DNA profiling is an effective method of identifying individuals in forensic casework. In sexual assault cases in particular, male donor DNA can be genotyped using Y chromosome short tandem repeat (Y-STR) analysis even in the presence of significant levels of female DNA. The recently released recommended national standard for sexual assault evidence collection from the victim is within 5 days after the sexual assault, with the probability of obtaining a male DNA profile diminishing significantly thereafter due to degradation and/or loss of sperm. The present study was designed to develop a method of obtaining a highly probative DNA profile with minute amounts of input male DNA through nested PCR. In this method, the DNA is first pre-amplified with custom designed primers, purified, and then re-amplified with a commercial secondary kit to amplify the pre-amplified products. In this way, trace amounts of male DNA are able to be amplified robustly, and the sensitivity (i.e. limit of detection) is greatly improved over a single amplification. Past studies conducted by the UCF NCFS Biology laboratory developed this method for the AmpFlSTR® Yfiler™ kit, which included 17 loci. This study aimed to improve the utility of the method by increasing the pre-amplified loci to include the Yfiler™ Plus PCR Amplification kit, which comprises 27 loci. The best kit to use for the pre-amplification was empirically determined, followed by optimization of the pre-amplification reaction via changes to primer concentrations and PCR cycling parameters. To verify that only male DNA was amplified, the new nested PCR method was tested with trace male DNA, female DNA, various mixture sets, environmentally compromised samples, and touch DNA. Using what is now termed the Y targeted pre-amplification (YTPA-v2) method, discriminatory Y-STR profiles were successfully recovered from as little as 5 pg (i.e. < 1 human diploid cell equivalent) of male DNA, even in the presence of male DNA from a different contributor or large quantities (up to 100,000 fold excess, i.e. 500 ng) of female DNA.


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Graduation Date





Ballantyne, John


Master of Science (M.S.)


College of Sciences



Degree Program

Forensic Science




CFE0008189; DP0023543





Release Date

August 2021

Length of Campus-only Access

1 year

Access Status

Masters Thesis (Open Access)

Included in

Chemistry Commons