Differential susceptibility of PCR reactions to inhibitors: an important and unrecognised phenomenon.
BACKGROUND
PCR inhibition by nucleic acid extracts is a well known yet poorly described phenomenon. Inhibition assessment generally depends on the assumption that inhibitors affect all PCR reactions to the same extent; i.e. that the reaction of interest and the control reaction are equally susceptible to inhibition. To test this assumption we performed inhibition assessment on DNA extracts from human urine samples, fresh urine and EDTA using different PCR reactions.


RESULTS
When copurified inhibitors were assessed using two different PCR reactions one reaction appeared to be inhibited whilst the other was not. Further experiments using various concentrations of unextracted urine to inhibit six different PCR reactions revealed that susceptibility to inhibition was highly variable between reactions. Similar results were obtained using EDTA as the PCR inhibitor. We could find no obvious explanation why one reaction should be more susceptible to inhibition than another, although a possible association with amplicon GC content was noted.


CONCLUSIONS
These findings have serious implications for all PCR-based gene expression studies, including the relatively new PCR array method, and for both qualitative and quantitative PCR-based molecular diagnostic assays, suggesting that careful consideration should be given to inhibition compatibility when conducting PCR analyses. We have demonstrated unequivocally that it is not safe to assume that different PCR reactions are equally susceptible to inhibition by substances co-purified in nucleic acid extracts.
Huggett
Jim F
JF
Centre for Infectious Diseases and International Health, Windeyer Institute for Medical Sciences, 46 Cleveland Street, University College London, London, W1T 4JF, UK. j.huggett@ucl.ac.uk
Novak
Tanya
T
Garson
Jeremy A
JA
Green
Clare
C
Morris-Jones
Stephen D
SD
Miller
Robert F
RF
Zumla
Alimuddin
A