**2.4 Mitochondrial DNA extraction**

G-spin™ Total DNA Extraction Kit (50 Preps) (REF: 17045) was used to extract mitochondrial DNA from blood of different species according to the manufacturer's protocol instructions.

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*Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool*

**Primer Conditions References**

30 95°C 30 sec

1 72°C 5 min

30 95°C 30 sec

1 72°C 5 min

30 95°C 30 sec

1 72°C 5 min

30 95°C 30 sec

1 72°C 5 min

59.3 °C 30 sec 72°C 130 sec

58.3 °C 30 sec 72°C 130 sec

58.3°C 30 sec 72°C 130 sec

59.3°C 30 sec 72°C 130 sec

1 95°C 2 min This study

1 95°C 2 min This study

1 95°C 2 min This study

1 95°C 2 min This study

*DOI: http://dx.doi.org/10.5772/intechopen.98248*

Homo 16S-F Homo 16S-R

Ovis 16S-F Ovis 16S-R

Capra 16S-F Capra 16S-R

Bos 16S-F Bos 16S-R

**Table 1.** *PCR conditions.*

**2.5 Agarose gel electrophoresis**

**3. Result and discussion**

Agarose gel was prepared by dissolving agarose powder in 1X TBE buffer. The amount of agarose can be dissolved depending upon the purpose in which agarose sheet used. About 0.7% agarose gel was used for visualization of the DNA after extraction while 1.5–2% agarose sheet was used for visualization of PCR product (amplicon). RedSafe (alternative for ethidium bromide) stock solution concentration was 10 mg/ml. Only 5 μl of RedSafe stock solution were added to 100 ml of

The primers were synthesized at Macrogen/Korea, were provided in a lyophilized form, which were re-dissolved with 300 nuclease-free water according to the institution of the manufacture company to reach to the final concentration (100 pmoles/μl). The working solution will be 10 pmoles/μl to be used directly in PCR [15, 16]. The PCR conditions were calculated using online Protocol Optimize writer

The four sets of designed primer pairs were submitted to specificity using Primer-Blast and the results revealed that, they are specific to amplify the 16S rRNA gene of humans (*Homo sapiens*), sheep (*Ovis aries*), goats (*Capra hircus*), and cows

melted agarose gel to get the final concentration of 0.5 μg/ml [13, 14].

**2.6 Primer pairs preparation and PCR conditions**

software. The conditions were illustrated in **Table 1**.


*Mitochondrial 16S rRNA Gene-Dependent Blood Typing as a Forensic Tool DOI: http://dx.doi.org/10.5772/intechopen.98248*

**Table 1.** *PCR conditions.*

*Forensic Analysis - Scientific and Medical Techniques and Evidence under the Microscope*

non-human using a species-specific primer design.

(*Ovis aries*), goat (*Capra hircus*), and cow (*Bos taurus*).

**2. Methodology**

**2.1 Study design**

**2.3 Primer design**

**2.2 Blood sample collection**

profiling methods used for human proof of identity and present their use in the chief cases of human identification from non-human [3–5]. Mitochondrial markers that are used for species identification are as follows: cytb gene, cytochrome c oxidase subunit I gene, 12S and 16S rRNA segment, and control region in wildlife [6–8]. A short fragment of the 12S rDNA was employed for DNA amplification leading to species identification. The mitochondrial DNA 16S rRNA gene is an advanced genetic marker for animal genetic diversity. Polymorphism sites, nucleotide variation, and haplotype variety were determined using whole sequences of the mitochondrial DNA 16S rDNA gene [9, 10]. Animal mitochondrial DNA (mtDNA) is commonly described as a small, circular molecule that is conserved in size, gene content, and organization [11]. The aim of this study is to design valuable species-specific-PCR tool to discriminate blood of humans from

The study design was experimental to design species-specific primer pairs for typing the blood samples and their assignment to human (*Homo sapiens*), sheep

Seventy-two blood samples were collected from humans, sheep, goats, and cows (18 blood samples for each). All blood samples were withdrawn by technicians and 5 ml were aspirated using an aseptic technique and transferred to EDTA-Na2 tubes and mixed well and stored in a refrigerator. The collection took 2 weeks (May 15,

2019–May 30, 2019). All samples were collected from Al-Diwanya city.

Homo 16S-R: ATCATTTACGGGGGAAGGCG (1200 bp); Ovis 16S-F:

(1060 bp); Capra 16S-F: GCCTGGTGATAGCTGGTTGT, Capra 16S-R: TCACCCCAACCAAAACTGCT (820 bp); and Bos 16S-F:

AGGCCTAAAAGCAGCCATCA, Ovis 16S-R: GCCCTTTTCTAGGGCAGGTT

CTAAGCAGCCCGAAACCAGA, Bos 16S-R: GGGCAGGGTTTTGTGTTGTC

G-spin™ Total DNA Extraction Kit (50 Preps) (REF: 17045) was used to extract mitochondrial DNA from blood of different species according to the manufacturer's

The gene selected for this study is the mitochondrial 16S rRNA gene. The NCBI data base was used to recover the sequences chosen for a primer design. The sequence ID of *Homo sapiens* (NC\_012920.1); sequence ID of *Ovis aries* (NC\_001941.1); sequence ID of *Capra hircus* (NC\_005044.2); sequence ID of *Bos taurus* (NC\_006853.1). Primer 3 software [12] was used to design the specific primer using the sequence of above-mentioned sequence IDs. The generated primers were as follows: Homo 16S-F: GCCTGGTGATAGCTGGTTGT,

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(1300 bp).

protocol instructions.

**2.4 Mitochondrial DNA extraction**
