MycoTOOL Mycoplasma Real-Time PCR (polymerase chain reaction) Assay. © 2018 Cold Spring Harbor Laboratory Press. Touchdown (TD) PCR offers a simple and rapid means to optimize PCRs, increasing specificity, sensitivity and yield, without the need for lengthy optimizations and/or the redesigning of primers. agencies have issued guidelines that provide protocols for. The use of touchdown PCR is essential when the sequence of the primer might not match that of the target-for example, if the sequence of the primer has been deduced from amino acid sequences, when the template DNA may contain several closely related targets, or when the target DNA is of a different species from that used to design the primers. To minimize mispriming during the early stages of the PCR, touchdown PCR should always be performed in conjunction with a hot start protocol. The annealing temperature in the early cycles is usually 3-5C. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. Touchdown PCR: In this type the annealing temperature is gradually decreased in later cycles. In subsequent cycles, the annealing temperature is gradually decreased by a small amount so that by the end of the PCR, the annealing temperature is 2☌-5☌ below the calculated T m of the primers. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. In touchdown PCR the temperature selected for the annealing step is initially set 5☌-10☌ higher than the calculated T m of the primers. The practical utility of any cloning method is predicated upon its reliability, rather than its convenience, price, or efficiency under optimum conditions. 'Touchdown polymerase chain reaction (PCR)' is a method to decrease off-target priming and hence to increase the specificity of PCRs. Numerous alternative approaches to PCR cloning have been developed, including TA cloning, ligation independent cloning (LIC), recombinase-dependent cloning, and PCR-mediated cloning.

Alternative to touchdown pcr protocol