A successful PCR reaction requires efficient amplification of the product. At normal temperatures nucleic acids fold into conformations (secondary structures) which have high negative free energy (∆G). The stability of these template secondary structures depends largely on their free energy and melting temperatures (T_m), and is extremely important for designing primers. If these secondary structures in the nucleic acids are stable even above the annealing temperatures, then the primers are unable to bind to the template DNA, and the yield of PCR product is significantly reduced. It is desirable to design specific primer pairs which do not assume secondary structures during the reaction, and this can be determined from a program called mfold server.

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