides are included in the primer sequence (Y,R,W,S,K,M,D,V,H,B or N), those nucleotides will be internally substituted prior to minimum and maximum Tm calculation.
Example: Primer sequence: CTCTRYCTWSCTCTCT Sequence for minimum Tm calculation: CTCTATCTAGCTCTCT Sequence for maximum Tm calculation: CTCTGCCTAGCTCTCT
ASSUMPTIONS:
Both equations assume that the annealing occurs under the standard conditions of 50 nM primer, 50 mM Na+, and pH 7.0. } ?> if ($_GET["formula"]=="Base-Stacking"){ ?>
SantaLucia J. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc Natl Acad Sci U S A. 1998 Feb 17;95(4):1460-5. NCBI
von Ahsen N, Oellerich M, Armstrong VW, Schütz E. Application of a thermodynamic nearest-neighbor model to estimate nucleic acid stability and optimize probe design: prediction of melting points of multiple mutations of apolipoprotein B-3500 and factor V with a hybridization probe genotyping assay on the LightCycler. Clin Chem. 1999 Dec;45(12):2094-101. NCBI } ?>