Anatomical and molecular insights into feasibility of large-amplicon PCR targeting MSH2 exon 12 in FFPE rectal tumors from Sri Lankan patients

Objective: To evaluate the feasibility of amplifying relatively a large (471 bp) MSH2 exon 12 PCR fragment from formalin-fixed paraffin-embedded (FFPE) rectal tumors, situating molecular feasibility within the context of rectal anatomy.
Methods: Archived FFPE rectal adenocarcinoma tissues (n = 12) and control rectal mucosa (n = 4) were analyzed. Tumour content was confirmed histologically, respecting anatomical boundaries of the rectum. DNA extraction was performed using a silica column-based method, and primer pairs specific for MSH2 exon 12 were optimized with gradient PCR. Strategies including increased MgCl₂, bovine serum albumin, DMSO, and pre-heating were applied to improve amplification from fragmented FFPE DNA. Amplicons were visualized via agarose gel electrophoresis, purified, and quantified.
Results: DNA yields varied widely (12.78–289.24 ng/µL), with most tumor samples showing adequate purity ratios. High-quality control DNA yielded consistent 471 bp products at annealing temperature of 64 °C. FFPE-derived DNA amplification was inconsistent, often producing faint or smeared products. Optimization improved yield for some samples but did not ensure reproducibility across all cases. Anatomical considerations including the rectum’s complex lymphatic drainage and mesorectal compartment were noted as potentially relevant to mutation patterns.
Conclusion: While the recovery of large-amplicon PCR products from FFPE rectal tumors remains challenging due to formalin-induced DNA damage, the study demonstrates methodological approaches to optimize yield in a real-world setting. Integrating molecular feasibility studies with rectal anatomical context may guide more precise surgical planning and targeted genetic screening in colorectal cancer management, particularly in resource-limited settings.