This protocol can be used for inserting new sequence or for replacing a region of DNA.
A region of DNA is amplified using two primers that have 5'-extensions corresponding to the sequences of junction regions at which the insertion is to be made within the target gene. PCR with these two primers generates a large DNA fragment with ends corresponding to the target gene.
These 5'-ends act as sticky feet that allow the heterologous DNA sequence to anneal to the target gene. The 3'-end of the annealed fragment acts as a primer in a standard DNA synthesis reaction of the remainder of insert plus vector.
Using a proofreading thermostable enzyme during the cooling from the denaturation temperature in PCR reaction should enhance specificity of priming and reduce the extent of primer fragment that reanneals to generate nonproductive double strands.
The single-stranded M13 template DNA can be prepared from a dut- ung-E. coli strain that leads to the incorporation of some dUTP instead of the normal dTTP in DNA synthesized within the cells.
Following primer-directed synthesis on the template, the newly synthesized strand contains no dU, but the original dU-containing template DNA strand is destroyed when the heteroduplex molecule is transformed into an dut+ ung+E. coli host, leading to efficient selection of insertion mutants.
For this procedure, both filamentous phage (such as Ml3) and phagemids (such as pBluescript and pUC-vectors) can be used to generate single-stranded template DNA.
Clackson, T, Winter, G (1989) Sticky feet-directed mutagenesis and its application to swapping antibody domains. Nucleic Acids Res. 17: 10163-10170.