Sauer:Stitching Genes by PCR
This method allows you to "stitch" genes or coding sequences together when there are no convenient restriction sites at the junction point. It is espcially useful when the target gene is flanked by other genes that can't be disrupted. The technique is by no means new, but this should keep people from asking me how to do it.
Refer to this figure:
- Step 1:
Scan your sequence and find restriction sites anywhere to the left and right of the gene (the left one can be in your gene). Make sure they are unique and they are not contained in the fragment you want to append to your gene. In the figure, they are marked "A" and "B".
- Step 2:
Design 4 primers. "A forward" primes toward your gene and anneals to the left of (or on) the restriction site "A"; "B reverse" primes back toward your gene and anneals past (or on) restriction site "B"; "stitch forward" anneals to amplify your appendage and has a tail that matches (anneals to) the left side of the fusion point (green in this case); 'stitch reverse" reverse primes your appendage and has a tail that encodes the region just to the right of the fusion point. The dashed lines show where the primers match.
- Step 3: