pcr based genotyping of cross breed cattle for a1 and

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BatchPrimer3. The SNP-flanking primer pair also can be designed together with AS primers or separately. The same primer selection algorithm is used.
PCR BASED GENOTYPING OF CROSS BREED CATTLE FOR A1 AND A2 BETA CASEIN TYPES

THESIS SUBMITTED TO THE NATIONAL DAIRY RESEARCH INSTITUTE, KARNAL (DEEMED UNIVERSITY) IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF

MASTER OF SCIENCE IN

ANIMAL BIOTECHNOLOGY BY

KAILASH PRASAD JAISWAL (B.Sc. in Biotechnology)

ANIMAL BIOTECHNOLOGY CENTRE NATIONAL DAIRY RESEARCH INSTITUTE (I.C.A.R.) KARNAL-132001 (HARYANA), INDIA

2012 Regn. No.2061010

Dedicated to My Family & Friends

ACKNOWLEDGEMENT It gives me an immense pleasure to place on record my highest esteem to my Major advisor Dr. Sachinandan De, Senior Scientist, Animal Biotechnology Center. My piece of work that lies in front of you is a blend of guidance of my major advisor. I remember sagacious guidance, indefatigable encouragement, close supervision, keen interest, critical appraisal, judicious planning of the project, calm and patient understanding of both of them, which not only inspired me to bring this project to a successful completion but also left certain experience which will guide me throughout of my life. I express my profound sense of gratitude to the members of my advisory committee, Dr. T.K. Datta, Principal Scientist, ABTC, Dr. D.Malakar, Senior Scientist, ABTC, Dr. B.R. Yadav, Principal Scientist, DCB and Dr. A. Kumaresan, Senior Scientist, LPM Division, for their valuable suggestions, throughout the research period. I am also thankful to Dr. A.K. Srivastava, Director, N.D.R.I. and Dr. Rameswar Singh, Registrar, N.D.R.I., for providing me the necessary facilities required for my study and research work and keeping my financial worries at bay in the form of NDRI fellowship. My heartfelt thanks to my lab seniors Biswajit sir, Moloya ma’am, Arpana ma’am, Rupinder ma’am, Ayan Sir, Shardul Sir, Debasish Sir, Gulshan Sir, Devender Sir, my friends Subas, S.Madhu, Mona Farazi, K.Sirisha, Nisha, Venkatesh, Prasant, Ravind, Nitya, Sonam, for their timely support when I needed. I also thank Shalini ma’am, Ritu ma’am, Ruchi ma’am, Sohan Sir, Monika ma’am, Neha ma’am, Suman ma’am, Dinesh sir, Sudarshan Sir, Manoj Sir, for their encouragement.

Abstract The dissertation work entitled “PCR based genotyping of cross bred cattle Karan Fries for A1, A2 beta casein types” was undertaken to distinguish between A1 and A2 type containing beta-casein and genotyping of hundred Karan Fries cross breed cattle. Casein is the main milk protein where it accounts for 80% of bovine milk protein and contains four fractions (alpha S1-CN, alpha S2-CN, beta-CN and k-CN). Beta casein contributes 25-35% of milk protein and many variants are reported (A1, A2, A3, B, C, D, E, F, G, H1, H2 and I) in different breeds of cattle. The beta casein variants A1 and A2 differs in the 67th amino acid position, the substitution of proline in A2 type with Histidine (in A1) is mainly due to a replacement of “C” nucleotide with “A” nucleotide in that corresponding nucleotide position. One hundred Karan Fries cross breed cattle were selected for genotyping of A1, A2 beta casein gene from the genomic DNA. The beta casein gene was amplified by Multiplex Tetra-Primer Amplification (T-ARMS-PCR). TARMS touchdown PCR and subsequent agarose gel electrophoresis could differentiate between the A1, A2 types of beta casein genes in these animals. The screening result showed three genotypes of animal in these 100 animals. The number of A2A2, A1A2 and A1A1 animals are 73, 19 and 8 respectively. The frequency of A2 and A1 alleles are 0.825 and 0.175 respectively.

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CONTENTS

Sl. No.

Chapter

Page No.

1.

Introduction

1

2.

