How homogous does a sequence need be for homologous recombination - (Sep/15/2014 )

Hello there,

I've been working on a knock-out construct for a couple of months.

Having troublesome PCR, troublesome digestion, troublesome ligation etc. I now finally have a construct which is far from perfect, but I want to know if it's good enough

The basic outline of my construct is:

Restriction siteExon1 \\ Intron // Exon2^^Selection marker^^Exon2Exon3

That what is red, is what is not perfect about this construct. Because the 3' flanking region could not be amplified from gDNA, but cDNA worked, so used that instead.. ofcourse now the intron is missing and I now have 25bp of Exon3 stuck to my exon 2.

That what is green is homologous.. the two sequences flanking the selection marker are about 350bp which should be enough for my target organism.

I'm wondering if the red areas would matter at all. My whole point is in disrupting the gene. Would the 6bp 5' of the homologous region and 25 bp 3' of the homologous region interfere with the recombination?

Hope you can help out.

Probably will not be of much effect.  If you are concerned, are you able to PCR out Exon1 \\ Intron // Exon2^^Selection marker^^Exon2 without the nonhomologous regions and use this as your donor insert?

Short answer is no - these bits shouldn't interfere with recombination.  People have been using this technique for a long time with success, using constructs that look very very similar to what you have there.

My suggestion would be to design some longer arms, the limits that people have worked with in the past (e.g. your 350 bp) are often difficult to get working, as this is a quite short sequence to get recombination with.