PCR GC-rich genomic DNA

Nancy J. Phillips, M.D. PHILLINJ at SLUVCA.SLU.EDU
Mon Jul 17 18:59:34 EST 1995


	I am having trouble obtaining a PCR product of a 70%GC region from
genomic DNA but not from a control plasmid with the target region. I have tried
1. Annealing 0 - 6 degrees above calculated (Rychlik-Rhoades) Tm 2. various
conc'ns formamide and DMSO 3. template, Mg++, primer conc'ns as for usual PCRs. 
Result: excellent 200 bp product from positive control plasmid template, no
200 bp band from human genomic DNA but a 500-1000 bp faint smear instead. These
primers are themselves 60-65% GC, 20-24 bp, Tms 71-73 degrees, no obvious
periodicities. Five primer pairs from five overlapping regions of this gene
have been tried , with similar lack of results, so I don't think that this is a
problem with a single primer. Next on my list is shearing or 4-cutter
restriction digest of my template before PCR. Anyone else have suggestions for 
amplification of GC-rich region from complex template? Algae or Gram+
researchers listening?
	Thanks
Nancy Phillips			phillinj at sluvca.slu.edu
Pathology
St. Louis University
St. Louis MO USA




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