Journal of Animal Research: v.5 n.2, p. 213-217. June 2015

DOI Number: 10.5958/2277-940X.2015.00036.4

Differentiation of Sheep and Goat Species by PCR-RFLP of

Mitochondrial 16S rRNA Gene

Deep Prakash Saikia 1* , Dhruba Jyoti Kalita 2 , Probodh Borah 1 , Satya Sarma 2 ,

Nagendra Nath Barman 3 and Rupam Dutta 1

1 Department of Animal Biotechnology, CVSc, AAU, Guwahati, Assam, INDIA

2 Department of Veterinary Biochemistry, CVSc, AAU, Guwahati, Assam, INDIA

3 Department of Veterinary Microbiology, CVSc, AAU, Guwahati, Assam, INDIA

* Corresponding author: DP Saikia; Email: saikiadeep17@gmail.com

Received: 20 January, 2015

Accepted: 30 May, 2015

ABSTRACT

The present study was carried out with an aim to develop a method for differentiation of sheep and goat meat using PCR-

RFLP. Tissue samples were collected randomly from ten animals of each species and used for mitochondrial DNA extraction.

PCR amplification of 600 bp fragment of 16S rRNA gene was done using universal primer. RFLP studies were carried out by

digesting the amplicons using restriction enzymes viz . Alu I and Hha I. Amplicons of Goat 16S rRNA gene was fragmented to

460bp and 140bp fragments by Hha I while the amplified gene of sheep was digested by Alu I into two fragments of 360bp and

240bp each. This resulting RFLP pattern of 16S rRNA could easily identify and differentiate meat of sheep and goat species.

Keywords: Mitochondrial DNA, PCR-RFLP, 16S rRNA gene.

Molecular methods developed during the past few years

based analysis is becoming more and more popular for

have been able to provide authentic and reliable tools for

the identification and characterization of different species

identification of different species of animals and birds.

(Meyer et al ., 1995). DNA has been extensively used

These latest techniques are able to overcome the drawbacks

for species identification due to its structural stability at

of many conventional methods used for characterization

high temperature and is conserved within all tissues of

of different species of animals. Species differentiation

an individual which helps in the development of species-

is highly essential to ensure the authenticity of meat

specific DNA probes (Chikuni et al ., 1990; Ebbehoj

and meat products . But such methods should be cheap,

et al., 1991), Polymerase chain reaction (PCR) assays

repeatable and rapid. Species identification is usually

(Chikuni et al ., 1994; Meyer et al ., 1994), Random

based on protein-isoelectric focusing, immunochemistry,

amplified polymorphic DNA (RAPD) (Meyer et al .,

immunoassay and electrophoretic methods (Zerifi et al.,

1994; Welsh et al ., 1990) and polymerase chain reaction-

1991) and determination of specific microscopic structural

restriction fragment length polymorphism (PCR-RFLP).

elements (Koolmees et al ., 1999). But all these methods

Mitochondria have their own genome and are evolved from

have their own drawbacks due to their dependency on

endosymbiotically incorporated organisms. Mammalian

protein characterization. Expression of protein is tissue

mitochondrial DNA is strictly maternally inherited and

dependent and they may denature on processing which

is circular-double stranded (16,569 bp) with 37 genes.

leads to subsequent loss of analytical specificity (Hunt

Among these 37 genes, 22 genes encode for t-RNAs, 2

et al ., 1997). Some other available techniques usually

genes encode ribosomal RNAs (12S rRNA &16S rRNA)

require blotting, staining, use of antibodies etc. These

and 13 genes encode enzymes involved in electron

limit their usefulness and for these reasons nucleic acid

transport chain of oxidative phosphorylation and ATP

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