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Two rounds of PCR can be used to construct insertion, deletion, mutation, or
fusion.
In the first round, primer pairs P1/P2 and P3/P4 are used separately
in two PCR reactions. The products are then mixed and the second round
PCR amplifies the desired product using primers P1 and P4.
Figure 1. Insertion
Figure 2. Deletion
Figure 3. Mutation
Figure 4. Fusion
1. Design primers.
2. Setup two independent PCR reactions, one with primer pairs P1/P2
and the other with primer pairs P3/P4.
3. Run agarose gel to check PCR products. If more than one band
is visible, purify the desired band.
4. Mix 1-5 ml the two PCR products (1:1 equal molar ratio).
Dilute 1:10 to 1:100.
5. Setup the second round of PCR using the primers P1/P4.
6. Run agarose gel to check PCR products. If more than one band
is visible, purify the desired band.
7. Clone the PCR product into desired vector. Restriction sites may
be selected/introduced in P1/P4 primers for easy cloning. Or clone directly
the PCR product.
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