Comprehensive review and meta-analysis of magnesium chloride optimization in PCR: Investigating concentration effects on reaction efficiency and template specificity

Optimizing the polymerase chain reaction (PCR) continues to be a major challenge in molecular biology, and obtaining the correct magnesium chloride (MgCl₂) concentration is key to a successful reaction. A clear understanding of how MgCl₂ affects PCR thermodynamics and kinetics is crucial for creating efficient and reliable protocols that work consistently. A systematic meta-analysis was conducted of 61 peer-reviewed studies published between 1973 and 2024. The study selection adhered to rigorous PICOS criteria, prioritizing experimental investigations that specifically examined the effects of magnesium chloride (MgCl₂) on key PCR parameters. Data extraction and subsequent analyses were performed using standardized methodologies, with particular emphasis on template characteristics, reaction conditions, and their interplay in influencing PCR efficiency and specificity. The analysis showed a strong logarithmic relationship between MgCl₂ concentration and DNA melting temperature, with an optimal ranges of 1.5 and 3.0 mM. Every 0.5 mM increase in MgCl₂ within this range was associated with a 1.2 °C increase in melting temperature. Template complexity significantly affected the optimal MgCl₂ requirements, with genomic DNA templates requiring higher concentrations than the more straightforward templates. This meta-analysis offers quantitative insights and evidence-based guidelines for optimizing magnesium chloride (MgCl₂) concentration using polymerase chain reaction (PCR). These results demonstrate that the precise modulation of MgCl₂ concentration, tailored to specific template characteristics, can significantly improve both the efficiency and specificity of PCR. These findings provide a robust theoretical framework for the development of template-specific optimization strategies and advance the design of more reliable and effective PCR protocols.