Original Article

Marker Assisted Molecular Investigation of Kappa-Casein Gene in Bos

Indicus Sindhi Genetic Group Using HINFI Restriction Enzyme
Iqbal Ahmed Memon1,2, Muhammad Asif Raza2, Waseem Ali Vistro3, Muhammad Farooque
Leghari4, Abdul Waheed Nizamani5, Najeebullah Lail2, Tanzeela Farooq3, Latif Ahmad4*
1
Department of Animal Breeding and Genetics, Sindh Agriculture University, Tando-Jam, Pakistan. 2Department of
Animal Sciences. 3Department of Bio-sciences, 4Department of Para-Clinical Studies, Baqai College of Veterinary
Sciences, Baqai Medical University, Karachi-Pakistan. 5Molecular Genetics, University of Windsor, Windsor, Ontario-
Canada.

ABSTRACT
Background: Present research aimed to determine molecular genotype of kappa-casein gene in female Red
Sindhi cattle. This gene has great influence on the technologically advanced milk properties. Methods: Blood
specimens (n=50) from females of this cattle breed at a well-managed farm in Sindh-Pakistan were collected
and commercial kit was employed for DNA extraction. Genotype determination of -casein gene and alleles was
done through PCR-RFLP technique by using primer; PCR products were digested upon HINFI restriction
enzyme. The digested fragments were analyzed by electrophoresis on agarose gel using ethidium bromide to
increase visibility. The bands were examined under ultra violet-light to study polymorphic locus on DNA
fragments. Results: Digestion upon HINFI restriction enzyme of 350bp fragment indicated three patterns. The
1st (homozygote genotype BB), 2nd (homozygote genotype AA) and 3rd (heterozygote genotype AB) patterns
yielded major fragment(s) of 1) 266bp, 2) 134bp and 132bp and 3) 134bp, 132bp and 266bp, respectively. Each
of the three patterns yielded one minor fragment of 84bp. The genotype frequency for homozygote AA and the
allelic frequency of allele A were higher than the same for homozygote genotype BB and the allelic frequency of
allele B, respectively. Conclusions: An accurate profile of genetic make-up and alternate forms of -casein
genes in Sindhi cattle is likely to help researchers, policy makers, immunologists, dietitians, neonatologists,
community physicians and managerial as well as production level officials to exploit it to full potential.

Key words: Genotype, Polymerase chain reaction, Restriction fragment length polymorphism, Alleles, Breeding

INTRODUCTION

Bovine farming is markedly linked with milk quality,
which in turn is most significantly dependent on genotype
and environment. Several constituents in bovine milk (e.g.,
vitamins, amino acids or building blocks of proteins,
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DOI:
10.21276/iabcr.2016.2.4.8
Received:15.11.16| Revised:10.12.16| Accepted:11.12.16
Corresponding Author
Dr. Latif Ahmad, Baqai College of Veterinary Sciences, Baqai
Medical University, Karachi-Pakistan.
Copyright: the author(s) and publisher. IABCR is an official publication of
Ibn Sina Academy of Medieval Medicine & Sciences, registered in 2001
under Indian Trusts Act, 1882. This is an open access article distributed under
the terms of the Creative Commons Attribution Non-commercial License,
which permits unrestricted non-commercial use, distribution, and
reproduction in any medium, provided the original work is properly cited.
proteins in water-soluble portion, casein (CN) and
inorganic substances including minerals) are linked with
good metabolic health e.g., more intake of dairy products is
connected with lesser danger of metabolic related disorders
and cardiovascular diseases.[1] CN portion of milk proteins
considerably enhances the physical as well as chemical
value of bovine milk. Progressively more constituents from
cow beestings and milk are being exploited as antimicrobial
agents for moneymaking. Likewise, vaccination techniques
to boost the natural concentrations of immune components
offer great potential in the development of hyper-immune
milk-derived products for prophylactic or therapeutic use in
humans.[2] In-vivo veterinary investigations point out that
whey protein and CN or their peptides partake immune-
modulatory effects.[1] Obese non-diabetic humans on high
fat mixed meal and whey protein isolate in the 4h
postprandial period have shown immune activation.[3]
Not only mammals are consuming dairy products, but other
creatures such as commercial poultry is also fed milk with
the notion to improve health and production. A large

