Environmental Monitoring & Assessment Flashcards

Which room arrangement best minimizes amplicon contamination in a PCR facility?

Answer: Separate reagent prep, sample prep, and amplification/analysis areas with one-way flow

Physical separation with unidirectional movement keeps amplified product from reaching clean areas.

For air pressure control, the pre-PCR (reagent preparation) room should ideally be:

Answer: At positive pressure relative to adjacent areas

Positive pressure pushes air out of the clean room, which keeps airborne amplicons from coming in.

UV irradiation in a PCR workstation decontaminates mainly by:

Answer: Forming pyrimidine dimers that block polymerase extension

UV-C causes thymine dimers and other crosslinks that keep DNA from serving as a template.

UV decontamination is LEAST effective against which type of contaminant?

Answer: Short amplicons (<200 bp), especially GC-rich ones

Short and GC-rich fragments have fewer adjacent pyrimidines, so UV damages them less often.

The dUTP/uracil-N-glycosylase (UNG) system prevents carryover contamination by:

Answer: Excising uracil from previous dU-containing amplicons before the new amplification

UNG removes uracil from earlier dUTP-made products, leaving abasic sites that break during heating.

Why must UNG be heat-inactivated at the start of thermal cycling?

Answer: To prevent it from degrading newly synthesized dU-containing amplicons

Active UNG would destroy the new products, which also contain uracil.

How often should a lab review trends in its environmental monitoring and NTC data?

Answer: On a scheduled, documented basis, with action and alert limits

Regular trend review against set limits catches contamination early, before it affects results.