© 2018 Cold Spring Harbor Laboratory Press. The touchdown PCR (TD-PCR) strategy relies on the use of annealing temperatures for the first PCR cycles that are a few degrees higher than the optimal primer. Touchdown PCR increased both specificity and yield by high and low annealing temperatures in two consecutive amplifications on various gel concentrations. 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. By then, the target sequence will have undergone several cycles of geometric amplification and therefore becomes the dominant product of the PCR. 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. Dpn I (1 µl) was used to digest the template, and 5 µl of the PCR product was used to transform XL10-Gold E. Mycobacterium tuberculosis complex in sputum samples. Annealing under conditions of high stringency favors the formation of perfect primer-template hybrids. Conditions for touchdown PCR were: 95☌ 3 min hot start 5 cycles of 94☌ 20 sec, 72☌ 5 min 25 cycles of 94☌ 15 sec, 68☌ 15 sec, 72☌ 2 min 72☌ 15 min. A multiplex touchdown PCR for detection of Streptococcus pneumoniae, Haemophilus influenzae type b and. The newer Taq polymerases are much faster, and allow annealing and extension steps as short as 10-15 seconds Further, in some assays, the annealing and extension steps can be run at the same temperature. In touchdown PCR the temperature selected for the annealing step is initially set 5☌-10☌ higher than the calculated T m of the primers. Traditionally, PCR melting, annealing and extension steps have been 30-120 seconds. Wax beads that contain the missing critical component and that melt again only when the temperature of the sample reaches 7580C. "Touchdown polymerase chain reaction (PCR)" is a method to decrease off-target priming and hence to increase the specificity of PCRs.
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