**2.1 Bone pre-processing**

The collected bone remains were washed using a rough part of a sterilized and UV irradiated dish sponge, and then washed three times in sterile bi-distilled water (Merck Millipore, USA), using a mild detergent (few ml of detergent, measuring up to 5% of the water). The washed bone fragments were left overnight to dry. After this, the bone powder was obtained by grinded the bones in small pieces at 30 Hz for 1–2 minutes, using the Bead Beater Mill Mix 20 (Tehtnica Domel, Slovenia).

**Figure 2.** *Skull recovered from the crime scene.*

*Genetic DNA Identification from Bone Remains in Kinship Analysis Using Automate… DOI: http://dx.doi.org/10.5772/intechopen.99587*

**Figure 3.** *Canine tooth after cleaning.*

After pre-processing, the Bone DNA Extraction Kit, Custom (Promega, USA) was used to perform the demineralization buffer, by following the FSC DNA IQ Maxwell protocol. 100 mg of pulverized bone powder was weighed on an electronic balance and transferred into 1.5 mL Eppendorf tubes (Eppendorf, Germany). The kit was used for the demineralization of the tooth and the rest of the bone remains, by preparing two cocktails lysis, A and B (**Figure 4**).

First, we prepared the bone lysis cocktail A in the following manner: 400 μL of demineralization buffer, 40 μL of proteinase K and 10 μL of 1-thyoglicerol were mixed by vortex for 10 seconds. After this, 400 μL of bone lysis cocktail A was transferred into a new, sterile 1.5 mL Eppendorf tube (Eppendorf, Germany). As per protocol, the tube was left to incubate in the Eppendorf thermomixer C (Eppendorf, Germany) for 2.5 hours, with intermittent shaking at 1000 rpm. Following this, the sample was centrifugated at 13.300 rpm for 5 minutes, after which the supernatant was transferred into another clean, sterile 1.5 mL Eppendorf tube.

Next, we prepared bone lysis cocktail B in the following manner: In a clean, sterile 1.5 mL Eppendorf tube, 990 μL lysis buffer was added to 10 μL 1-thyoglicerol. From the bone lysis cocktail B, 800 μL was transferred into another clean, sterile 1.5 mL Eppendorf tube and vortexed for 10 seconds to ensure proper mixing.

The entire volume of the previously obtained eluate was added to well 1 of the Maxwell 48 RSC Instrument (Promega, USA). In wells 8, the plungers and 50 μL of the elution buffer were added in 0.5 mL elution tubes supplied by the producer. The

FSC DNA IQ Casework protocol was executed from the instrument, to obtain the DNA from the bone fragments and the tooth. After the run was complete, the obtained DNA samples were stored at T = 4°C, till the time of analysis. The extraction of reference DNA samples from relatives of the deceased was performed on the automate Maxwell® 48 RSC instrument (Promega, USA), using the Maxwell® FSC DNA IQ™ Casework (Promega, USA).

After DNA extraction, biological samples were kept at �86°C for 24–72 hours till the genetic analysis.

#### **2.2 Quantification of the extracted DNA samples**

The Power Quant™ System Kit (Promega, USA) was used for the quantification of the DNA samples. In this case, following the manufacturer's recommendations, for each sample, a mix solution with a final volume of 18 μL, consisting of 10 μL of Power Quant 2 x Master Mix, 7 μL of Amplification grade water and 1 μL of Power Quant 20 x Primer Mix was prepared. The quantification was performed on a 7500 real time PCR system (Thermo Fischer Scientific, USA), using the HID Real-Time PCR Analysis Software v 2.0.6. The DNA concentrations in the saliva, bone fragments and tooth are presented in **Tables 1**–**3**.


**Table 1.**

*DNA concentrations from saliva and femur bone fragment.*

*Genetic DNA Identification from Bone Remains in Kinship Analysis Using Automate… DOI: http://dx.doi.org/10.5772/intechopen.99587*


**Table 2.**

*DNA concentrations from saliva and petrous pyramid.*


#### **Table 3.**

*DNA concentrations from saliva and canine tooth.*

#### **2.3 Amplification of the DNA samples**

The DNA samples were amplified for the STR markers using the Global Filer™ PCR Amplification Kit (Thermo Fischer Scientific, USA). The analysis was done as per the recommendations of the manufacturers. In this step, the amplification of the DNA samples was performed on a Pro Flex PCR System (Thermo Fischer Scientific, USA). The PCR reactions of the DNA samples were carried out in a total volume of 25 μL, which contained: Master Mix: 7.5 μL, Primer Set: 2.5 μL, nuclease-free water: 12.5 μL and template DNA: 2.5 μL. The Global Filer PCR Amplification Kit (Thermo Fischer Scientific, USA) contains 21 autosomal STR markers, as follows: D1S1656; D2S441; D2S1338; D3S1358; D5S818; D7S820; D8S1179; D10S1248; D12S391; D13S317; D16S539; D18S51; D19S433; D21S11; D22S1045; CSF1PO; FGA; TH01; TPOX; vWA; SE33, plus DYS391 and the gender-specific marker, amelogenin. In all amplification reactions, positive and a negative PCR controls were used.

#### **2.4 Capillary electrophoresis of the amplified DNA samples**

During the capillary electrophoresis, the samples were analyzed on a 3500 Genetic Analyzer (Applied Biosystems, USA), as per the manufacturer's recommendations. For the autosomal STR markers, 1 μL of the amplified PCR product (for each DNA sample) and 1 μL of the allelic ladder (AL) were used. These were added into a mix containing 9.6 μL of Hi-Di™ Formamide (Applied Biosystems, USA), 0.4 μL Gene Scan™ and 600 LIZ™ Size Standard v.2.0 (Applied Biosystems, USA). Gene Mapper ID-X Software version 1.4 (Applied Biosystems, USA) was used to analyze the obtained data.

#### **2.5 Statistical analyses**

The genetic profiles obtained from bones and the reference sample obtained from salivas, were compared with an estimation of the familiar relationships. In these cases, the calculation of likelihood ratio (LR) and posterior probability (PP) were performed using the DNA VIEW software and the allele frequencies from the Romanian population [10]. The genetic profiles obtained from the bones were also

compared with the genetic profiles belonging to the persons included in the elimination database to monitor the possible contamination.
