Profound inhibition of the PCR step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes.
Supplies, such as bags of plastic reaction tubes, are sometimes left in the laminar flow hoods unintentionally while the ultraviolet (UV) lamp is illuminated overnight. In addition, UV irradiation is used for sterilization and amplicon inactivation to avoid contamination. The oligonucleotide ligation assay (OLA) is a unique approach to mutation detection of point mutations, small deletions, and small insertions. Recently, we encountered problems with this assay and peak heights were much lower or disappeared. After going through systemic trouble-shooting, we found that profound inhibition of the polymerase chain reaction (PCR) step of CF V3 multiplex PCR/OLA assay by the use of UV-irradiated plastic reaction tubes. When UV-irradiated tubes used throughout the assay, tubes exposed for 8 weeks at 0.7 m from the UV source gave a reduction of 60% and 67% in the assay products on the basis of sum of peak heights. Tubes exposed for 3 weeks at 0.1 m from the UV source totally eliminated assay product yielding no peaks. Further experiments showed that the inhibition happened mostly in the PCR step. Burgess and Hall had reported that inhibition of PCR of human glyceraldehydes-3-phosphate dehydrogenase transcripts after UV irradiating the tubes. This showed that the inhibition was not assay-specific. The reason that the inhibition of PCR was more profound could be due to a multiplex PCR assay and small reaction volume. The mechanism of PCR inhibition by UV irradiation is not clear. In conclusion, plastic reaction tubes intended for PCR/OLA assays should not be exposed to UV.
Fox
David H
DH
Molecular Pathology Laboratory, Department of Pathology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA.
Huang
Chih-Kang
CK
Du
Juan
J
Chang
Tylis Y
TY
Pan
Qiulu
Q
D016477:Artifacts; D004247:DNA / Q000528:radiation effects*; D006801:Humans; D015345:Oligonucleotide Probes / Q000528:radiation effects*; D010969:Plastics / Q000528:radiation effects*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000528:radiation effects*; D014466:Ultraviolet Rays
D015345:Oligonucleotide Probes; D010969:Plastics; D004247:DNA