How to Use Gene2Oligo

Input Sequence

The input sequence can be made of either a single line or many lines of different length. Only A, T, G or C characters are permitted. They could be either upper or lower case, or a mix of both. The sequence should be oriented 5' to 3'.

Choose a design mode

There is a choice between three design modes. For inexperienced users, we strongly recommend to use the length priority mode with the software optimized T_m or the T_m priority mode if there is a need to focus on a special temperature.
Setting both hybridization unit size and T_m in the length priority mode requires a special attention to avoid selecting mutually exclusive values. For example, there is no possibility to get any oligonucleotides if the temperature selected is too high or too low for a given oligonucleotide size (a length of 15 nucleotides and a T_m of 80°C are not compatible).
It is also important to allow a certain degree of freedom to the system to succeed in the design. If the T_m range required is too narrow, say +/- 1°C, there is almost no chance to obtain a set of oligonucleotides. From our experience, 3 to 4°C seems to be a good compromise.

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 T_m-avg calculated for the given median size and a dT_m 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 T_m-avg and dT_m, but again, be extremely careful for not choosing mutually exclusive parameters.

Melting temperature priority mode

In this mode, one can set a value for T_m-avg and d_Tm. The program will automatically compute which median hybridization unit size will lead to the closest T_m to the input T_m. 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 T_m.

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 T_m. No header sequence will be added, and only a short tail sequence will be use to get the correct size for the last oligonucleotide.

Tm calculation

The DNA and sodium concentrations should reflect those used during hybridization either during LCR or assembly PCR. They are bothe used to predict the Tm of the hybridization units. Please note that the DNA concentration is expressed in nM and the sodium concentration in mM.

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.