EFFECT OF CELLULAR PHYSIOLOGY ON PCR AMPLIFICATION EFFICIENCY

Culture conditions, and other variables that modulate a cell's physiol ogy, can bias a polymerase chain reaction (PCR) amplification against generating a representative population profile. Two Pseudomonas putida nahR alleles were constructed in pUC19 that differ solely in a 31-bp internal segment whose sequence has been inverted. After PCR amplifica tion, the products could be distinguished on the basis of a change in a unique restriction site. When an Escherichia coli strain carrying on e nahR allele is submitted to different growth conditions, the consequ ences of such variations on the relative PCR amplification of whole ce lls can be ascertained through coamplification with a strain carrying the other allele and subsequent restriction analysis. Cells in station ary phase displayed improved amplifiability while cells grown at 42 de grees C were equally amplifiable as compared to tells grown at 37 degr ees C. However, sublethal levels of tetracycline or growth in minimal medium made the PCR target in these cells relatively less amplifiable. When cells are completely lysed and the plasmid DNA is purified befor ehand, the coamplification bias is eliminated. These results suggest t hat mixed populations containing cells in different physiological stat es may not be representatively amplified by PCR unless a DNA extractio n step is included.