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Real-time PCR (RT-PCR) is a special form of PCR, quantifying the amount of amplified DNA present after each round of PCR cycling via measuring fluorescence signals (Wilhelm J and Pingoud A, 2003). As the fluorescence increases proportional with the amount of DNA, after each cycle the amount of DNA can be detected in the exponential phase. Signals are either indicated by fluorescently tagged PCR primers or a DNA-intercalating fluorescent dye is added to the PCR mixture, of which the most popular is SYBR Green I binding to double- stranded DNA. A big drawback of the SYBR Green I method is the low specificity as no differentiation between PCR products can be achieved. Only when performing an additional melting point analysis after PCR, the fragment lengths and thus the specificity of the PCR products can be detected and authentic PCR products can be distinguished from occurring unspecific primer dimers. During a melting point analysis DNA is melted via raising the temperature continuously from 50°C to 95°C. At fragment-specific melting temperatures the double-stranded DNA molecules denature, whereas the fluorescence dye is released and a

decrease in fluorescence intensity is detected. As specific PCR products have a higher melting point as unspecific primer dimers, a differentiation is possible.

Real-time PCR using the Stratagene Mx 3000P thermocycler. Real-time PCR was applied on microdissected ancient bone samples of Egyptian mummy material. RT-PCR was performed to quantify the amount of extracted DNA originating from microdissected ancient bone samples of Egyptian mummy material. Real-time PCR was applied to sample mummy4, exemplary for all of four mummy samples, in a Stratagene RT-PCR cycler (Stratagene Mx 3000P, Stratagene, La Jolla, CA, USA) using the QuantiFast SYBR Green I PCR kit (QIAGEN GmbH, Hilden, Germany) for 2-step PCR according to the protocol recommended by the manufacturer. RT-PCR DNA amplification rates of sample mummy4, were compared to RT-PCR amplification rates of 1 ng/µl, 500 pg/µl, 100 pg/µl, 50 pg/µl and 20 pg/µl male and female starting DNA target material concentrations serving as reference probes and internal target amount standards. 1 µl of these standard concentrations was used for analysis, while just 0.5 µl of extracted mummy DNA was used due to the scarcity of mummy material. The mummy sample was filled up to 1 µl with sterile water (Ampuwa, Fresenius, Bad Homburg, Germany). In a 96-well plate (ABgene PCR Plates, Thermo Scientific, Epsom, Surrey, UK) 1 µl of DNA sample solution was mixed with 24 µl of RT-PCR master mix, containing 12.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x), 2.5 µl of 10 µM primer solutions Amel1 and Amel2 (final 1 µM per primer) and 6.5 µl of RNase-Free water. Primer sequences were listed in the appendix, chapter 9.2. 2-step PCR conditions were used according to the manufacturer’s protocol recommending 5 min initial denaturation at 95°C, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec and subsequent melting curve analysis. Conditions for generating these dissociation curves were 95°C for 1 min, 55°C for 30 sec, slowly ramping the temperature from 55°C to 95°C, and final denaturation time of 95°C for 30 sec. Data was obtained during ramping, while continuously fluorescence data was collected. Data analysis was performed via appropriate software for the Stratagene Mx 3000P “MxPro – Mx3000P v3.00” (Stratagene, La Jolla, CA, USA).

Real-time PCR using the lab-on-a-chip integrated Fluorescence Reader. The Fluorescence Reader module of the lab-on-a-chip system comprised a blue LED (λmax = 470±2 nm) for excitation light (LUXEON Rebel LXML-PB01-0023, 3.4 V forward bias, 0.7 A operating current), filter sets, a self-made LED power control box, a trigger signal break-out box (NI SCB-68 with the PCI ADC/DAC, Quick Reference Label, S-Series Devices, National Instruments Germany GmbH, Munich, Germany) and a CCD camera as detection device for

capturing emitted light (Rolera-XR, QImaging, Surrey BC, Canada). Filter sets included an excitation filter with λmax = 482 nm (spread 36 nm = 464-500 nm excitation spectrum) and an emission filter with λmax = 536 nm (spread 40 nm = 516-556 nm emission spectrum) (Interferenzfilter of BrightLine series, AHF Analysentechnik AG, Tübingen, Germany). For automatic picture taking, a self-programmed LabVIEW-based software was used “Grand_NIVision_Intensity_Consec_Subtract_Loopback_NewCamera.VI” (LabVIEW 8.6, National Instruments Germany GmbH, Munich, Germany). The software was adapted for taking pictures manually (named “Norbert.VI”). For real-time PCR operations, excitation and emission devices of the Fluorescence Reader were directed to reaction center B on the LOC chip surface.

