Sequence to be added
(1) When a PCR product is inserted into a vector, a sequence that can be recognized by a restriction enzyme has to be added to each of two primers. An additional redundant sequence is also provided on the outer side of the sequence to be recognized: otherwise, the restriction enzyme cannot recognize the target sequence since the target sequence is located too close to a terminal of a linear DNA fragment. Fig. 1 illustrates how the sequences are added to the primer.
(2) The calculation of the Tm does not take the added sequences into consideration, since the sequences do not contribute to annealing of the primer.
(3) When the PCR product is inserted into the vector, a sequence containing uracil (U) may also be added. After a PCR amplification in this system, the resulting product is treated with uracil-DNA glycosylase and then with alkali to form a single-stranded region on the 3'-terminal side (the uracil-DNA glycosylase causes abasic sites to be formed, which are hydrolyzed with alkali).
(4) If a primer having a Tm suitable for PCR has a sequence of continuous Ts or continuous As at its terminal, the sequence should be removed. Such a terminal is not stable and interferes with the integrity of the PCR process.