This program is created for researchers who want to design cloning primers with correct sequences orientation as well as optimal melting temperature.
First, it is to generate correct sequences orientation of reverse primers based on users' input. Reverse primers have to be inverted the input sequences. That means sequence orientation has to be inverted in backwards. Also, all A and T have to be swapped mutually. Same as G and C.
Secondly, pairs of primers should have similar melting temperatures (Tm) since annealing during PCR occurs for both strands simultaneously, and this shared Tm must not be either too much higher or lower than the reaction's annealing temperature. So total primer Tm between 58 degree to 61 degree is the most optimal in most of the case. Part of the program is to help users to calculate the input sequence Tm and indicate whether users have to shorten or increase base pair(s) to generate the optimal primer sequences.