Mitochondrial 16S rRNA gene-dependent Blood typing as a Forensic Tool

Background: Mitochondrial DNA is an important tool for human identification and is used to differentiate between human and animal blood at the crime scene, because in extreme conditions nuclear DNA is severely destroyed while Mitochondrial DNA contains multiple copies (200-2000) in per cell as well as resists harsh and more stable conditions. Methodology: Seventy-two blood samples were collected from human ( Homo sapiens ), sheep (Ovis aries), goat ( Capra hircus ) and cow ( Bos taurus ) (Eighteen blood samples for each). All blood samples were withdrawn by technician and 5ml were aspirated using aseptic technique and transferred to EDTA-Na2 tube and mixed well and stored in refrigerator. The collection takes 2 weeks (15th May 2019 to 30th May 2019). All samples were collected from Al-Diwanyia city. Results: The results of PCR reveal that, the primer pairs were specific and non-specific products not appear for all samples. The amplification of Homo sapiens mitochondrial DNA with primer pairs of other ( Ovis aries, Capra hircus and Bos taurus ) and amplification of each with primers pair of another genus gave negative results and this a primary evidence for primer pairs specificity. The amplicon of 16S rRNA gene of Homo sapiens were 1200bp , Ovis aries were 1060bp ,Capra hircus were 820bp , and Bos taurus were 1300bp. The sequencing revealed that no cross-reactivity of designed primer pairs and the PCR assay based on the designed primer pairs will be simple, fast, sensitive, specific, and cost-effective. Conclusion: Sensitivity, specificity and accuracy of the designed species specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood sports or evidence


Introduction
Mitochondria have their possess small spherical genome, mtDNA, which encodes for the thirteen important subunits of the electron transport chain and ATP synthase together with 22 tRNAs and 2 rRNAs necessary for mitochondrial protein synthesis [1,2] . Mitochondrial DNA presents several characteristics valuable used for forensic studies, especially attendant to the absence of recombination, to a great copy number, and to matrilineal inheritance. Mitochondrial DNA typing founded on sequences of the control region otherwise filled genomic sequence is used to examine a variation of forensic mtDNA profiling methods used for human proof of identity and present their use in the chief cases of human identification from non-human [3][4][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][7][8] . A short fragment of the 12S rDNA was employed for DNA amplification leading to species identification. The mitochondria DNA 16S rRNA gene is an advanced genetic marker for animal genetic diversity. Utilizing gene mitochondrial DNA 16S rRNA. Polymorphism sites, nucleotide variation, and haplotype variety were determined using whole sequences of the mitochondria 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 vaulable species specific-PCR tool to distriminate blood of human and non human Species specific-primer design.

Methodology Study Design
The study design was experimental to design species specific primer pairs for typing the blood samples and their assignment to human (Homo sapiens), sheep (Ovis aries), goat (Capra hircus) and cow (Bos taurus).

Blood Sample Collection:
Seventy two blood samples were collected from human, sheep, goat and cow (Eighteen blood samples for each). All blood samples were withdrawn by technician and 5ml were aspirated using aseptic technique and transferred to EDTA-Na2 tube and mixed well and stored in refrigerator. The collection take 2 weeks (15th May 2019 to 30th May 2019). All samples were collected from Al-Diwanya city.

Mitochondrial DNA extraction
G-spinTM 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.

Agarose gel electrophoresis
Agarose gel was prepared by dissolving agarose powder in 1X TBE buffer. The amount of agarose which can be dissolved depending upon the purpose in which agarose sheet used. 0.7% agarose gel used for visualization the DNA after extraction while 1.5%-2% agarose sheet visualization of PCR product (amplicon). RedSafe (alternative for ethidium bromide) stock solution concentration was 10 mg/ml. Only 5μl of RedSafe stock solution were added to 100ml of melted agarose gel to get final concentration 0.5μg/ml [13,14] .

Primer pairs preparation and PCR conditions
The primers were synthesized at (Macrogen/ Korea), were provided in a lyophilized from, which were re-dissolved with 300 nuclease-free water according to institution of 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 software. The conditions were illustrated in table (1).

