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Sex determination of cattle meat by polymerase chain reaction amplification

of the amelogenin (AMELX/AMELY) gene 1

Article in Veterinary World · January 2012

DOI: 10.5455/vetworld.2012.526-529

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Vet. World, 2012, Vol.5(9): 526-529 RESEARCH

Sex determination of cattle meat by polymerase chain reaction

amplification of the amelogenin (AMELX/AMELY) gene
1
P. Gokulakrishnan, R. R. Kumar, B. D. Sharma, S. K. Mendiratta, D. Sharma , O. P. Malav

Division of Livestock Products Technology,

Indian Veterinary Research Institute, Izatnagar-243 122, Uttar Pradesh, India
1. Genome Mapping Laboratory, Central Avian Research Institute, Izatnagar-243 122, Uttar Pradesh, India.
Corresponding author: P. Gokulakrishnan, e-mail:[email protected]
Received:11-02-2012, Accepted: 25-03-2012, Published Online: 10-06-2012
doi: 10.5455/vetworld.2012.526-529

Abstract
Aim: The objective of this study was to develop a simplified, efficient, and accurate protocol for sexing of cattle meat based on
the amelogenin (AMELX/AMELY) gene using PCR technique which is superior to earlier work in terms of band patterns.
Materials and Methods: Based on the amelogenin gene located on the conservation region of X and Y chromosomes, a pair
of primers was designed and the system of PCR was established to amplify a 241-bp fragment from the X chromosome in
female cattle, and a 241-bp fragment from X chromosome and 178-bp fragment from the Y chromosome in male cattle,
respectively. The accuracy and specificity of the primers was assessed using DNA template extracted from cattle meat
samples of known sex. The protocol was subjected to a blind test showed 100% concordance, proving its accuracy and
reliability.
Results: PCR products of cattle meat samples after electrophoresis showed two bands (241, 178-bp) for tissue from male
while female tissue resulted in only one (241-bp) band.
Conclusions: Our findings show that the PCR assay based on the amelogenin gene is reliable for sex determination in cattle
meat.
Keywords: Amelogenin gene, Cattle meat, PCR, Sex determination

To cite this article: Gokulakrishnan P, Kumar RR, Sharma BD, Mendiratta SK, Sharma D, Malav OP (2012) Sex
determination of cattle meat by polymerase chain reaction amplification of the amelogenin (AMELX/AMELY)
gene, Vet World, 5(9): 526-529, doi: 10.5455/vetworld.2012.526-529

Introduction –MS/MS) [3] and gas chromatography-mass

spectrometry (GC-MS) [4]. Most of the methods
Determination of sex origin of cattle meat has
proved to be highly specific and sensitive, but were not
been always of public interest in country like India
where slaughter of cow (female cattle) is banned performed on a regular basis for meat sexing due to the
because of religious beliefs and laws thereby. The technical limitations or the expensive equipments
economic aspect allied with such discriminatory required.
slaughter policy also gets support as male beef is Over the last few decades, DNA-based techniques,
designated to be of higher quality than cow meat and especially polymerase chain reaction (PCR)-based
therefore yield higher prices [1] particularly in methods for meat sexing have received particular
European countries. To implement the regulations attention, and have proved to be reliable, sensitive, and
pertaining to such issues and to assure consumers of fast [5,6]. DNA regions that differ between male and
accurate labeling meat analysts need to have reliable, female individuals are essential features in PCR sex
authentic and simple method for determining the sex determination. In general, these methodologies were
of cattle meat. developed mainly based on amelogenin gene
To date, a range of different methodologies have (AMELX and AMELY) [7-9], zinc finger gene (ZFX
been developed for determining the sex of meat, and ZFY) [10-13] and the sex determination region of
mainly based on detecting either hormone or DNA [1]. the Y chromosomal gene (SRY) [14-16]. Alternatively,
In general terms, hormone-based methods include the bovine-specific repetitive sequence BRY-1 [17]
immunochemical determination using ELISA [2], and and the single copy sequence BOV97 M [18] are also
chromatographic techniques with mass-spectrometric used in the analysis for the sexing of cattle. More
detection, such as high performance liquid chromato- recently, based on real time PCR, both SYBR Green
graphy-mass spectrometry/mass spectrometry (HPLC [19] and TaqMan [20] technology for bovine sex
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 Sex determination of cattle meat by polymerase chain reaction amplification of the amelogenin gene

