The Folmer COI mtDNA universal primers that are considered standard for DNA barcoding of life contain so many mismatches against the target sequences of vertebrate origin that they often end in failure to amplify many of vertebrate DNA extractions. This discrepancy favors for the selection and designing of new metabarcode primers that can be used to identify all individuals of vertebrates or at least all individuals represented in a class of Vrtebrata such as Class Mammalia. The current study embarks on such an endeavor. In this study development of new mtDNA metabarcode (16SrRNA) that can be used as universal primers to amplify almost all species of Class Mammals for different forensic and molecular biodiversity analyses.
Mitochondrion, the energy coins for the cell, performs the function of the oxidative phosphorylation and the formation of ATP also called energy coins for the cell. Mammalian mitochondrial genome (mtDNA) is a double stranded, circular, covalently closed molecule of approximately size of 16.4 kb. The mtDNA is inherited from the mother as a haploid and heteroplasmy has been found hardly.This fact makes it potentially relevant in the identification of maternal relationships, absence of recombination and the fast rate of evolution Blood/tissue samples were collected from Class Mammals (one specimen from every order reported to be present in Pakistan). DNA was extracted from the collected specimens through standard organic method, qualified and quantified and then PCR-amplified using novel universal primers selected from aligned mtDNA sequences originating from all mammalian mitochondrial DNA genomes submitted to different online sequence databases such as NCBI nucleotide database. The sensitivity of PCR also be assessed using a range of DNA concentrations. The amplified products were sequenced on ABI Genetic Analyzed following Sanger’s dideoxy method of
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sequencing. The correctness of obtained mtDNA sequences were examined visually in Chromas Lite 2.1 software and then alignment of these sequences were performed against highly similar DNA sequences in NCBI nucleotide databases using BLAST in order to identify the origin of unknown mtDNA sequences. Both sequencing experiments and phylogenetic studies confirmed the specificity of the universal primer set developed and present a novel metabarcode found in this region of genome (16SrRNA) for species level identification of large number of mammalian species. So, in future, this barcode can be used for species identification in various fields of study such as meat adulteration, illegal trade, food mislabeling and molecular estimation of biodiversity.