Review of literature

4

2.1

Indian cattle population

4

2.2

Milk production in India

5

2.3

Mammalian milk

6

2.4

Milk Protein

7

2.5

Casein

8

2.5a

Importance of casein protein in milk as a food

9

2.6

Bovine beta-casein

9

2.6a

Structure of beta casein gene

9

2.6b

Variants of beta casein

11

2.6c

Beta casomorphin

13

2.6d

Beta-casomorphin 7 and it health implication in

14

Human and Animal 2.6e

Animal Trials

15

2.7

Mutation Detection

16

2.7a

Mutation detection techniques

17

3.

Materials and methods

21

3.1

Chemical and equipments

21

3.2

Experimental animals included in the study

21

3.3

Preparation of reagents, glass ware and Plastic

21

ware

3.4

Isolation of Genomic DNA from Blood

22

3.5

Quality check and Quantification of genomic DNA

23

3.5a

Spectrophotometric quantification of DNA

23

3.5b

Quality checked by Agarose gel electrophoresis of

23

DNA 3.6

Primer designing for Tetra-primer ARMS PCR

24

3.7

Optimization of Annealing Temperature of tetra-

25

primer ARMS–PCR 3.8

Agarose gel electrophoresis of PCR Product.

27

4.

Results and Discussion

28

4.1

Isolation of genomic DNA from Blood

28

4.2

Quality check and quantification of genomic DNA

28

4.3

PCR amplification of beta-casein 7th exon for

34

differentiation of A1&A2 4.4 5.

Gene and genotypic Frequency

35

Summary and Conclusion

41

Bibliography

i-ix

Appendix

LIST OF TABLES Table

Title

Page No.

No. 1.

Comparative composition of milk of different

6

species 2.

Protein composition of Ruminant milk

7

3.

Distribution of casein fraction in buffalo and

9

bovine milk 4.

The bovine beta- casein gene present in

10

chromosome-6 5.

Beta casein detail of mRNA and Amino acid in

10

Ruminants 6.

Chromosomal location of casein gene cluster in

11

different species 7.

First complete sequencing of milk mature protein

11

and corresponding genes 8.

Discovery of genetic variants of beta-casein

13

9.

Composition of the PCR mixture for master mix

26

10.

Touchdown thermal cycle Parameters

27

11.

Quantity and Quality of isolated DNA using

29

Nanodrop 12.

A1,A2 genotypes and their frequencies in Indian Cross breed cattle (Karan fries) in NDRI

40

LIST OF FIGURES Fig. No.

Title

Page No.

1.

Schematic illustration of primer design for the 19 Tetra-primer ARMS PCR

2.

Schematic illustration of primer design for the 25 Tetra-primer ARMS PCR

3.

Agarose gel electrophoresis (1%) of genomic 30 DNA isolated from peripheral blood leukocyte

4A

Agarose gel electrophoresis in (1%) of isolated 31 genomic DNA in 1xTAE buffer ,A,

4B, C

Agarose gel electrophoresis in (1%) of isolated 32 genomic DNA in 1xTAE buffer B,C

4.D, E

Agarose gel electrophoresis in (1%) of isolated 33 genomic DNA in 1xTAE buffer, D,E.

5.

Agarose gel electrophoresis (2%) of allele 34 specific PCR product

6.

Agarose gel electrophoresis (2%) of allele 35 specific PCR product.

7.A, B

Agarose gel electrophoresis (2%) of allele 36 specific PCR product ,A,B,

7.C, D

Agarose gel electrophoresis (2%) of allele 37 specific PCR product, C,D.

7.E, F

Agarose gel electrophoresis (2%) of allele 38 specific PCR product, E,F.

