Abstract
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) per cell and resists harsh and more stable conditions. Seventy-two blood samples were collected from humans (Homo sapiens), sheep (Ovis aries), goats (Capra hircus), and cows (Bos taurus) (18 blood samples for each). All blood samples were withdrawn by a technician and 5 ml were aspirated using an aseptic technique and transferred to EDTA-Na2 tubes. They were mixed well and stored in a refrigerator. The collection took 2 weeks (May 15, 2019–May 30, 2019). All samples were collected from Al-Diwanyia city. The results of PCR testing revealed that the primer pairs were specific and non-specific products did 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 primer pairs of another genus gave negative results, and this is primary evidence for primer pair specificity. The amplicon of 16S rRNA gene of Homo sapiens was 1200 bp, Ovis aries was 1060 bp, Capra hircus was 820 bp, and Bos taurus was 1300 bp. 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. There is sensitivity, specificity, and accuracy in the designed species-specific primer pairs and applicability of the designed primer pairs in forensics to investigate blood spots or evidence belonging for human, sheep, goat, and cow.
Highlights
Mitochondria possess a small spherical genome, mitochondrial DNA (mtDNA), which encodes for the 13 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 typing founded on sequences of the control region otherwise filled genomic sequence is used to examine a variation of forensic mtDNA
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
Summary
Mitochondria possess a small spherical genome, mtDNA, which encodes for the 13 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 which have the potential to be valuable for forensic studies, especially attendant to the absence of recombination, to a large 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. 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]. 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 non-human using a species-specific primer design
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