Development of a Microwave-based DNA Extraction Method to Increase the Success of Direct and Rapid PCR Technique

Authors

Fabiana Taglia, Florida International UniversityFollow

Document Type

Dissertation

Degree

Doctor of Philosophy (PhD)

Major/Program

Chemistry

First Advisor's Name

Bruce McCord

First Advisor's Committee Title

Committee chair

Second Advisor's Name

Steven B. Lee

Second Advisor's Committee Title

Committee member

Third Advisor's Name

Yuan Liu

Third Advisor's Committee Title

Committee member

Fourth Advisor's Name

Kathleen Rein

Fourth Advisor's Committee Title

Committee member

Fifth Advisor's Name

Brian Young

Fifth Advisor's Committee Title

Committee member

Sixth Advisor's Name

Jeffrey Wells

Sixth Advisor's Committee Title

Committee member

Keywords

Forensic Genetics, DNA typing, DNA extraction

Date of Defense

3-30-2022

Abstract

The goal of this project was to develop a fast, microwave-based extraction technique that could be employed for direct and rapid DNA analysis. The hypothesis was that the use of a microwave could increase the yield of DNA by opening the cell membrane, rendering the DNA available without the use of any other chemical treatment, and improving results from very low quantity samples.

At present rapid human DNA analysis is mainly restricted to genotyping saliva and sometimes blood samples. We hypothesized that microwave processing could expand the types of samples assessable to these procedures and increase sensitivity.

There were two different microwave ovens employed: a commercial one and a computer-driven microwave. The samples came from three different body fluids: saliva, semen, and blood. Control samples were prepared using the same samples that were not subjected to microwave extraction. The quality and quantity of DNA profiles obtained from these experiments were assessed using a novel PCR-based analysis, a homemade multiplex consisting of 9 simultaneously amplified mini STR loci. A direct PCR method was developed to analyze samples rapidly with minimal pretreatment and no extraction. Once optimized, this procedure was developed for touch DNA samples and mixtures. The optimized microwave protocols were also assessed using a completely automated commercial rapid DNA instrument. We hypothesized that preprocessing by microwave digestion could widen the application of these commercial systems, which are generally designed to analyze oral swabs.

The evaluation of the methodologies was performed using statistical tests (ANOVA or t-test) on allele call rates, peak heights, and allelic concordance for all loci. Overall, the use of a microwave coupled with rapid direct PCR represents a valuable addition to an analyst’s toolkit, increasing the DNA yield as well as the speed of the experiments. Moreover, it is less expensive and less toxic than other pretreatments making it an excellent procedure for onsite analysis.

This project was supported by the Irregular Warfare Technical Support Directorate (IWTSD, formerly CTTSO) of the US Department of Defense. The research started in October 2019 and was completed in May 2021

Identifier

FIDC010677

Creative Commons License

This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Recommended Citation

Taglia, Fabiana, "Development of a Microwave-based DNA Extraction Method to Increase the Success of Direct and Rapid PCR Technique" (2022). FIU Electronic Theses and Dissertations. 4977.
https://digitalcommons.fiu.edu/etd/4977