LIST OF ABBREVIATIONS %

Percentage

g

microgram

µl

microlitre

µM

micromolar

AMP

Adenosine monophosphate

ARMS

Amplification refractory mutation system

AS

Allele specific

ATP

Adenosine monophosphate

BMC-7

Betacasomorphin-7

CN

Casein

cm

centimeter

SDS

Sodium-dodesyle sulfate

DEPC

Diethylpyrocarbonate

P:C:I

Phenol: chloroform: Isoamylalcohol

DNA

Deoxyribonucleic acid

DNase

Deoxyribonuclease

dNTPs

Deoxynucleoside triphosphate

OD

Optical Density

EDTA

Ethylenediaminetetraacetate

CSN

Casein

KCl

Potassium chloride

kb

Kilo base pair

bp

base pair

mg

milligram

MgCl2

Magnesium chloride

ml

milliliter

mM

millimolar

Mo

Molybdenum

Etbr

Ethidiumbromide

OD

Optical density

OH⁻

Hydroxyl radical

PCR

Polymerase chain reaction

T-ARM

Tetra primer refractory mutation system

DNA

Ribonucleic acid

RNase

Ribonuclease

DNA

Deoxy ribonucleic acid

TAE

Tris-acetate EDTA

TF-II D

Transcription factor-II D

TNF

Tumor necrosis factor

Tris-HCl

Tris-base hydrochloric acid

U

Enzyme unit

UV

Ultra-violet

V

Volt

Pm

Pico mole

PCR

Polymerase chain reaction

IEP

Isoelectric point

CDS

coding sequence

ACE

Angiotensin I converting enzyme

SNP

Single nucleotide polymorphism

SSCP

Single strand conformation polymorphism

DGGE

Denaturing Gradient Gel Electrophoresis

HRM

High resolution melting Curve Analysis

RFLP

Restriction fragment length polymorphism

CDGE

Constant denaturing gel electrophoresis

TGE

Temporal temperature gradient gel electrophorasis

Introduction

1. INTRODUCTION

The total milk protein component of milk is composed of numerous specific proteins. Two major milk protein groups are caseins and whey proteins (ß-lactoglobulin and alpha-lactalbumin). Bovine milk contain four caseins (alpha s1-CN 15-18%, alpha s2-CN8-11%, beta-CN25-35% and kCN 18-15%, Eigel, et al 1984; Roginsiki 2003). All other proteins found in milk are grouped together under the name of whey proteins. Bovine beta casein contains a total of 209 amino-acid residues. Bovine beta-casein gene is highly polymorphic. A total of 12 protein variants are known so far. Initially three variants of bovine beta casein were discovered and denoted as A, B and C. It was later found that A variants could be further resolved into A1, A2 and A3 by gel electrophoresis. The 12 genetic variants of bovine beta caseins are A1, A2, A3, B, C, D, E, F, H1, H2, I and G (Kaminiski, et al. 2007). Out of these, A1 and A2 variant forms are important in dairy cattle (Farrell et al., 2004). The nucleotide sequence change in 67 th amino acid position of the beta-casein reading frame, from CCT to CAT, causes substitution of proline (A2) by histidine (A1, B) in the amino-acid sequence. This might cause a change of secondary conformation in the protein structure and affect the physical properties of casein micelle and vulnerability to enzymatic digestion. During this enzymatic process, betacasein opioid peptide beta-casomorphin 7 is released exclusively from A1 and B variants (Hartwig, 1997, Jinsmaa and Yoshikawa, 1999, Cieoelinska et al., 2007 and De Noni, 2008). The B-Casomorphin-7 (BCM-7), a 1

Introduction

bioactive seven-amino-acid peptide, can be released by digestion in the small intestine of A1 b-casein with pepsin, leucin amino peptidase and elastase.

The term ‘opioid’ refers to chemical substances that have

morphine-like activity in the body. Some of them are known to play an important role in the response to stress and pain, and the control of food intake. These agents act by binding to opioid μ-receptors, which are found principally in the central nervous system and the gastrointestinal tract (Teschemacher 2003). BCM-7 has opioid and cytomodulatory properties. Synthetic BCM-7 can inhibit responses of lymphocytes to stimulants in vitro (Elliott et al, 1997). Elliott et al (1997) reported that NOD (Nonobese diabetic) mice fed with A1 b-casein did not develop diabetes if they were also given naloxone (the morphine antagonist). They suggested that appearance of diabetes in genetically susceptible NOD mice fed A1 b-casein not those fed with A2 b-casein might be due to release from A1 b-casein of the bioactive peptide, BCM-7 which had a strong inhibitory effect on immune function. The debate about A1 and A2 milk types has been in the public arena for over a decade. There have been claims and counter claims about whether ‘ordinary milk’, which is a mixture of A1 and A2 milk, is linked to a range of disease conditions, and whether selecting cows that produce only A2 milk can avoid these problems. About 75% of the world’s 300 million strong dairy herd, produces milk that contains the A1 beta casein. There is a somewhat controversial claim, backed by 16 years of research, that this milk, which is drunk by most people in the western world, could be a cause of diabetes, heart disease, autism and schizophrenia in people with immune 2

