Length priority mode
In the length priority mode, one can choose the median size for the hybridization unit. By default, the software will use the Tm-avg calculated for the given median size and a dTm of +/- 4°C to search for a set of oligonucleotides satisfying all the conditions described above. For higher flexibility, it is also possible to force the program to use user-defined values for Tm-avg and dTm, but again, be extremely careful for not choosing mutually exclusive parameters.
Melting temperature priority mode
In this mode, one can set a value for Tm-avg and dTm. The program will automatically compute which median hybridization unit size will lead to the closest Tm to the input Tm. Then all computation will be the same as describe above. This could be seen as the length priority mode using the optimal median size for this Tm.
Basic cutting mode
We also offer a tool to chop down the sequence into oligonucleotides of equal length. There is no optimization in this mode to avoid non-specific hybridization between different oligonucleotides or to ensure a good uniformity of Tm. No header sequence will be added, and only a short tail sequence will be use to get the correct size for the last oligonucleotide.
Melting temperatures are computed using the Nearest-Neighbor model using DNA parameters published by Dr. J. SantaLucia (Proc Natl Acad Sci U S A (1998) 95: 1460-5). Published data are for a 1M sodium concentration in the buffer. To compute the Tm, I use the following formula:
where H is the enthalpy, S the entropy at a given salt concentration,
R the gas constant (1.9872 cal/K.mol) and [DNA] the DNA concentration.
The salt correction for a concentration different from 1M is done by using the following formula:
where [Na] is the sodium concentration of the reaction buffer and
N is the total number of phosphates in the duplex divided by 2.