3. After incubation, centrifuge the column assembly to separate the swab cutting from the lysed cells.
4. Add 4 µL glacial acetic acid to the lysed cells in NaOH to neutralize.
5. Perform a 1/5 dilution of the neutralized sample by adding 20 µL sample to 80 µL low Tris-EDTA (TE). Alternatively, 400 µL of the low TE can be added directly to the neutralized sample.
6. Remove 2 µL of the diluted sample and add to Quantifi ler Trio real-time PCR reaction. Use the standard protocol for the Quantifi ler Trio kit.
Proof of concept for Y-Screen assay Mock samples were created by making serial dilutions of either male saliva or seminal fl uid and adding 50 µL of sample to female buccal swabs (see Table 1). A small cutting taken from each swab was processed with the Y-Screen assay. The remainder of the swab was processed with a Maxwell 16 nucleic acid extraction instrument (Promega Corp., Fitchburg, Wis.). Purifi ed samples were quantifi ed and genotyped with an Applied Biosystems AmpFlSTR YFiler assay and/or AmpFlSTR Identifi ler Plus assay.
As shown in Table 1, with 50 µL neat saliva from a male donor added to a buccal swab from a female donor, the assay was positive for both the small autosomal target and the Y target. Post-extraction, the purifi ed DNA was quantifi ed at 14.5 ng/µL with a male:female ratio of 1:1.4. The resulting Identifi ler Plus STR profi le (Figure 1) shows the expected mixture profi le. As the saliva input dilution decreased, the amount of male DNA decreased, as expected. With the 1:1000 dilution, the Y-Screen assay was still positive with subpicogram levels of male DNA.
After DNA extraction, 1.6 pg/ µL male DNA was recovered and no alleles were detected with the YFiler assay (Figure 2). For the dilutions to 1:50,000, no male DNA was obtained with the Y-Screen assay and no information was obtained with STR following purifi cation. In all mock samples tested, the Y-Screen assay was predictive of downstream results; a positive male quantifi cation result with Y-Screen correlated with a positive male quan- tifi cation result post-extraction.
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Figure 2 – YFiler results for purifi ed DNA from a buccal sample from a female donor containing an equivalent of 0.05 µL of saliva sample from a male donor.
AMERICAN LABORATORY • 43 • AUGUST 2015
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