Introduction

deficiencies. It is also claimed that the protein beta casein A2 is benign in this respect. Cows in the well-known dairy breeds can produce either or both of the beta casein proteins. Genotyping has shown that about 80% of Indian (Bos indicus) cows produce only beta Casein A2 type. Keeping in view the importance of beta casein genotyping the dissertation work is proposed with the following objectives:

1. To perform a PCR based method to distinguish A1 and A2 type of beta casein.

2. To genotype Karan Fries cattle for A1 and A2 beta casein type.

3

Review of Literature

2. REVIEW OF LITERATURE

Domestication of cattle took place as early as 7000 B.C. and more than 1200 million cattle are kept today, chiefly as sources of milk, meat and hide. About 800 different cattle breeds are recognized (Felius 1995; http://dad.fao.org; http://www.ansi.okstate.edu/breeds), many of which are genetic isolates. Selection has not only been directed primarily towards production of milk and meat, but often also towards their appearance (coat colour, horns etc.). There has been inherent selection for local disease resistance and docility, except in breeds used for bullfighting. Most developed taurine breeds have superior production, but zebu (Bos indicus) breeds are able to survive dry and warm climates better. Taurine dairy breeds have been commonly crossed to local zebu (Bos indicus) breeds in the tropics, but the favorable effects of heterosis are mainly in the first generation (Mc Dowell et al., 1996).

2.1 Indian Cattle population: India is home to about 185 million cattle i.e.15% of the world cattle population (17th Livestock Census).Out of the total livestock population in the country around 38.2% are cattle (17th Livestock Census).Thirty Indigenous

breeds

contribute

to

the

genetic

diversity

in

cattle

population(Basic Animal Husbandry Statistics, Ministry of Agriculture, Government Of India, 2010). Cross breed cattle constitute 13.3% of the total 4

Review of Literature

cattle population and 86.7% are indigenous cattle (17th Livestock Census).However, over the years there has been a declining trend in cattle population. During 1997-2003, there was a decline of 4.3% while the crossbred milch cattle population has increased by 34.4%(17th Livestock Census).Dairy farmers are replacing low producing native breed of cow with a smaller number of high yielding crossbred animals.

2.2 Milk Production in India Agriculture has been the mainstay of the Indian economy. All the same the dairy sector has emerged as one of the largest contributors to the GDP. Where poverty and unemployment still ails the Indian economy, over 40 million households in India at least partially depend on milk production. So, dairying has been considered as one of the activities aimed at alleviating poverty and unemployment especially in the rural areas in the rain-fed and drought-prone regions. The milk production in the country quadrupled from 23 million tons in 1973 to 100 million tons in 2007, with the remarkable annual growth rate of 4.5 per cent as against the world's average of about one per cent. Today, India has emerged as the largest world producer of milk with an annual production of 100 million tons in 2007 accounting for more than 13% of world's total milk production. It is also the world's largest consumer of dairy products, consuming almost 100 per cent of its own milk production. The Indian cattle contribute about 22467 tons while the buffalo accounts for about 59201 tons and cross bred cattle contribute about 253058 tons (Basic Animal husbandry Statistics, ASH series-12, 2010) of milk.

5

Review of Literature

2.3 Mammalian Milk: Milk is valued because it is an important source of many of the nutrients essential for the proper development and maintenance of the human body. There is a large variety of Milch / milk-producing animals reared in our country viz., cows, buffaloes, goats and camels. Milk of different species has different composition. Table -1: Comparative composition of milk of different species. Component

Goat

Sheep

Human

Fat (%)

3.80

7.62

3.67-4.70

Solid-not-fat (%)

8.68

10.33

8.90

9.02

Lactose (%)

4.08

3.7

6.92

4.78

Protein (%)

2.90

6.21

1.10

3.23

Casein (%o)

2.41

5.16

0.40

2.63

Whey proteins (%)

0.43

0.81

0.70

0.60

Total Ash (%)

0.79

0.90

0.3 1

0.73

Ca (%)

0.194

0.160

0.042

0.184

P (%)

