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Authors: Sneha Choudhary
Advisor: A.K. Kataria
Title: Use of Mitochondrial DNA for Animal Species Identification from Various Biological Sources
Publisher: Rajasthan University of Veterinary and Animal Sciences Bikaner-334001
Language: en
Type: Thesis
Agrotags: null
Abstract: Authentication of animal species in legal and forensic cases is very important but the type of sample obtained in these cases is difficult to be validated by only physical examination so a molecular detection program for animal species identification is needed. The use of DNA based approaches like PCR amplification with species specific PCR primers and DNA sequencing for species identification in case of animal persecution, biodiversity conservation and geographical origin is now used in crimes such as illegal collection/smuggling, poaching and illegal trade of species. Use of mitochondrial DNA in DNA based animal species identification systems has been widely used because mitochondria is highly abundant in cells and present in degraded or ancient DNA sample. In the present study a whole set of DNA templates from different animal species was isolated from the tissue samples. Phenol-chloroform-isoamyl alcohol (PCIA) based DNA extraction protocol was used for bone samples whereas DNA extraction from blood and meat/muscle tissue samples was phenol based, using separate protocols specific for both samples. The extraction method for bone and cooked meat/muscle tissue needed higher concentrations of proteinase K as compared to its counterparts. Initially presence of mitochondrial (mt) DNA in the total DNA isolated from the different types of tissue samples was confirmed by amplification of total DNA with universal primers based on mt cyt b region. A 307 bp fragment was obtained from PCR amplification confirming the presence of mt DNA in the sample. A conventional PCR based individual animal species identification procedure was designed for horse, sheep, cattle, goat, pig, chicken, buffalo, emu and quail, intended to amplify different species specific mtDNA genes/regions generating different size amplified fragments specific for particular species. A fragment of 242 bp was amplified from buffalo and a fragment of 229 bp was amplified from emu mt cyt b region while a fragment of 96 bp was amplified from mt D-loop region from quail DNA sample. Fragments of size 439 bp, 398 bp, 331 bp, 274 bp, 227 bp and 157 bp were amplified from horse, pig, sheep, cattle, chicken and goat DNA samples using primers based on conserved region of mt cyt b region. These regions in all species were amplified by common forward primer and species specific reverse primers, so a multiplex PCR was tested on goat, chicken, cattle, sheep, pig and horse and a duplex PCR was tested on cattle and horse samples. Each set of individual species specific primers were tested on all of the animal species used in the study to determine the specificity of the primers. The primers were specific to the particular species as there were no cross-reactions. Two universal primer pairs were also tested based on mitochondrial cyt B region and 16S rRNA region designed specifically using the consensus sequences of all 13 animal species tested in this work and generated 469 bp and 512 bp PCR amplified fragments. These fragments were used for PCR sequencing for individual animal species identification. The sequences obtained after sequencing were BLAST at NCBI and confirmed as part of mt cyt b gene and mt 16S rRNA gene sequences. The sequences available at NCBI GenBank were used for comparison of obtained sequences and database sequences of particular species by BLAST. The no.1 BLAST hit corresponded to the first alignment result with the highest identity with the mitochondrial genes while no.100 BLAST hit corresponded to the last alignment result (out of 100 results) with the lowest identity with the mitochondrial genes.
Subject: Animal Biotechnology
Theme: Use of Mitochondrial DNA for Animal Species Identification from Various Biological Sources
These Type: Ph.D
Issue Date: 2016
Appears in Collections:Thesis

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