Samples of saliva corresponding to 250,000; 125,000; 62,500; 12,500; 2500; 625; 125; 62; 32; 16; 8; and 4 epithelial cells were put into two series of tubes containing a cotton swab (SelefaTrade, Spånga, Sweden) used to swab 4 cm2 of clean, sterilized window glass. Cells were counted using a Bürker chamber (Hawksley, Sussex, UK). Sterile saline was used for dilution and moistening of the swabs. DNA was extracted using Chelex (14). One negative extraction control was added for each extraction batch. The extraction volume was 200 µL. A singleplex real-time PCR assay amplifying a 156-bp gene fragment was used (16,17). The polymerases investigated were AmpliTaq Gold (modified Taq), Bio-X-Act Short (undisclosed blend of enzymes) (Bioline, London, UK), ExTaq Hot Start (modified Taq) (Takara Bio Inc., Shiga, Japan), KAPA2G Robust (Taq mutant) (KAPA Biosystems, Cape Town, South Africa), OmniTaq (Taq mutant) (DNA Polymerase Technologies, St. Louis, MO, USA), PicoMaxx High Fidelity (a mixture of recombinant Taq and cloned Pyrococcus furiosus polymerase) (Stratagene, La Jolla, CA, USA), rTth (recombinant Tth) (Applied Biosystems), Taq, and Tth (both, Roche Diagnostics, Mannheim, Germany).
Each reaction contained 1 U polymerase, 1× polymerase-specific PCR buffer, 0.2 mM dNTP (Roche Diagnostics), 3.5 mM MgCl2 (in total), 0.5 µg/µL BSA (Roche Diagnostics), 0.3 µM forward primer (RB1 80 F), 0.3 µM reverse primer (RB1 235 R), and 0.2 µM TaqMan MGB probe (RB1 212 MGB, Fam-labeled). Autoclaved MilliQ water (Millipore) was added to a total master mix volume of 12 µL for each reaction. Eight microliters of DNA template was added, giving a final reaction volume of 20 µL. Negative amplification controls were used. Primers were purchased from MWG Biotech AG (Ebersberg, Germany) and the TaqMan MGB probe from Applied Biosystems. A LightCycler 2.0 (Roche Diagnostics) was used for thermal cycling and detection, using the following PCR program: 95°C for 1 min; 50 cycles of 95°C for 0 s, 60°C for 20 s, and 72°C for 20 s; and 40°C for 30 s. For Bio-X-Act Short, the extension temperature was 68°C. For AmpliTaq Gold and PicoMaxx High Fidelity, the initial 95°C step was lengthened to 10 min.
Results are given as crossing points (Cp)—that is, the fractional cycle number at which the second derivative of the amplification curve is a maximum. The two saliva dilution series were amplified in duplicate, giving four results for each level of dilution. Standard curves were prepared by plotting Cp values against the logarithm of the cell concentration (cells/µL) in the DNA template. The slope of the standard curve within the dynamic range of amplification was estimated using the regression tool in Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). The slope was used to calculate the PCR efficiency using the equation E = 10(−1/slope)−1. A slope of -3.32 gives the ideal efficiency of 1.0. The amplification efficiency was calculated for each of the four data sets. Two detection limits were defined: 100%, the lowest dilution giving positive results for all four replicates; and 50%, the lowest dilution giving positive results for two of the four replicates.STR analysis of inhibited crime scene saliva samples
Thirty-two crime scene saliva samples, previously giving no or incomplete DNA profiles, were amplified in duplicate using the AmpFlSTR SGM Plus primer set with each of the three best-performing DNA polymerase–buffer systems from the first study (i.e., Bio-X-Act Short, ExTaq Hot Start, and PicoMaxx High Fidelity), and AmpliTaq Gold as reference. Sixteen samples were extracts from cigarette butts, and 16 were extracts from cans, bottles, or foodstuffs collected with cotton swabs. For AmpliTaq Gold, the reaction mix was prepared according to the manufacturer's recommendations (AmpFlSTR SGM Plus PCR Amplification Kit User's Manual), and 10 µL DNA template was used with 15 µL mastermix in the PCR. For Bio-X-Act Short, ExTaq Hot Start, and PicoMaxx High Fidelity, reaction mixes were prepared using 2.5 U polymerase, 1× polymerase-specific PCR buffer, 0.2 mM dNTP, 0.25 µg/µL BSA, and 5.5 µL AmpFlSTR SGM Plus primer mix. For Bio-X-Act Short, 1 mM MgCl2 was added (ExTaq Hot Start and PicoMaxx High Fidelity buffers contain MgCl2). Autoclaved MilliQ water was added, giving a final master mix volume of 16.5 µL, of which 15 µL was used together with 10 µL DNA template in each reaction. Thermal cycling was performed on a GeneAmp PCR System 9700 (Applied Biosystems) using the standard PCR program (AmpFlSTR SGM Plus PCR Amplification Kit User's Manual), with the exception of Bio-X-Act Short, for which the following program was used: 94°C for 5 min; 28 cycles of 94°C for 60 s, 59°C for 60 s, and 68°C for 60 s; 68°C for 45 min; and store at 10°C. An ABI 3130xl Genetic Analyzer (Applied Biosystems) was used for fragment separation, and DNA profiles were evaluated using GeneMapper ID software, Version 3.1 (Applied Biosystems).