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 Memon IA, et al.: Marker assisted molecular investigation of kappa-casein gene in Bos indicus
number of intervention investigations are underway to
evaluate the influence of dairy milk proteins or peptides
(especially CNs) on metabolic fitness.[1] It is because many
genetic variants of CN have been described for cattle, other
ruminants and camel etc. and have got tremendous
importance in allergenicity potential of milk; genetic
variants of CN also have differences in their hypertension
and pain relieving, dipeptidyl peptidase-4 inhibitory and
bone mineralization, osteoblast differentiation and
related activities.[4] Moreover, predisposition to infectious
diseases depends on the genetic basis for cytokines and
bovine CN peptides can assist host defense counter to
pathogens through cytokine release. Differences within
cytokine genes have been associated with diversity within
individuals in their immune response and in resistance to
multiple pathogens.[5] The weakened immune system
invites a number of pathogens to invade the body. For
example Pausterella multocida is such a secondary invader
and causes several, financially important illnesses e.g.,
bovine hemorrhagic septicemia, enzootic pneumonia,
avian/fowl cholera and swine atrophic rhinitis.[6] We can
upgrade the milk genetically if we know the functions of
specific genes of milk proteins in every population of milch
animal and combine genotyping as measure of livestock
selection strategies.[7]
The CN comprise about 80% of milk proteins in bovines.
The genetic variants of CNs are set up to be alpha()S1-, S2-
, beta()- and kappa()-CNs. There is little if any additive
effects of either CN-genes on milk production (quantity).
This feature of CN-genes is advantageous, given that either
allele can be selected to improve the milk quality or
quantitative traits, including fat, protein, and/or other solid
contents, without compromising milk production.[8] The
CN-genes are strongly connected and inherited as a bunch
so they put a latent importance and can perform a vital
function in marker-assisted choice for milk traits.[9] These
are situated on chromosome six within a 200kb fragment in
the order S1, , S2 and . The S1-, -, and S2- CN-genes
are directly connected and form an evolutionarily
associated family, whereas the -CN gene is at smallest
amount 70kb away from them.[10] The -CN constitutes just
about 12% of the CN and is situated on chromosome 6q31.
The -CN is one of the most important milk proteins,
controlled by means of a gene with five exons and four
introns. The -CN gene is one of 4 groups of major genes
in milk-CN fraction, with 19,800 Dalton molecular weight
and 169 amino acids; it exists on chromosome number 6 in
bovines and chromosome number 4 in sheep and goats. The
total length of -CN gene is near to 13KD, but the majority
of the coding sequences for the mature -CN protein are
enclosed in the 4th exon.[9]
The B variant of -CN gene results due to point mutation
(T/C) in exon 4, which causes increased efficiency of
production of cheese from milk. It is generally accepted
that milks from -CN-BB cows have higher proteins
(including CNs) than the milks from -CN-AA or AB
animals.[9] The CN is precipitated in the cheese curd. It has
also been shown that B variant of -CN is associated with
www.iabcr.org International Archives of BioMedical and Clinical Research | Oct-Dec 2016 | Vol 2 | Issue 4
increased -CN concentration in milk.[11] The -CN gene
has been frequently calculated due to its influence on the
technologically advanced milk properties. Moreover,
majority of milk allergenic patients showed a strong
humoral and cellular response to - and -CNs. Fourteen
variants of -CN have been explained i.e. A, A1, B, B2, C,
D, E, F1, F2, G1, G2, H, I, and J, the most common being
the A and B alleles. The -Casein A is the most frequent
variant in the bulk of breeds, followed by -CNs B and H.
The chief carbohydrate-free component of milk is -CN A,
which is foreseen as the reference protein. Other -CN are
also carbohydrate-free. The -CN is soluble in the presence
of calcium and in contrast to the other caseins not calcium
sensitive.[4]
Main aim of the present research was to find out the
genotypes and allelic frequency for -CN gene by the
Polymerase Chain Reaction-Restriction Fragment Length
Polymorphasim (PCR-RFLP) technique in Red Sindhi
cattle. The Sindhi cattle varies in body color from deep
reddish brown to a yellowish red, but normally a deep
red.[12] They are the supreme standard of all Zebu (Bos
primigenius indicus) milch types. This breed is also known
as Bos indicus Sindhi genetic group, Red Karachi and Red
Maliri cattle; it originated in the Sindh province of
Pakistan. Due to resilience to harsh environment and
diseases, this breed is widely kept for milk production
across Africa, South Asian and other countries (e.g., UK,
Canada, Indonesia, Thailand, Brazil). Crossing of this
breed with those originating from temperate regions of the
world is being practiced to combine their tropical
adaptations.[13]
METHODS
Study settings and design
The primer and probes for the amplification and
hybridization of the variable fragments of bovine -CN
gene were designed from the published nucleotide
sequence of the cattle. Chi-Square test was applied to the
results and expected values and significance test for
deviation were calculated according to Hardy-Weinberg
equilibrium for Red Sindhi cattle. The statistical program
Tools for Population Genetic Analysis (TFPGA) was used
in the estimation of heterozygosity.
Selection and Description of Experimental Subjects
Blood specimens were obtained from the jugular veins of
50 Red Sindhi cows maintained at a Breeding Farm in
Tando Muhammad Khan-Sindh, Pakistan. Following
methods were applied.
DNA EXTRACTION
Exactly 5mL of collected blood was immediately
transferred from each syringe to K-EDTA coated sterile
vacutainer tubes. These blood specimens were stored at -04
C. DNA was extracted by using commercial kit GF-1
(Vivntas) according to the manufacturer directions. Finally
the value and amount of DNA was assessed by Nano-drop
Spectrophotometer.
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 Memon IA, et al.: Marker assisted molecular investigation of kappa-casein gene in Bos indicus

