Annealing Temperature Calculator

Melting temperature is the temperature at which half of a primer-template duplex population is dissociated, and it follows from duplex thermodynamics rather than from base counting. The nearest-neighbor model treats stability as the sum of stacking interactions between adjacent base pairs, so each of the ten unique dinucleotide steps carries its own enthalpy and entropy term, plus helix initiation terms for the terminal bases. Tm is then delta-H divided by the quantity delta-S plus R times the natural log of Ct over 4, minus 273.15 to convert from Kelvin, where R is 1.987 cal per mol per K and Ct is total strand concentration; the factor of 4 applies to non-self-complementary oligos. The concentration term is why Tm is not a fixed property of a sequence: doubling primer concentration shifts it by roughly a degree. Salt matters because the phosphate backbone is polyanionic. Monovalent cations screen that charge and raise Tm by roughly 16.6 times the log of sodium molarity in the older empirical form, and divalent magnesium does so more efficiently still, which is why a correction on the order of 8 times the log of magnesium molarity is applied. Free magnesium, not total magnesium, is what counts, since dNTPs chelate it roughly mole for mole. Practical primer design keeps length at 18 to 25 bases, GC content at 40 to 60 percent, and the two Tm values within about 5 degrees C of each other, then sets annealing about 5 degrees C below the lower Tm. Mismatches, secondary structure, and non-standard buffers all shift the real optimum, so a gradient run remains the arbiter.

The polymerase chain reaction was conceived by Kary Mullis at Cetus Corporation in 1983 and published with colleagues in 1985 and 1986; Mullis shared the 1993 Nobel Prize in Chemistry for it. The first protocols used the Klenow fragment and required fresh enzyme each cycle. The switch described by Saiki and colleagues in Science in 1988 to the thermostable polymerase from Thermus aquaticus, the hot-spring bacterium isolated by Thomas Brock and Hudson Freeze at Yellowstone in 1969, made thermal cycling automatable and turned annealing temperature into a parameter that had to be chosen deliberately for every primer pair. The estimation methods predate PCR itself. The simple 2 degrees per A or T plus 4 degrees per G or C rule is credited to R. Bruce Wallace and coworkers, who published on short oligonucleotide hybridization in Nucleic Acids Research in 1979; it remains adequate only for probes under about 14 bases. Nearest-neighbor thermodynamics grew out of optical melting studies by Ignacio Tinoco and colleagues in the 1970s and was given widely used DNA parameter sets by Kenneth Breslauer and coworkers in PNAS in 1986. Competing parameter sets disagreed until John SantaLucia Jr. reconciled seven of them into the unified parameters published in PNAS in 1998, which is the set most Tm calculators now implement. Salt corrections were refined further by Richard Owczarzy and colleagues in the 2000s, and a magnesium correction from work by Oliver von Ahsen and coworkers around 2001 is commonly layered on top.