Objectives: 1) To shed light on the perception and practices of certified lab professionals regarding PCR protocol implementation. 2) To decipher the association of perception with implementation of PCR protocol. 3) To provide a review of the steps of qualitative PCR based on the current guidelines.

Study design: Cross-sectional survey based study (KAP)

Settings: Non-probability convenience sampling was used to collect data from qualified lab professionals working at various Karachi-based diagnostic / research facilities equipped with PCR.

Materials and Methods: The method of data collection was mixed (quantitative and qualitative). A self-structured questionnaire comprising 18 questions on PCR thermal cycles, DNA polymerase, PCR primers, DNA extraction methods, gel electrophoresis and lab environment etc. was disseminated among participants. Measure of central tendency, frequency distribution, chi-square test of independence and thematic analysis were performed.

Results: The mean temperatures chosen for annealing, denaturation and extension phases of PCR cycle were 55°C, 93°C and 71°C.  The mean thickness of agarose gel and the mean size of DNA ladder chosen for gel electrophoresis was 4 mm and 127 bp. PCR performance showed a statistically significant association with the decision to aim for DNA high purity standards (p 0.008). The decision to follow the manufacturer's protocol had a very significant association with the decision to alter cycle temperature (p 0.001). Thematic analysis revealed the hindrances in construction of a PCR based laboratory, the preferred manufacturers of PCR kit, and the preferred lab sterility methods.

Conclusion: We discerned the preferences and practices of lab professionals for PCR. We also elucidated the working of qualitative PCR in light of internationally accepted guidelines.

Keywords: Base Composition, DNA, Complementary, DNA Primers, Laboratories, Nucleic Acid Denaturation, Polymerase Chain Reaction.