'Basic' probably refers to the 2(AT)*4(GC) formula. 'Salt-adjusted' probably refers to that formula plus a correction term (I think it is 16.6*logM in the case of monovalent cations) for the variation in Tm with salt concentration. 'Nearest neighbor' is a model where oligonucleotides are treated like sequences of dinucleotides, with experimentally derived thermodynamic parameters for each nucleotide pair (this accounts for stacking effects, the influence of sequence, rather than composition, fraying, mispairing, etc). Nearest neighbor is by far the most accurate.
Please remember that Tm calculations always make a number of assumptions, the first of which is to assume that annealing or dissociation is a two-state process (which it is not, especially as sequence length increases). Also remember that association exhibits first-order kinetics, and so Tm will change with primer concentration.
'Basic' probably refers to the 2(AT)*4(GC) formula. 'Salt-adjusted' probably refers to that formula plus a correction term (I think it is 16.6*logM in the case of monovalent cations) for the variation in Tm with salt concentration. 'Nearest neighbor' is a model where oligonucleotides are treated like sequences of dinucleotides, with experimentally derived thermodynamic parameters for each nucleotide pair (this accounts for stacking effects, the influence of sequence, rather than composition, fraying, mispairing, etc). Nearest neighbor is by far the most accurate.
Please remember that Tm calculations always make a number of assumptions, the first of which is to assume that annealing or dissociation is a two-state process (which it is not, especially as sequence length increases). Also remember that association exhibits first-order kinetics, and so Tm will change with primer concentration.
So while designing a primer for RT-PCR, which Tm should be considered?
Thanks,
Actually for PCR it doesn't matter much which one you choose. Tm is only a starting point from which to adjust annealing temperature according to your results, as annealing/melting reactions do not reach equilibrium, Mg++ and primer concentration vary between cycles, etc. Also, most times the exact conditions of the reaction do not match exactly those used in the calculations.
It is more important to avoid self-complementary primers, primers dimers, false priming sites etc. than it is to obsess about accurate Tm.
Basic melting temperature is the most commonly used .but the least preferred method.For oligonucleotides of the size upto 40 nt 'nearest neighbour method ' is more accurate.For most primers other than sequencing primers (which is 100% complementary) it is better to stick to salt adjusted melting temperature.So for cloning primers I would go with 'salt adjusted method' .But as pointed out by Alejandro Martin, these are all estimates.
Hello all.. I am also designing some primers. I have come across some different Tm methods like santa lucia, breslauer and nearest Nbr (which is discussed above). All of them are showing different Tm values for the same nucleoetide sequence. Which one should I choose? Ideal Tm must be 60-65 deg.. But i am unable to get any appropriate value.
Thank you.
It may refer to different sets of thermodynamic parameters for each possible dinucleotide, published by Breslauer in '86 (I think) and Santa Lucia in '98. Or may also refer to different salt correction formulas, or to both. Hard to say without a link to the website you are using.
- Badges
- Science method