Overlap PCR

The application of overlap PCR is widespread, and the mechanism is actually simple: suppose you are going to link two gene or a promoter and a gene, the primary method is cutting out by enzyme, but frequently we fail to find suitable restriction enzyme sites, or the enzyme is too special or expensive, in either situation purchasing this enzyme can turn out to be a waste. Overlap PCR affords a better alternative. Here is an illuminating example: suppose we have two genes, namely A and B, whose sequences are 5-atgcatgctagctagaacgctacgctgactaccccctgatc-3 and 5-atgctagtagctagccccccccaggggataattttttaaaacg-3 separately. At the beginning we should design primer, and the sequences of primer is assumed as follows:
A1:5-ATGCATGCTAGCTAGAACGCT-3
A2:5-ggggggctagctactagcatgatcagggggtagtcagcgt-3
B1:5-acgctgactaccccctgatcatgctagtagctagcccccc-3
B2:5-cgttttaaaaaattatcccct-3
Our aim is to link A and B with PCR. If we observe primer A2 and B1 carefully, we will find this two primers are much longer than the others. The reason is that we add 20 sequences from the 5’ end B to the 3’ end of A2, 20 sequences from the 3’ end of A to the 5’ end of B1 when we design our primer. Here is the steps of overlap PCR :
（1）amplify A with A1 and A2, B with B1 and B2
（2）recycle gene A and B
（3）Amplify A+B with template A and B, primer A1 and B2. Because A and B have complementary bases, they can combine each other at annealing, thus we can get A+B by overlap PCR.
The third step is diverse. Some individuals add template A, B, dNTP, Buffer and water first, then amplify genes within 3-5 cycles, and finally add primer A1, B2 and Taq enzyme. The advantage of this method is that we can get distinctive amplifications, whereas the most obvious disadvantage is it is too trivial. Another alternative is to add primers, double templates, enzymes, dNTP and all other components to PCR cubes, which consumes far less time.
Currently overlap PCR has been applied widely, like changing the specific sequence of DNA; although there have been convenient many kits inducing mutants, they are too expensive;
Synthetizing gene, whose basic technique has utilized the methodology of overlap PCR; linking the promoter and the target gene; linking two distinctive expression kits: as we know, we frequently need to link several kits to observe their effects, yet the wide existence of intros prevent exons from linked together, which can be easily handled by overlap PCR.