Melting Temperature

Basic Melting Temperature (Tm) Calculations

Two standard approximation calculations are used.

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.

Base-Stacking Melting Temperature (Tm) Calculations

This aproximation uses Thermodynamical concepts to compute T_m. The following references were used to develop the script:

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, Schutz 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