DNA AMPLIFICATION AND ELECTROPHORESIS Figure 1: The Formulae Used In Statistical Analysis
-CN gene was enhanced in PCR with primers JK5: (5- (a) (b) (c)

ATC ATT TAT GGC CAT TCC ACC AAA G-3) and JK3
(5-GCC CAT TTC GCC TTC TCT GTA ACA GA-3).[13]
The PCR reaction mixture contained 12.5l Master Mix
(Green Master Mix, Promega, Madison, WI, USA),10pmol
of every primer, 50 mg of DNA template and residual
amount, nuclease free H2O was added[13] to full the amount
of 25l PCR Mixture. PCR enhanced in thermal cycler
(Applied Biosystem2720, USA) under conditions detailed Where Hi is the
Where q is the Where Hi is the numeral numeral of
in Table 1. allele frequency of heterozygous cattle heterozygous cattle
and n is the total and n is the total and n is the total
number of samples numeral of cattle
Table 1: Detail of thermo-cycling conditions for PCR tested.
[15] [16]
numeral of cattle
[16]
investigated. investigated.
Initial Final
Cycle 45
denature extension
94 C/30 60 C/30 72 C/30
94 C/5 min 72 C/7 min
sec sec sec

The expected heterozygosity designed (based on the

TAE buffer is solution comprising of a blend of Tris base,
acetic acid and EDTA. The PCR product was run on the
1% Agar gel (AG) and 2l ethidium bromide (EtBr) was
used to make it visible in TAE as the running buffer at 80V
and 100V current for 35 minutes and the bands were
watched under ultra violet-light to study the fragment
measured by 50kb ladder (Range Ruler S/N# SM-0613).
Restriction Fragment Length Polymorphism
Following the PCR, the product was further digested by
haemophilus influenza serotype F restriction enzyme
(HINFI) purchased from Fermentas. Final reaction volume
of 30L containing 20L of each PCR product, 2L of
buffer 10x, 2L HINFI enzyme (10 unit L-1) and 6L
nuclease free water was added. The reaction mixture was
first incubated at 37C for overnight and then incubated at
65C for 20 minutes to deactivate the enzyme. The
resulting digested fragment was electrophoresed on 3% AG
stained with 2L EtBr in 1X TAE as the running buffer.
The AGs were run at 80 volts and 100 volts for 70 minutes
and the band was watched under UV-light to examine the
polymorphism by the magnitude of alteration in DNA
fragments.
Ethical issues
The synopsis of this research work was approved by the
code of ethics at Sindh Agriculture University, Tando-Jam-
Sindh, Pakistan.
Statistical analysis
Estimation of the allele frequencies of -CN was studied
through alleles, which were divided by 2n and the standard
error of the allele frequencies were designed by applying
the formula as given in Figure 1(a)
The probability of Hardy-Weinberg equilibrium as dealing
with the experimental genotypic frequencies was gained
using the test for each breed composition and the exact
probability test.[17] The pragmatic heterozygosity was
calculated by formula given in Figure 1(b), which in a
straight line determined the frequency of heterozygosis.
pragmatic/observed allelic frequencies) was known by
formula given in Figure 1(c). The statistical program
TFPGA was used in the estimation of these parameters.[18]
RESULTS
The extracted DNA samples were used for the templates
for PCR amplification i.e.,
increase in the frequency of replication of DNA segment.
The product was digested with restriction enzyme HINFI.
In this way, DNA fragments get separated and become
visible as bands. The restriction absorption analysis of
350bp PCR products of -CN indicated three restriction
patterns. We observed 2 bands/fragments (266bp and 84bp)
in 1st pattern (BB), 3 (134bp, 132bp and 84bp) in 2nd
pattern (AA) and 4 (266bp, 134bp, 132bp and 84bp) in 3rd
pattern (AB). The 3rd pattern is the coupling of the 1st and
2nd patterns i.e., it was heterozygous. The restriction
patterns and fragment sizes of the restriction digestion
analysis of PCR product are shown in Table 2 and Table 3,
respectively. Tables 4 to 5 and Plates 1 to 2 (agar gel
photograph of PCR product on -CN) have been discussed
below.
Genotype and Allele Frequency of -CN in Red Sindhi
cattle
The genotype frequencies of -CN gene in Red Sindhi
cattle was analyzed and results are shown in Table-4. The
table reveals that the frequency of homozygous genotype
AA (0.25) of -CN gene is superior to that of homozygous
BB (0.12) and similar to that of heterozygous AB (0.36),
while the genotypic frequency of heterozygous AB is
superior to the frequency of homozygous genotype BB.
The allelic frequency of -CN in Red Sindhi cattle were
analyzed and reported in Table-4. The table reveals that the
frequency of allele A (0.7) is superior to that of the allele B
(0.3). The allele frequency of -CN for A- allele is two
times greater than frequency of B-allele.

