The stability of a particular double stranded sequence is related to the number of hydrogen bonds formed. GC rich sequences pair with greater stability than AT rich sequences, but sequence ordering is also a component of stability. For example, the duplex AA/TT forms a more stable pair than the duplex AC/GT due to the more favorable interaction of neighboring bases in the duplex. SeqBuilder Pro uses the nearest neighbor model to measure the stability of a given duplex. It is based upon the free energy of adjacent dinucleotides and calculated from dinucleotide entropy and enthalpy data presented in Bresslauer et al. for DNA, and Freier et al. for RNA.

The calculation of free energy (∆G) is ∆G = ∆H - T∆S, where ∆H is enthalpy, ∆S is entropy and T is temperature. The value of T is 25°C.

The calculation of ∆H and ∆S is where the dinucleotide data come in. For a given sequence, S(abcde): ∆H is the sum of dinucleotide ∆H values [∆H(abcde) = ∆H(ab) + ∆H(bc) + ∆H(cd) + ∆H(de) + ∆H ini], where ∆H ini is a helix formation constant.

∆S is the sum of dinucleotide ∆S values [∆S(abcde) = ∆S(ab) + ∆S(bc) + ∆S(cd) + ∆S(de) + ∆S ini], where ∆S ini is a helix formation constant.

SeqBuilder Pro uses ∆G values computed over the entire template sequence and ∆G values computed in pentamer windows to evaluate the stability of a given primer. Selected primers will have ∆G values near the mean ∆G computed over the entire template, pentamer ∆G values lower than the mean at the 3’ end, and for pair scoring, a stability profile of pentamer ∆G values above the mean near 5’ and descending toward 3’.

Wherever possible SeqBuilder Pro uses the nearest neighbor method for computing the ∆G and the Tm of primers. These models are valid for sequences of less than 50 bases. For long primers, probes and products a less accurate method of computing free energy is used, which is based upon the GC content of the sequence.

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