Figure-1. Alignment of the AMELX and

AMELY gene sequences (GenBank
Accession no. M63499.1 and
M63500.1). The primers AML-1F and
AML-1R are indicated by bold letters.
Dashes indicates gaps in alignment.

determination have been reported. between X and Y chromosome. The primers were
Amelogenin has been targeted by number of expected to yield a PCR fragment of 241 bp with the
workers, where samples from male show two bands AMELX gene as target sequence and 178 bp with AMELY
and female show only one band. Ennis and Gallagher gene, respectively. (Figure-1). The PCR primers were
[7] reported the primer pairs which yielded 280 and synthesized by Eurofins Genomics India Pvt Ltd,
217 bp, whereas the primer pairs, suggested by Chen et Bangalore.
al [8] resulted in amplified product of 417 and 340bp,
PCR amplification: Polymerase chain reaction
in case of cattle. The purpose of the present work was
to develop a protocol for sexing of cattle meat based on (PCR) was performed in 25 µl of reaction mixture
the amelogenin gene (AMELX/AMELY) using PCR containing 50 ng of genomic DNA, 200 µm of each
technique which is superior to earlier work in terms of dNTP, 1.5 mM MgCl2, 5 pmoles of each primer, 1 unit
band patterns. GoTaq® Flexi DNA Polymerase (Promega, Madison,
WI, USA), 1x PCR- colored buffer (Promega,
Materials and methods Madison, WI, USA) and nuclease free water to make a
Sample selection and DNA extraction: A total of final volume. Amplification was performed on a PTC-
16 muscle tissue samples of cattle (8 male and 8 200 DNA Engine® thermal cycler, (Bio-Rad, USA)
female) were collected from local market of Kerala using 0.2 ml reaction tubes. The PCR programme
and West Bengal, India. The collected samples were consisted of 5 min denaturation at 94°C, followed by
transported to the laboratory under refrigeration, and 34 cycles of denaturation (94°C, 45 sec), annealing
were stored frozen at - 20°C prior to analysis. Genomic (60°C, 45 sec) and primer extension (72°C, 60 sec).
DNA was extracted from the samples using the The final cycle was followed by extension at 72°C for
®
DNeasy blood and tissue kit (QIAGEN, Germany) 10 min and indefinite hold time at 4°C.
according to the manufacturer's instructions.
Gel electrophoresis: The PCR product obtained was
Subsequently, the quality of genomic DNA was
assessed by agarose gel electrophoresis using 0.8% analyzed by agarose gel electrophoresis in 2% agarose
agarose gel (AMRESCO, USA) stained with ethidium gels (AMRESCO Inc., USA) stained with ethidium
bromide. The purity and concentration of DNA was bromide. A 100 bp DNA ladder (O'Gene Ruler, Fermentas)
® was electrophoresed simultaneously in order to assess
estimated spectrophotometrically using Nanodrop
the size of amplification product. The gels were
ND-1000 spectrophotometer (Thermo Scientific) at
260 and 280 nm. The DNA sample showing the OD visualized in automatic gel documentation system
260:280 nm value of 1.70-1.90 was considered as (MiniBis, DNR Bio-Imaging Systems) and the size of
good quality. the amplicon was determined using software available
with the gel documentation system.
Design of PCR primers: As target for PCR primers
Results and Discussion
the amelogenin gene was chosen in this study. Based
on the information obtained from the alignment of PCR products of cattle meat samples after
AMELX gene (GenBank accession No. M63499.1), electrophoresis showed two bands (241, 178-bp) for
AMELY gene (GenBank accession No. M63500.1) tissue from male while female tissue resulted in only
sequences, a pair of primers AML-1F (GGCCAACA one (241-bp) band (Figure-2).
CTCCATGACTCCA), AML-1R (TGGGGAATAT The objective of this study was to develop a
YGGAGGCAGAG) was designed with homology cattle meat sexing assay based on the amelogenin gene
www.veterinaryworld.org Veterinary World, Vol.5 No.9 September 2012 527
 Sex determination of cattle meat by polymerase chain reaction amplification of the amelogenin gene