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 Memon IA, et al.: Marker assisted molecular investigation of kappa-casein gene in Bos indicus

Table 2: Restriction pattern of the restriction digestion analysis of PCR product of -CN
Base Pair AA BB AB UNCUT
350
266
132/134
84

Table 3: Fragment size of the restriction digestion analysis of PCR product of -CN
Genotype Fragment size in base type No. of genotype

Uncut PCR Products 350 50

AA 132 and 84 26
BB 266 and 84 6
AB 266, 134, 132 and 84 18

Table 4: Genotype Frequency of -CN Gene in Red Sindhi cattle

Genotype No. Observed Frequency Frequency %
AA 26 0.52 52
BB 6 0.12 12
AB 18 0.36 36
TOTAL 50 1 100

Table 5: Allelic Frequency of -CN in Red Sindhi cattle

Allele No. Observed Frequency Frequency %

A 70 0.7 70
B 30 0.3 30
TOTAL 100 1 100

Table-6 reveals the observed expected values of AA, BB two major fragments (134bp, 132bp) and a minor fragment
and AB genotypes. According to Hardy-Weinberg (84pb); (AB) has three major fragments (266bp 134bp,
principles for calculation of expected values relating of the 132bp) and a minor fragment (84bp). The A and B variants
Chi-Square test results (one degree of freedom at 0.05 change in the 136th and 148th positions of amino acids. At
levels) are presented in Table-6 revealing the null 136th position, threonine is removed by isoleucine while at
hypothesis that the population in Hardy-Weinberg 148th position, aspartic acid is removed by alanine, for A
frequencies in Red Sindhi cattle is not rejected. and B respectively. Alternative phenotypes observed in
present study coincided with the findings of previous
Table 6: Chi-Square of Frequency of -CN in Red Sindhi studies in 2007 and 2008 in Friesian cattle and buffalo,
cattle respectively.[19,20]
S. Observed Expected Chi-
No.
Genotype
Values Values Square
The genotype frequency of homozygous genotype (AA)
1. AA 26 25 (0.52) was found to be greater than the homozygous
2. BB 6 5 genotype BB (0.12) and the allelic frequency of allele A
0.44
3. AB 18 20
Total 50 50
(0.7) was two times higher than the allelic frequency of
allele B (0.3) in Red Sindhi cattle population. Allelic
frequency of -CN in a study in 2007 for A-allele (0.72)
DISCUSSION was superior compared to B-allele (0.27) in Jersy crossbred
The extracted DNA samples from blood specimens of dairy bulls.[19] -CN had also been reported to be useful
females of Red Sindhi cattle were digested with the marker for milk production traits on which bulls can be
restriction enzyme HINFI to detect the polymorphism. evaluated and chosen for upcoming breeding programs. In
Digestion upon HINFI restriction enzyme of 350bp a study in Brazil greater frequency of A-allele (0.70) as
fragments, the homozygous genotype BB, the homozygous compared to B-allele (0.30) in Red Sindhi cattle was
genotype AA and the heterozygous genotype AB have two, observed.[15] The findings of previous studies in 2007 and
three and four fragments, respectively. BB has a major 2008 are in agreement with the present research.[19] The
fragment (266bp) and a minor fragment (84bp); AA has findings of own research are in concordance with the

