An innovative transfer DNA experimental design and qPCR assay: Protocol and pilot study

Abstract

Forensic “touch” DNA samples are low‐quantity samples that are recovered from surfaces that have been touched by single or multiple individuals. These samples can include DNA from primary contributors who directly touched the surface, as well as secondary contributors whose DNA was transferred to the surface through an intermediary. It is difficult to determine the type of transfer, or how often and under what conditions DNA transfer occurs. In this paper, we present an innovative protocol that combines (1) a paired male and female transfer DNA experimental design in which the presence of male DNA indicates secondary transfer and (2) a cost‐effective quantitative PCR (qPCR) assay of a sex‐specific region in the Amelogenin gene to detect male and female DNA. We evaluate the ability of the Amelogenin qPCR assay to detect low concentrations of male and female DNA in mixed samples. We also test experimental DNA samples using our transfer DNA protocol to differentiate primary and secondary DNA transfer. Male DNA was detected in the majority of known mixed samples, even in samples with 4× more female DNA—this result demonstrates the ability to detect low concentrations of male DNA and the presence of secondary transfer DNA in our experimental design. Primary DNA transfer was detected in 100% of our experimental trials and secondary DNA transfer was detected in 37.5% of trials. Our innovative protocol mimics realistic case scenarios to establish rates of primary and secondary DNA transfer in an inexpensive and simplified manner.

An innovative transfer DNA experimental design and qPCR assay to identify primary, secondary, and tertiary DNA transfer

“…This study utilized the innovative transfer experimental design and Amelogenin qPCR assay developed and tested in McCrane and Mulligan [33] to detect secondary and tertiary DNA transfer on an expanded sample. We identified substantial levels of secondary (50%) and tertiary (27%) DNA transfer across two trials, demonstrating the common occurrence of this phenomenon.…”

“…Biological sex, age, self‐identified ethnicity, and any skin conditions affecting the hands were recorded for all participants. The AR‐15 pistol grip (Hogue AR‐15 pistol grip from http://brownells.com) and a standard ceramic coffee mug were sterilized by complete submersion in a freshly diluted 10% sodium hypochlorite (bleach) solution for 15 min, rinsed with DNA grade sterile water for several minutes, and left to air dry in an ancient DNA (aDNA) clean laboratory per recommendations [33, 34]. The pistol grip and coffee mug were randomly swabbed six times each after sterilization to serve as negative sample controls; all 12 negative control swabs were tested with the same qPCR protocol as experimental samples and showed no DNA presence, demonstrating that the sterilization procedure adequately removed any existing DNA prior to experimental trials.…”

“…All extraction blanks were tested twice for contamination and all extraction blanks were negative. [33], qPCR was performed using the C_990000001_10 Amelogenin (Sex Determination) Assay by Thermofisher (Waltham, Massachusetts), in accordance with manufacturer-recommended protocols. In this assay, the sexspecific polymorphic regions were labeled with fluorescent dyes FAM (Y chromosome sequence) and VIC (X chromosome sequence).…”

“…A total of 22 experimental trials were conducted, consisting of 44 participants (22 males and 22 females). Experimental trials followed the transfer DNA experimental design tested in McCrane and Mulligan [33], including the optional additional steps needed to test for tertiary DNA transfer (Figure 1). More specifically, in each trial, a biological male picked up and held a sterilized AR-15 pistol grip for 30 seconds and then put it down on a sterilized benchtop surface standing on its base.…”

“…In this study, we utilize an innovative approach to generate data on the frequency and conditions under which primary, secondary, and tertiary DNA transfer occurs [33]. In contrast to existing protocols that often utilize STR amplification to match samples to specific individuals, this protocol combines (1) a paired male and female transfer DNA experimental design in which male DNA indicates secondary or tertiary DNA transfer with (2) an Amelogenin quantitative PCR (qPCR) assay to detect male and female DNA.…”