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**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.**