Homologous recombination - (Jan/25/2007 )
We’re trying to replace a copy of a gene with a mutated version via homologous recombination. We only know the sequence for an internal fragment (about 3kb) of the gene we are targeting. The mutation is within this internal fragment, but the fragment does not contain the start and stop codons.
We have 2 questions regarding the design of a homologous recombination vector:
1) For homologous recombination to work, must the homologous fragment in the vector include the start codon or the transcription start site, or will an internal sequence flanking the mutation be sufficient?
2) Must the homologous fragment in the vector be “in-frame” with the rest of the target gene?
Thanks very much for any advice you can give.
The recombination system has no knowledge of the location, frame, or boundaries of the coding sequence. It operates entirely on the DNA sequence. So, the recombination region need not include the translation start or stop codon. The recombined sequence must read, when translated, to produce in-frame the codons of the target protein. So, you must alter, add, or remove triplets of bases within the recombination region to maintain the proper frame for the C terminal region of your protein.