Result and Discussion
The four sets of designed primer pairs were submitted to specificity using Primer-Blast and the results revealed that, they are specific to amplify 16S rRNA gene of Human (Homo sapiens), Sheep (Ovis aries), Goat (Capra hircus ) and Cows (Bos taurus) (table 2). 16S rDNA region is highly conserved region among mtDNA [17] . mtDNA can be easier to retrieve from low-quantity and/or degraded DNA samples, as it is present at many copies per cell, thus providing a clear advantage over nuclear genome-based methods of species identification [18][19][20] . The results of PCR reveal that, the primer pairs were specific and non-specific products not appear for all samples. The amplification of Homo sapiens mtDNA with primer pairs of other (Ovis aries, Capra hircus and Bos taurus) and amplification of each with primers pair of another genus gave negative results and this a primary evidence for primer pairs specificity. The amplicon of 16S rRNA gene of Homo sapiens were 1200bp ( Figure 1A), Ovis aries were 1060bp( Figure 1B), Capra hircus were 820bp ( Figure 1C), and Bos taurus were 1300bp ( Figure 1D). PCR amplification and sequence analysis of mitochondrial 16S rRNA gene for their use in differentiation/identification and subsequently evaluating their application in solving the forensic cases [21] . Mitochondrial 16S is suitable for the differentiation Cont... Table (2): Primer-Blast of designed primer pairs of 300 mammalian species. 16S rDNA gene is common mitochondrial gene for detection of blend mutton and pork at high sensitivity. The mitochondrial 16S rRNA genes have been used as molecular markers to identify mammals, birds, shrimp, and other species using species-specific primers that amplify the 12S rRNA or 16S rRNA gene regions from mtDNA [17,22] . Gene loci on the mitochondrial genome have been used in species identification. These include the 12S and 16S rRNA loci. The D-loop (displacement loop) has been used less in species identification but more in intraspecies identification. Due to the greater sequence variation at this non-coding locus, it is now being used as a tool for identifying the presence of particular species within mixture of many species [23,24] .
The secondary and confirmatory assay for specificity of primer pairs used in study is sequences of PCR products. Eight amplicons from each were sent for sequencing using Sanger technique (Macrogen/Korea). The retrieved sequences firstly must be trimmed to remove unwanted sequences before submitting them for BLASTN. The trimming performed by Bioedit to get the finally processed sequences. Abbreviation of homo sapiens sequences were used as (HIS-1 to HIS-8), Ovis aries sequences be (IOA-1 to IOA-8), Capra hircus sequences be (IBCH-1 to IBCH-8) and Bos taurus sequences be (IBT-1 to IBT-8).
The identity percentage and alignment results of amplified 16S rRNA gene of homo sapiens, Ovis aries, Capra hircus and Bos taurus with database were illustrated in table (3,4,5,6) respectively.     The sequencing of the 16S rRNA has revolutionized the study and identification of human and non-human in forensic. Many study development a simple method using universal primers for species identification based on direct PCR sequencing using primer sets were designed based on the conserved regions of the 16S rRNA loci detected by the comprehensive sequence comparison among 30 animals whole [25] . Mitochondrial DNA the method could be a dominant tool for mammalian species identification, especially in forensic cases in which many unidentified biological samples must be analyzed such as blood spots [25,] . The 16S and 12S sequences allowed identification of most species to the genus level Faster-evolving DNA regions are required to identify closely-related animals species [26] . the successfully used forensically informative nucleotide sequencing analysis of the 16S rRNA mitochondrial DNA to identify before unknown biological specimens of human and animals [27] . The mitochondrial 12S rRNA and 16S rRNA genes, including those from fish and amphibians to mammals including human beings. Therefore, universal primers were designed to amplify sequences in the fastevolving animal mtDNA [17] . the PCR amplifications of mitochondrial 16S rRNA followed by sequencing and analysis showed to be very efficient for identification of species origin of species [21] . The 12S rRNA and 16S rRNA gene sequences of animals reveal the fitting level of interspecific variation but the great level of intraspecific homogeneity [28] .
The results showed no cross-reactivity of designed primer pairs and the PCR assay based on the designed primer pairs will be simple, fast, sensitive, specific, and cost-effective.

Registration of Sequences in GenBank:
All the 32 sequence of 16S rRNA gene were submitted to GenBank for registration. After checking and revision the following accession numbers were donated:

Conclusion
Sensitivity, specificity and accuracy of the designed species specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood sports or evidence belonging for human, sheep, goat and cow.
Ethical Clearance: The project plan displayed on the scientific committee and scientific ethical committee of the department of Biology-college of science at university of Babylon and get approval Source of Funding: There is no funding source and it is completely covered by authors

Conflict of Interest:
There is no conflict of interest