An improved method of elimination of DNA from PCR reagents.
OBJECTIVE
The presence of exogenous DNA in commercially available polymerase chain reaction (PCR) reagent preparations is a serious problem when amplifying conserved regions of bacteria. The preferred and currently in-use method of decontamination using 8-methoxypsoralen (8-MOP) and UVA requires re-standardization of decontamination with increasing concentrations of 8-MOP and UVA irradiation timings, if the DNA load of reagents is high due to lot-to-lot differences. The objective of this study was to develop a decontamination method, which would (i) work at the minimum reported concentration of 8-MOP and UVA irridation timings; and (ii) take care of inter-batch DNA-load variability of reagents.


METHODS
The improved method described here was formulated after studying the exact molecular mechanism of action of 8-MOP with DNA. The successful working of the method was experimentally proven and validated with 6-7 new batches of PCR reagents. The sensitivity of eubacterial PCR, after using the new method of decontamination, to be used clinically was checked with both the spiked specimens and the actual clinical specimens.


CONCLUSIONS
The new method was found to work at the same starting parameters of 8-MOP and UVA in such situations. The increased efficiency of this method was found to be due to the synergistic effect of both the selective treatment of Taq DNA polymerase and the split-irradiation approach.
Rowther
Farjana B
FB
Research laboratories, P.D. Hinduja National Hospital and MRC, Mumbai, India.
Rodrigues
Camilla
C
Mehta
Ajita P
AP
Deshmukh
Minal S
MS
Kapadia
Farhad N
FN
Hegde
Ashit
A
Joshi
Vinay R
VR
D001419:Bacteria / Q000235:genetics; D004247:DNA / Q000097:blood / Q000235:genetics / Q000302:isolation & purification* / Q000528:radiation effects; D006801:Humans; D007202:Indicators and Reagents; D008730:Methoxsalen; D016133:Polymerase Chain Reaction / Q000379:methods*; D012016:Reference Values; D018805:Sepsis / Q000097:blood; D014466:Ultraviolet Rays
D007202:Indicators and Reagents; D004247:DNA; D008730:Methoxsalen