PCR in Forensic Science: A Critical Review

Abstract

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.

Physical Simulation-Based Calibration for Quantitative Real-Time PCR

“…After running the qPCR process, the amplification curves obtained are shown in Figure 16. The uniformity of Ct values (𝛥𝐶 ) can be calculated according to Equation (4), and the precision of Ct values (𝑅𝑆𝐷 ) can be calculated according to Equation (5). The results of the uniformity test are presented in Table 8.…”

“…Fluorescence quantitative PCR (qPCR) instruments are utilized for various nucleic acid detection and gene analysis characterized by the polymerase chain reaction (PCR) technique, aimed at detecting DNA/RNA with extensive applications in clinical diagnostics [1][2][3], animal quarantine [4], forensic identification [5], adulteration identification and species identification [6][7][8], in vitro diagnostics [9], and pharmaceutical research and development [10]. Particularly, amidst the global outbreak of the COVID-19 pandemic, qPCR method has emerged as the gold standard [11] for nucleic acid detection, leading to a larger-scale deployment of qPCR instruments in the field of molecular biology.…”

Looking into the Quantification of Forensic Samples with Real-Time PCR

“…In this work, we aim to revisit in a detailed manner the phase through which the qualitative and quantitative presence of human DNA is verified in extracts from forensic samples. In every accredited forensic laboratory, the DNA present in a sample must be quantified before it progresses to STR PCR for DNA profiling [ 2 ]. The importance of this analytical phase is underscored by the early guidelines of the forensic community.…”

“…The amount of fluorescence detected correlates directly with the amount of human DNA present in the reaction, as the greater the amount of DNA, the more reporter dye molecules are cleaved, and the greater the fluorescence detected. Typically, as the qPCR program progresses, and the DNA increases exponentially, an amplification curve is generated using these fluorescence data, which (at the end of the qPCR program) are then used to calculate a DNA concentration [ 2 ]. Measured DNA concentrations are used to normalise the amount of DNA template to an optimal range for STR amplification reactions, ensuring STR profiles are in the linear range of the capillary electrophoresis instrument with minimal artefacts and stochastic effects.…”