Help:PCR GC rich region

Elizabeth Bates bates at cismsun.univ-lyon.fr
Wed Aug 9 08:13:35 EST 1995


I have a problem with a cloned cDNA that I am trying to amplify by PCR 
for site-directed mutagenesis. I'm using large (20-30 base) oligos with 
single or double base mutations in the coding regions and 100% 
homologous oligos as external oligos.
The problem seems to be that an area of this gene rich in GC somehow 
locally blocks PCR amplification and prevents certain fragments from 
being produced : For example I can sometimes (read in some 
circumstances) amplify the whole fragment and can amplify regions away 
from the GC rich zone but can't amplify fragments which cover this zone.


-A-	    -C-	                                      -H- 	-E-
5'----------------------------------------------------------------------
----------------------------------------------------------------------5'
			    	-D-   -G-        'GC rich'	                   	-F-          	-B-

Fragments A to B amplify as do fragments A to D and E to B. However 
fragments C to F do not amplify. Oligos C and F work as fragments C to G 
and H to F amplify. Apart from this GC rich zone I can't see why the 
fragments refuse to amplify. I've tried all the standard changes of 
temperature and shortening the cycle length (the fragment in total is 
800 bp and the GC rich area about 100 bp). and even DMSO and Formamide. 
No difference is seen with Pfu rather than Taq. (In fact overall the 
amplifications are weaker with Pfu). Hot (Mg2+) start does help A to C 
amplification, but still no C to F.
To cap it all the gene in question hates high copy number vectors and 
does its best to escape when cloned into commercial mutagenesis-type 
plasmids. Also I have only one unique site upstream of the problem 
region.
Has anyone dealt with this type of problem before and does anyone have 
any suggestions? Email me and I'll post a resume of the replies to the 
newsgroup.

I'm really begining to hate PCR.

Liz.





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