0.270

0.145

0.06

0.235

Cl (%)

0.154

0.270

0.060

0.105

Vitamin A (IU /g fat)

39.00

25.00

32.00

21.00

Vitamin B, (mg/ 100 ml)

68.00

7.00

17.00

45.00

Vitamin B,(mg /100 ml)

210.00

36.00

26.00

159.00

Vitamin C (mg /100 ml)

20.00

43.00

3.60

2.00

Vitamin D (IU g fat)

0.70

ND

0.27

0.70

Energy (Cal. / 100 ml)

0.00

ND

68.00

9.00

Posati and Orrs (1976); Saini and Gill ( 1991).

6

Cow 3..67

Review of Literature

2.4 Milk proteins The proteins of milk have been categorized into caseins and whey proteins, on the basis of their structure and physico-chemical behaviour (Fox et al., 1975). Casein accounts for 80% of the total protein in the milk. There are four caseins derived directly as the gene products; αs1, αs2, β and κcaseins (Swaisgood et al, 1975). Each of the four caseins exhibits variability referred to as micro heterogeneity due to variability in degree of phosphorylation,

glycosylation,

disulphide

bonding

and

genetic

polymorphism (Fox et al., 1992). The whey proteins (20% of the total milk protein) represent a rich source of biological active molecules that are able to influence a range of physiological functions such as its role in uptake of trace elements and vitamins. However, increasing attention is being focused on physiologically active peptides derived from these proteins. Milk-derived bioactive peptides are considered as prominent candidates for various healthpromoting functional foods targeted at heart, bone and digestive system health as well as improving immune defense (Korhonen et al., 1998). Such peptides are inactive within the sequence of the parent protein and can be released by digestive enzymes during gastrointestinal transit or by fermentation or ripening during food processing. Table -2: Protein composition of Ruminant milk Component(g/100g)

Bovine1

Ewe1

Goat1

Buffalo2

Protein

3.2

4.6

3.2

4.6

Casein

2.6

3.9

2.6

4.5

Whey Protein

0.6

0.7

0.6

0.5

1.Bovine, ewe, goat (Velisek, 1999) 2.Buffalo milk (Formaggioni, 1999).

7

Review of Literature

2.5 Casein Casein is the major protein fraction present in milk. The four main types of casein comprise approximately 80% of the total protein in milk. The white and opaque appearance of milk which is combined with calcium and phosphorus as clusters of casein molecules is called micelles. The caseins, as proteins, are made up hundreds of individual amino acids, each of which may have a positive or a negative charge, depending upon the pH of the milk system. At some pH value, all the positive charges and all the negative charges on the casein protein will be at equilibrium, so that the net charge on the protein will be zero. That pH value is known as the isoelectric point (IEP) of the protein and is generally the pH at which the protein is least soluble. For casein, the IEP is approximately 4.6 and it is the pH value at which acid casein is precipitated. In milk, which has a pH of about 6.6, the casein micelles have a net negative charge and are quite stable. The casein component of milk is relatively heat-stable, capable of surviving pasteurization at ~62-71 °C. Conversely, the whey protein component is denatured at these temperatures. In general, caseins have limited α-helix and β-sheet secondary structure. They tend to be rich in proline residue content and have very few disulfide bonds. Casein solubility is pH dependent and is also affected by ionic strength and composition. Addition of sodium chloride will affect solubility differently depending on when it is added to the solution during pH adjustment (Strange et al.,1994).

8

Review of Literature

2.5a Importance of casein protein in milk as a food Casein is a class of bovine milk protein that may provide effects beyond nutrition. It is used as a binding agent in processed foods and supplements all eight essential amino acids the human body cannot create. The number of amino acids released by whey in that short period of time is much higher than those released by casein. This fast and efficient metabolism has led to claims that whey is superior to casein, which takes much longer to be metabolized. The slow metabolism of casein may be useful, especially when combined with whey. Casein has anti-catabolic properties, which slow the process of protein breakdown but is not as effective as whey at promoting protein syntheses. The anti-catabolic effects of casein mean that it may help to prevent muscle loss while promoting muscle recovery after exercise (Rose Elliot et al., 2006). Table-3: Distribution of casein fraction in buffalo and bovine milk (Ng-Kwai-Hang 2002 and Grosclaude, 2003).

