OPTIMIZING DNA EXTRACTION METHODS FOR MAJOR HISTOCOMPATIBILITY COMPLEX GENE STUDIES IN BLACK FRANCOLIN (Francolinus francolinus) USING SOFT TISSUE SAMPLES

Background: Robust immunogenetic profiling of wild birds requires high-quality genomic DNA (gDNA) isolated from multiple tissue types. The Major Histocompatibility Complex (MHC) serves as a key immunogenetic indicator for evaluating adaptive capacity and population health in birds. However, successful multi-locus polymerase chain reaction (PCR) and high-throughput sequencing of these highly variable genes depend critically on the quantity and purity of extracted DNA. To date, there is a lack of literature on DNA extraction from Black francolin (Francolinus francolinus), as well as information about the comparison of different extraction methods using different tissue types. Hence, this study evaluated three DNA extraction techniques (Phenol-Chloroform-Isoamyl Alcohol extraction, PCI; Silica-Membrane Spin-Column purification, SC; Magnetic Bead-based isolation, MB) using skeletal and cardiac muscle tissues from the Black francolin. 
Methods: DNA yield and quality were assessed via spectrophotometry, agarose gel electrophoresis, and functional validation through PCR amplification of MHC Class II exon 2. An assessment was conducted on the hands-on time and cost-effectiveness of each protocol, and the degree of DNA degradation was also assessed using the TaqMan PCR method.
Results: The PCI yielded a high DNA concentration. However, spectrophotometric and PCR analyses showed moderate DNA quality. The SC produced the best DNA purity with a moderate cost-benefit ratio and low time consumption in comparison to other extraction methods. PCR amplification of MHC Class II exon 2 from skeletal muscle was specific and reproducible, with no primer-dimers or non-specific amplification, across all extraction methods. Despite lower yields, SC and MB methods produced sharp ~270 bp amplicons comparable to PCI, indicating that DNA integrity rather than quantity determines successful amplification. 
Conclusions: The SC extraction technique was identified as the most effective DNA extraction approach for eliminating myofibrillar proteins and PCR inhibitors, making it superior for accurate MHC genotyping in the Black francolin.