Ultrahigh Speed Direct PCR: A Method for Obtaining Y-STR and STR Based Genotypes in Under 20 Minutes

• Main Author: Gibson-Daw, Georgiana C
• Format: Others
• Published: FIU Digital Commons 2018
• Subjects: Chemistry; Physical Sciences and Mathematics
• Online Access: https://digitalcommons.fiu.edu/etd/3722; https://digitalcommons.fiu.edu/cgi/viewcontent.cgi?article=4698&context=etd

There are many situations in forensic DNA typing where the time it takes to identify an individual is critical. Examples include suspects under arrest and individuals detained at ports of entry. It is also important to identify victims quickly when dealing with mass disasters such as terrorist attacks, airplane crashes and natural disasters in order to notify families. The goal of this project was to develop an ultrafast method for screening saliva samples through the use of rapid direct PCR coupled to microfluidic separation and detection. Fast amplification was achieved through the use of high speed thermocyclers and the experimental optimization of PCR reactants and polymerases. High processivity mutant polymerases were tested at increased speeds along with PCR enhancers. Experimental optimizations and designs were used to ultimately achieve amplification of a 4 loci Y-STR multiplex in 12.5 minutes and a 7 loci STR multiplex in 6.5 minutes. The use of direct PCR not only decreases the overall time of sample processing through the elimination of the extraction, but also, by the removal of the extraction step cuts down on the possibility of contamination to the sample. By using inhibitor resistant fast polymerases such as Omnitaq, BSA and the PEC-1 enhancer, and optimizing cycling conditions, a direct PCR method was developed where amplification from diluted saliva was successfully achieved in 13.5 minutes. The products from these ultrafast amplifications can be coupled to a microfluidic chip for analysis. The electrophoretic system separates the PCR products and provides complete genotypes for both Y and autosomal STR multiplexes in 80 seconds. This permits complete sample processing in under 15 minutes for the rapid direct PCR protocol. This screening method can exclude individuals who do not match evidentiary material and test evidence to see if it is be viable for full analysis. The result of this work was a reliable and robust method for the rapid genotyping of forensic samples which can be used in situations where rapid single sample DNA analysis is necessary.