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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.

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