Figure-2. Electrophoretic pattern of

amplified fragments from the cattle meat
DNA using AML-1 F and AML-1R primers.
Samples are: male (Lanes 1,3 and 5) and
female (Lanes 2,4 and 6). M - Marker of
Molecular weight, 100 bp ladder (O’Gene
Ruler, Fermentas), N= Non-template control.

(AMELX/AMELY) using PCR technique which is AMELX and AMELY, this gene is used as a target for
superior to earlier work in terms of band patterns. The sex determination in mammals like humans [24],
most common approach in sexing cattle meat involves cattle [25], horses [26] and sheep or goats [27].
the co amplification of the Y-chromosome specific
Conclusions
sequence containing the Y-linked genes (SRY) and an
autosomal sequence that acts as a control for the presence In conclusion our findings show that the PCR
of DNA [21]. In the present study, we employed assay based on the amelogenin gene is reliable for sex
primers derived from a sequence for X and Y specific determination in cattle meat. Due to the relatively
amelogenin, and verified the accuracy of the assay by short size (<250-bp) of the products compared to
evaluating genomic DNA from 8 males and 8 females. earlier work, this method can be successfully applied
The overall amplification products obtained showed to sex determination of cattle meat samples from
100% accuracy. This assay provides a rapid and highly degraded DNA and also in a shorter period of
sensitive method for sexing, because of the presence time. The PCR product comparatively shorter in size,
of the X chromosome band. This result was superior to size difference between band from AMELX and
those reported by other authors [7,8]. Ennis and AMELY was more prominent on resolution, ensuring
Gallagher [7] reported the primer pairs which yielded no ambiguity. Also, the advantage of this assay is that
280 and 217 bp, whereas the primer pairs, suggested neither additional control amplicons with a second
by Chen et al [8] resulted in amplified product of 417 locus-specific autosomal primer pair nor restriction
and 340bp in cattle. However we have attempted to endonuclease steps are necessary for sex determi-
explore the amelogenin gene for designing the nation and control of the PCR reaction. The method
primers, which could amplify the relative bands of proved to be reliable, cheap and is potentially suitable
smaller size and our self-designed primers effectively for routine analyses.
amplified the PCR products below 250 bp.
Author’s contribution
Amelogenin gene encodes for a protein found in
developing tooth enamel which belongs to the family P. Gokulakrishnan carried out the experiment and
of extra cellular matrix proteins [22]. In most mammals drafted the manuscript. R.R. Kumar, B.D. Sharma,
the amelogenin gene is located on both X and Y S.K. Mendiratta guided during the research and helped
chromosomes (AMELX and AMELY) [23], but a 63 in drafting of manuscript. D. Sharma provided the
bp deletion in exon 5 of the AMELY gene in comparison laboratory facilities and guided the study. O.P. Malav
to the AMELX homologue yields two different size helped in collection of sample. All authors read and
bands in male and two similar size bands (which approved the final manuscript.
appear as a single band on resolution) in female [22]. Acknowledgements
Due to an insertion in the region amplified in the X
specific gene (AMELX) or a deletion in the Y specific The authors gratefully acknowledge Indian
gene (AMELY), the amplified fragments are of Veterinary Research Institute (IVRI) and Central
different sizes. Due to length polymorphism between Avian Research Institute (CARI), Izatnagar, India for
www.veterinaryworld.org Veterinary World, Vol.5 No.9 September 2012 528
 Sex determination of cattle meat by polymerase chain reaction amplification of the amelogenin gene

providing necessary facilities to accomplish this research. Amplification and application of the HMG box of
bovine SRY gene for sex determination. Animal
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