Optimization of PCR Conditions for Improved Amplification Efficiency and Specificity on PfHRP2/3 Genes Deletion in Plasmodium falciparum
Yusuf A.M.*
Dept. of Biology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
Umar A.M.
Dept. of Biological Sciences, Faculty of Life Sciences, Federal University Dutsinma, Katsina State (PMB 5001), Nigeria
Eberemu N.C.
Dept. of Biological Sciences, Faculty of Life Sciences, Federal University Dutsinma, Katsina State (PMB 5001), Nigeria
Auta T.
Dept. of Biological Sciences, Faculty of Life Sciences, Federal University Dutsinma, Katsina State (PMB 5001), Nigeria
Wagini N.H.
Dept. of Biology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
Gidado S.M.
Dept. of Biology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
Zainab A. Yar’adua
Dept. of Biology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
Matazu N.U.
Dept. of Biochemistry, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
Suleiman M.
Dept. of Biology, Faculty of Natural and Applied Sciences, Umaru Musa Yar’adua University Katsina, Katsina State (PMB 2218), Nigeria
DOI: https://doi.org/10.54083/BioResToday/6.6.2024/327-335
Keywords: Annealing temperature, DNA template dilution, HotStart PCR, MgCl2 titration, PCR optimization, Primer dimer
Abstract
This study aimed to optimize PCR conditions for improved specificity and sensitivity by investigating the effects of DNA template dilutions, magnesium chloride (MgCl2) titration concentrations, annealing temperatures and primer dimer formation. Real-Time PCR (RT-PCR) assays were conducted and agarose gel electrophoresis and polyacrylamide gel electrophoresis were employed for visualization and analysis of PCR products. For DNA template dilutions, RT-PCR revealed a concentration-dependent decrease in cycle threshold (CT) values, indicating higher initial copy numbers in undiluted DNA samples. Melting curve analysis confirmed single-species template DNA, while agarose gel electrophoresis demonstrated decreasing band intensities with dilution, alongside nonspecific amplification products in all samples. MgCl2 titration concentrations showed optimal amplification at 1.5 mMol and 2 mMol, with weaker amplification at lower concentrations and nonspecific products at higher concentrations. Annealing temperature optimization revealed optimal efficiency at 58 °C and 55 °C, with reduced amplification at extremes and nonspecific products at higher temperatures. Primer dimer formation was observed, affecting amplification specificity, with the lower peak denaturing at a higher temperature indicative of primer dimerisation. Optimization strategies such as HotStart PCR and the use of monoclonal antibodies for Taq polymerase inhibition were discussed for improved specificity. Overall, systematic optimization of PCR conditions is crucial for achieving reliable and reproducible results, with considerations for DNA template concentration, MgCl2 titration, annealing temperature and primer dimer formation essential for enhancing PCR performance.
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