Towards a smart PCR process

• Published in: Australian journal of forensic sciences Vol. 57; no. 2; pp. 220 - 231
• Main Authors: McDonald, Caitlin, Taylor, Duncan, Linacre, Adrian
• Format: Journal Article
• Language: English
• Published: Sydney Taylor & Francis 04.03.2025; Australian Academy of Forensic Sciences; Copyright Agency Limited (Distributor)
• Subjects: Analysis; Annealing; Denaturation; Deoxyribonucleic acid; DNA; DNA amplification; DNA fingerprinting; Forensic sciences; Likelihood ratio; Parameter modification; Polymerase chain reaction; Real time; STR amplification; AUSTRALIA
• ISSN: 0045-0618; 1834-562X
• DOI: 10.1080/00450618.2024.2309924

PCR has transformed DNA profiling, providing the opportunity to analyse trace DNA. Trace DNA often contains degraded DNA, such that many samples generate no meaningful STR data. The PCR process has changed little since first use despite the drive for greater sensitivity: the same steps of denaturation, annealing and extension used in the first STR amplifications are recognizable in current processes. Our aim is to move towards a smart PCR that can monitor amplification and amend cycling conditions. To achieve this, DNA amplification needs to be performed on a real-time PCR machine. Initially, this was the adjustment of the time at the denaturation and annealing steps. Standard STR profiling was performed using the GlobalFiler TM kit on an open-source qPCR machine and reduced in step changes the denaturation time to 2 s from 10 and time of annealing from 90 s to 30. The comparison of the STR profiles resulting from the modified amplification parameter to those of the control set indicated a loss of allelic amplification although the resulting likelihood ratios were extremely high. We identify the groundwork required to attain the ambitious goal of creating a smart PCR system that can respond in real time.