Abstract
The present study describes the use of PCR and PCR-RFLP methods to discriminate between blood samples obtained from domestic camel, buffalo and sheep. The DNA was extracted from 3 blood samples of each species at 3 different ages and subjected to PCR amplification of a mitochondrial cytochrome b (cyt b) gene segment (358 bp) using universal primers. Subsequent cleavage with 4 restriction endonucleases (AluI, HaeIII, HinfI and Taq I) gaverise to a species-spe- cific pattern on an agarose gel. Results of cleavage by AluI and HaeIII were 2 fragments of different sizes in all animals; Hinf I produced 2 fragments in camel and sheep and 3 fragments in buffalo; Taq I produced 2 bands in camel and sheep while no fragmentation was generated for buffalo. The results suggest that the method of PCR-RFLP using these restriction enzymes can reliably identify chosen species. Moreover, PCR-RFLP is a rapid and simple method for identification of animal species.
Highlights
The RFLP analysis of the cyt b gene region of the three different animal species revealed high specificity and discrimination, even there was no common fragment shared among the studied species
The more modern techniques allow the identification of species specific markers or repeats (SSR), mitochondrial DNA restricted fragment length polymorphism, and random amplified polymorphic PCR (RAPD-PCR) which have the major advantages over protein analysis and that the samples heated to as high as 120°C can still be analysed and discriminated between species (Minarovič et al, 2010; Linacre, 2012; Muhammad and Yasmeen, 2015)
The PCR amplification of the cyt b arovič et al (2010) who reported that Alu I enzyme gene, using universal primers (L14816 and H15173) generated different digestion patterns enabling idengenerated a specific 358 bp product with DNA sam- tification of Mustelavison (81 bp, 109 bp and 169 bp), ples of all the three animal species tested, which is in Mustelaputoriusfuro (169 bp and 190 bp), Susscrofadoaccordance to the report of (Parson et al, 2000).The mesticus (115 bp and 244 bp), Oryctolaguscunninculus resulting PCR amplified products when digested with is not cleaved by AluIso it has whole 359 bp fragment four restriction endonucleases (AluI, HaeIII, Hinf I on agarose gel, Anseranser (130 bp and 229 bp)
Summary
Determining the species origin of different kind of samples (blood, meat, skin, etc.) is important in forensic purpose for species differentiation and identification (Sherryn et al, 2015) and it is an integral part of food regulatory issue as adulteration and substitution of meat has always been a concern for various reasons such as public health, religious factors, wholesomeness and unhealthy competition in meat market (Rashid et al, 2014; Hou et al, 2015).Various methods like traditional morphological or protein identification methods, species specific markers or repeats (SSR), mitochondrial DNA restricted fragment length polymorphism (mtDNA-RFLP), and random amplified polymorphic PCR (RAPD-PCR) are employed for detection of the species origin of different forensic samples and meat, and among these DNA based assays are gaining popularity (Cheng et al, 2014; Lin et al, 2014). The mitochondrial DNA (mtDNA) has been the most widely studied region of eukaryotic genomes which has played a critical role in development of population and evolutionary genetics (Abou-Hadeed et al, 2011; DeMasi et al, 2015). It has several advantages over nuclear genome for diagnostic studies of animals because of greater abundance in sample extracts and higher copy number making the amplification of mtDNA seg-. The cyt b gene is used as an important utility tool in studies of legal medicine and molecular evolution (Prusak et al, 2004; Abou-Hadeed et al, 2011). This gene has been completely sequenced or partially sequencedfor many species of mammals, birds (Bravi et al, 2004; Andrzej and Kamila, 2005), reptiles, amphibian and fishes (Chow et al, 1993; Ram et al, 1996; Quinterio et al, 1998; Lidstrom, 1999; Parson et al, 2000) and some invertebrates (Lee et al, 2009a)
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