Abstract Title

Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assualt Kit Swabs

Presenter Name

Francine Brignac

RAD Assignment Number

1307

Abstract

Sexual assault kits comprise 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming, and requires the use a dangerous chemical known as Phenol:Chloroform:Isoamyl Alcohol (PCIA). The purpose of this study is to assess the relative efficacy of the PCIA method when compared to three other currently available differential extraction methods.

A single male volunteer and a single female volunteer donated semen and saliva, respectively. Aliquots of semen were serially diluted such that three decreasing concentrations of semen could be assessed alongside a consistent concentration of saliva. From these three different mixtures, swabs were made and allowed to dry in a 37 °C drying oven for two weeks, then at room temperature for an additional four weeks in order to simulate aged samples. Three days prior to DNA extraction and purification, another set of swabs were created to simulate fresh samples. The aged and unaged samples were tested in triplicate for each of the four extraction methods.

The methods to be compared include two manual and two automated methods. The manual methods include the standard differential (SD) and the Lounsbury Method, which is a modified version of the SD. The two automated methods include the AutoMate ExpressTM DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA), and a method employing the use of two of Qiagen’s DNA platforms: the QIAcube and the Qiagen EZ1® Advanced XL (Qiagen®, Hilden, Germany).

Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an all around great choice for labs. All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA), and RStudio® (RStudio® Inc., Boston, MA).

Presentation Type

Poster

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Comparison of Four Differential DNA Extraction Methods for Casework Analysis of Sexual Assualt Kit Swabs

Sexual assault kits comprise 40-50% of a typical Forensic Laboratory caseload. The traditional method to process these samples is time-consuming, and requires the use a dangerous chemical known as Phenol:Chloroform:Isoamyl Alcohol (PCIA). The purpose of this study is to assess the relative efficacy of the PCIA method when compared to three other currently available differential extraction methods.

A single male volunteer and a single female volunteer donated semen and saliva, respectively. Aliquots of semen were serially diluted such that three decreasing concentrations of semen could be assessed alongside a consistent concentration of saliva. From these three different mixtures, swabs were made and allowed to dry in a 37 °C drying oven for two weeks, then at room temperature for an additional four weeks in order to simulate aged samples. Three days prior to DNA extraction and purification, another set of swabs were created to simulate fresh samples. The aged and unaged samples were tested in triplicate for each of the four extraction methods.

The methods to be compared include two manual and two automated methods. The manual methods include the standard differential (SD) and the Lounsbury Method, which is a modified version of the SD. The two automated methods include the AutoMate ExpressTM DNA Extraction System (ThermoFisher Scientific, Carlsbad, CA), and a method employing the use of two of Qiagen’s DNA platforms: the QIAcube and the Qiagen EZ1® Advanced XL (Qiagen®, Hilden, Germany).

Results indicate that as sperm sample concentration decreases, automated methods produce superior results both in DNA quantity obtained and in quality of STR profiles produced. Automated methods reduce hands-on time, facilitate higher through-put of samples, and reduce analyst contact with hazardous chemicals such as PCIA, making it an all around great choice for labs. All Real-Time PCR and electropherogram data were analyzed using Microsoft® Excel (Microsoft® Corp., Redmond, WA), and RStudio® (RStudio® Inc., Boston, MA).