Calibration of fluorescence intensities. The fluorescence signal was calibrated using the QuantiFast SYBR Green I PCR kit (QIAGEN GmbH, Hilden, Germany). Decreasing amounts of DNA were used to synthesize dilution series, whereas 0.1 µl of 10x concentrated DNA was mixed with 0.9 µl of 2x QuantiFast SYBR Green I PCR Master Mix. DNA concentrations of 100 ng/µl, 50 ng/µl, 10 ng/µl, 5 ng/µl and 1 ng/µl of male and female reference DNA were used, resulting in final concentrations of 10 ng, 5 ng, 1 ng, 500 pg and 100 pg present in prepared dilutions. 1 µl of each dilution was placed on reaction center B of a LOC chip, covered with 5 µl of Sealing Solution (Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany) and centered to the detection path of the CCD camera. This whole setup was darkened by capping it totally with a black cloth in order to exclude interfering ambient light. Pictures were taken manually via LabVIEW-based software “Norbert.VI”. Increasing exposure times were chosen starting with 200 ms, to 400 ms, 600 ms, 1000 ms, 2000 ms and 4000 ms. Measurements were performed at room temperature as well as at 55°C and 72°C. Measurements were repeated several times and pictures concerning fluorescence intensity were analyzed visually.

Experimental setups of performing real-time PCR. Real-time PCR was performed using the QuantiFast SYBR Green I PCR kit for 2-step PCR (QIAGEN GmbH, Hilden, Germany) according to the protocol recommended by the manufacturer, but reaction volumes were adapted to low-volume PCR applications. 1 µl total LV-PCR reaction mix contained 0.5 µl of 2x QuantiFast SYBR Green I PCR Master Mix (final 1x), 0.1 µl of 10 µM primer solutions β-Actin up and β-Actin down or Amel1 and Amel2 respectively (final 1 µM per primer), 0.2 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) and 0.1 µl of 10x concentrated input DNA (final 1x). Primer sequences were listed in the appendix,

5 ng/µl and 1 ng/µl were used, resulting in final concentrations of 1 ng, 500 pg and 100 pg present in 1 µl total reaction mix. 1 µl of prepared master mix was placed on reaction center B of a LOC chip, covered with 5 µl of Sealing Solution (Advalytix AG/Beckman Coulter Biomedical GmbH, Munich, Germany) and centered to the detection path of the CCD camera. This whole setup was darkened by capping it totally with a black cloth in order to exclude interfering ambient light. 2-step PCR cycling conditions recommended by the manufacturer’s protocol were slightly changed concerning temperature hold times: 5 min initial denaturation at 95°C, 40 cycles of 95°C for 30 sec and 60°C or 55°C respectively for 60 sec. Increasing fluorescence intensities were recorded by taking pictures at the end of each annealing and extension step at 55°C or 60°C during 35-45 cycles in total. Pictures were either taken manually via LabVIEW-based software “Norbert.VI” or automatically via LabVIEW-based software “Grand_NIVision_Intensity_Consec_Subtract_Loopback_NewCamera.VI”. Chosen exposure times chosen ranged from 200 ms, to 400 ms and 600 ms. PCR reactions were repeated several times and pictures concerning fluorescence intensity were analyzed visually. Real-time PCR was also performed using the QuantiTect SYBR Green I PCR kit for 3- step PCR (QIAGEN GmbH, Hilden, Germany) according to the protocol recommended by the manufacturer, but reaction volumes were adapted to low-volume PCR applications. 1 µl total LV-PCR reaction mix contained 0.5 µl of 2x QuantiTect SYBR Green I PCR Master Mix (final 1x), 0.1 µl of 10 µM primer solutions β-Actin up and β-Actin down or Amel1 and Amel2 respectively (final 1 µM per primer), 0.2 µl of sterile water (Ampuwa, Fresenius, Bad Homburg, Germany) and 0.1 µl of 10x concentrated input DNA (final 1x). 3-step PCR cycling conditions recommended by the manufacturer’s protocol were slightly changed concerning temperature hold times: 15 min initial denaturation at 95°C, 35-45 cycles of 95°C for 30 sec, 55°C for 60 sec, 72°C for 30 sec, and final product extension at 72°C for 7 min. Conditions for picture taking and real-time PCR performances were according to the 2-step PCR performance just described.