Optimization of PCR Conditions for BAT-25 and BAT-26 Amplification Using Commercial DNA Extraction Kits: A Methodological Approach from Sri Lanka.

Objective:Colorectal cancer (CRC) is the third most common cancer worldwide, with 7,694 CRC cases diagnosed in Sri Lanka between 2001 and 2010. The aim of this study was to optimize polymerase chain reaction (PCR) conditions for the amplification of two selected microsatellites, BAT-25 and BAT-26, using a commercial FFPE DNA extraction kit in a selected Sri Lankan population. This study integrates molecular analysis with anatomical pathology by utilizing FFPE colonic tissue, emphasizing the anatomical and histological integrity required for reliable DNA amplification.

Materials and Methods: Ten colorectal cancer patients and ten non-colorectal cancer controls were selected from the National Cancer Institute, Maharagama, and the Colombo South Teaching Hospital. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tissues using the QIAamp® DNA FFPE Extraction Kit. DNA quality and concentration were assessed using a NanoDrop spectrophotometer. PCR conditions for BAT-25, BAT-26, and the  β - actin housekeeping gene were optimized. Amplified products were analyzed by agarose gel electrophoresis on a 2% gel, and DNA bands were visualized under UV illumination.

Results: PCR conditions for BAT-25 and BAT-26 were successfully optimized using FFPE-derived DNA. Clear and specific amplification bands of expected sizes were obtained at an annealing temperature of 56 °C. No nonspecific amplification or primer-dimer formation was observed. The clarity and reproducibility of the bands confirmed the reliability of the optimized PCR conditions and the commercial DNA extraction kit.

Conclusion: Optimized PCR conditions for BAT-25 and BAT-26 using FFPE-derived DNA from colorectal cancer patients provided consistent and reproducible amplification of microsatellite markers in Sri Lankan laboratory settings.