Do overlapping in the middle of the sequence - (Jun/19/2006 )

Hi, this is a basic theoric question.

If I have a DNA sequence, let's say, 300bp and another 200bp. To the second one I join a overlapping sequence (21bp )to the first one. This overlapping sequence is complementary of the 260-280 section of the first one. If I do PCR with no primers, will I obtain either templates plus a longer one as a result of the linkage of both sequences? Is this possible or the overlapping must be at the end of the primer sequence? Must melting temperature of the PCR be calculated as if we have only a 21bp primer?

what you're asking about is 'crossover' pcr, 3part pcr or sandwich pcr - something like that. Basically, if you have overlapping fragments, they will find each other at some frequency if denatured and reannealed. Its a useful techniqe for connecting 3 seperate nucleic acid fragments using PCR. For example, to use for directed genomic integrations (fungal, bacterial, etc.)

What you can do is amplify flanking regions for arms with overlapping sequence to a marker on a plasmid or another fragment of interest. You can put all 3 fragments together and do standard pcr rxn. The flanking arms act as primers for the full length 3 part fragment. In the final amplification rxn you can add outside primers, and amplify the final 3 part construct for cloning or linear tranformations. (Its easier to explain with a diagram, but it works.)
I helped construct the largest know deletion strain set for A. nidulans (fil. fungi) using this technique. I had the joy of doing it again for A. fumigatus...