PCR gene splicing: help!

Kevin Morano ez005528 at bullwinkle.ucdavis.edu
Fri Oct 7 13:51:42 EST 1994


Hi- I'm trying to splice two genes together using PCR and I'm having no 
luck at all. Can a PCR guru help? My situation:

1. I synthesized fragment 1 and fragment 2 from plasmid templates and 
designed the primers such that the 3' end of fragment 1 has 16 bp overlap 
with the 5' end of fragment 2.

2. I have performed numerous amplifications using equimolar amounts of 
both fragments as the template, with only the flanking primers. (5' 
primer from frag. 1 and 3'(downstream) primer from frag. 2)

3. I am expecting a spliced version of these two, for a product of 1.5 kb.

4. All I am getting is a product of the same size as fragment 2

5. If I eliminate the 5' primer, I still get this product. Sans 3' 
primer, no product; no primers, no product.

Questions: Why can't I get the spliced product? How does the reaction 
actually produce a "linked" splice molecule from the primers? Shouldn't 
50% of my products be produced using the annealed fragments themselves as 
the primers? I am really keen on getting this to work, and would greatly 
appreciate any help. I will post responses in a summarized form.

Thanks!
Kevin
---------------------------------------------------------------------------
Kevin A. Morano
Internet:kamorano at ucdavis.edu    
Bitnet:kamorano at ucdavis   
Section of Microbiology, University of California, Davis



More information about the Methods mailing list