Assay for numerical and graphical detection of Native American mitochondrial haplogroup B with real-time PCR

Mitochondrial haplogroup B is characterized by a 9-base-pair deletion (CCCCCTCTA) located between nucleotide positions 8,272 to 8,289 of the human mitochondrial genome. Conventional molecular approaches for detecting this deletion include PCR amplification followed by polyacrylamide gel electrophoresis, or PCR coupled with capillary electrophoresis. In this study, we propose a numerical and graphical method based on real-time PCR that combines melting temperature (Tm) determination with melting-curve analysis to distinguish between haplogroup B and non-B individuals. Using 1 ng of DNA, samples belonging to haplogroup B exhibited a mean theoretical Tm of 76.1550 °C, whereas non-B sample exhibited a mean Tm of 76.8250 °C. Across different DNA concentrations, the mean experimental Tm of haplogroup B samples differed from that of non-B samples by 0.7258 ± 0.0526 °C. Statistical analysis identified the optimal DNA concentration range for this technique as 0.7-2 ng per real-time PCR reaction. These observations indicate that 0.5-1 ng of DNA per assay yield consistent and reliable results. It is recommended that each assay include minimum one pair of haplogroup B and non-B samples as controls. The proposed real-time PCR method enables accurate identification of the 9-base-pair deletion characteristics of haplogroup (macrogroup) B, while posing lower health risks, requiring less analysis time and fewer resources than conventional techniques. This approach is applicable in evolutionary, clinical, and forensic investigations.