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 Memon IA, et al.: Marker assisted molecular investigation of kappa-casein gene in Bos indicus
findings of other previous studies in 2007 and 2008, that
reported frequency of genotype AA to be greater than
frequency of genotype BB and allelic frequency of allele A
was two times greater than the frequency of allele B in
cattle.[21,22]
Expected values and significance test for deviation were
calculated according to Hardy-Weinberg equilibrium for
Red Sindhi cattle. The observation in the present study has
shown that population of Red Sindhi cattle was not in
Hardy-Weinberg equilibrium. According to previous
workers, every breed and genetic groups were originated to
be in Hardy-Weinberg equilibrium.[15,23] The observations
in the present study might have been varied from the
findings of the workers in previous decade, because of
smaller sample size, small sampling area, possibly strict in-
breeding within Red Sindhi population and/or
environmental variations/alterations.
The -CN gene can be used as a marker gene for milk in
animals because of its positive effects on milk protein and
milk production. The PCR-RFLP technique can be applied
as a quick, exact and little cost method for detection of -
CN gene. PCR technique, amplified a deoxyribonucleic
acid fragment of -CN gene with 350 BP. The results of the
RFLP analysis showed three fragments 266bp,
134bp/132bp and 84bp after restrictions with enzyme with
HINFI that recognize changes in codon 148. The higher
allelic frequency of -CN A in Red Sindhi cattle is
advantageous given that -CN A is anticipated as the
reference protein due to being the major carbohydrate-free
component of milk.[4]
Plate-1: Agrose gel photograph of uncut PCR product of -
CN in Red sindhi cattle.
Plate-2: Agrose gel photograph of HINFI digestion of -CN
product of Red Sindhi cattle.
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CONCLUSION
In the present study, genotypes of -CN gene and alleles in
Sindhi cows were determined by electrophoresis on agar
gel using EtBr after digesting upon HINFI restriction
enzyme. The results revealed that -CN BB genetic variant
had one major fragment (266bp) and one minor fragment
(84bp); -CN AA genetic variant had two major fragments
(134bp, 132bp) and one minor fragment (84pb); -CN AB
genetic variant had three major fragments (266bp 134bp,
132bp) and one minor fragment (84bp). The frequency of
allele A of -CN is two times greater than frequency of B-
allele. The A and B variants change in the 136th and 148th
positions of amino acids. At 136th position, threonine is
removed by isoleucine while at 148th position, aspartic acid
is removed by alanine, for A and B respectively. The
researchers, policy makers, immunologists, dietitians,
neonatologists and managerial as well as production level
officials can utilize the outcome of the present study
according to their domains.
ACKNOWLEDGEMENTS
Deepest gratitude to Prof. Dr. Abdul Hussain Nizamani
(Late), Prof. (Retd.) Dr. Muhammad Khaskheli and Dr.
Bachal Bhutto for their precious guidance, help and
valuable suggestions during research work preparation and
the management. Special thanks to Islamic Educational,
Scientific and Cultural Organization (ISESCO) and
ISESCO Center for Promotion of Scientific Research
(ICPSR) Morocco for facilitating research work at Red
Sindhi Cattle Breeding Farm, Tando Muhammad Khan as
well as Dr. Abdullah Arijo, and all staff members Faculty
of Animal Husbandry and Veterinary Sciences, Sindh
Agriculture University, Tando-jam, for facilitating the work
at university laboratories.
ETHICAL APPROVAL
The Bos indicus Sindhi genetic group subjects used in this
research were those maintained at Red Sindhi Cattle
Breeding Farm in Tando Muhammad Khan-Sindh,
Pakistan. The synopsis of this research work was approved
by the code of ethics at Sindh Agriculture University,
Tando-Jam-Sindh, Pakistan and the supervisory committee
headed by Prof. Dr. Abdul Hussain Nizamani (Late),
Department of Animal Breeding and Genetics, Faculty of
Animal Husbandry and Veterinary Sciences of the said
university.
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How to cite this article: Memon IA, Raza MA, Vistro WA,
Leghari MF, Nizamani AW, Lail N, Farooq T, Ahmad L. Marker
Assisted Molecular Investigation of Kappa-Casein Gene in Bos
Indicus Sindhi Genetic Group Using HINFI Restriction Enzyme.
Int Arch BioMed Clin Res. 2016;2(4):34-39.DOI:
10.21276/iabcr.2016.2.4.8
1.
Source of Support: Nil, Conflict of Interest: None
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