AlphaS1-

AlphaS2-

casein

casein

Buffalo

30.2%

Bovine

38.4%

Beta- casein

Kappa- casein

17.6%

33.9%

15.4%

10.5%

36.5%

12.5%

2.6 Bovine Beta-Casein: 2.6a Structure of bovine casein genes All four bovine casein genes is part of a casein gene cluster of four casein genes (alpha-S1-casein, alpha-S2-casein, beta-casein and kappacasein) located on chromosome six, mapped to bovine chromosome six at 9

Review of Literature

q31 to 33 by in situ hybridization and residing on 200 kb to 300 kb of DNA (Ferretti et al., 1990).

Table -4: The bovine beta-casein gene present in chromosome 6 Species

Gene size

Bos taurus

Exon No.

8506bp

Amino Acid

9

224

Gene

mRNA

Accession

Size

No, AC_00163

1126bp

Protein ID

NP_851351

(Source: NCBI, GenBank)

-

Equus caballus

-

Bubalus bubalis

9

Bos indicus

9

Accession No.

Bos grunniens

mRNA

1-15

No.

9

Accession

Sus scrofa

Protein

9

Size

Capra hircus

60-104 105-731 60-734

mRNA

9

Amino acid

Ovis aries

CDS

9

Peptide

Exon

Bos taurus

Mature

Species

Signal peptide

Table -5: Beta-Casein Detail of mRNA and Amino acid in Ruminants

224aa

1126bp NP_851351

1-222

222aa

1088bp NP_001009373.1 NM_1009733

1.>188

188aa

574 bp ABQ52487

16-232

1-232

232 aa

1120 bp NP_999599.1

-

-

1..>498

1-15

-

1-15

1-15

-

16-222

-

1..>498 AEY63645.1 233 aa

103bp AEB71414.1

362

AER27665.1

10

EF558564.1 NM_214434.2 JN655525.

1131 bp NP_001075321.1 NM_001081852

304

(Source NCBI, GenBank ).

NM_181008

GU975754.1JN051276.1

Review of Literature

Table-6: Chromosomal location of casein gene cluster in different species. Species

Chromosome no.

Reference

Rabbit (Oryctolagus cuniculus)

OCU-12

Gellin et al., 1985

Cattle(Bos taurus)

BTA-6

Threadgill et al.,1990

Human(Homo sapiens )

HAS-4

Menon et al.,1992

Sheep (Ovis aries)

OAR-6

Hayes et al., 1993

Goat (Capra hircus)

CHI-4

Popescu, et al., 1996

Buffalo(Bubalus bubalis)

BBU-7

Iannuzzi et al.,2001

Table-7: First complete sequencing of milk mature protein and corresponding genes (Gronen and Vender Poel et al., 1994) Complete Sequencing of

Complete Sequencing of

Protein

gene

Alpha s1-CN 199aa.

Merceir et,al, 1971.

Koczen et al., 1991.

Alpha s2-CN, 207aa

Brignon et al, 1977.

Groenen et al., 1993.

Beta-CN 209aa.

Rebadeau Dumas et al., 1972.

Bonsing et al., 1988.

Kappa-CN169aa

Merceir et al, 1973.

Alexander et al., 1988.

Alpha La 123

Brew et al., 1970.

Vilotte et al., 1987.

Beta Lg 162aa

Braunitzer et al., 1972.

Alexander et al., 1992.

Casein Protein Size

2.6b Variants of Bovine beta casein The convention for the nomenclature of the variants for each protein is a progressive alphabetical order corresponding to the chronological order of the discoveries. The nomenclature is unified for the four species of Bos genus, i.e. B. taurus ("common" bovine), B. indicus (zebu), B.grunniens (yak),B. javanicu s(banteng of Bali).There are 12 genetic variants (A1,A2, A3, B, C, D, E, F, H1, H2, I and G) of CSN2 based on 11

Review of Literature

changes of certain amino acids in the beta-casein protein that alter its properties. Beta-casein may be present as one of two major genetic variants: A1 and A2 (Formaggioni et al, 1990). A2 beta-casein is recognized as the original beta-casein protein because it existed before a mutation caused the appearance of A1 beta-casein in European herds a few thousand years ago (Elliott et al. 1999).The major difference between the A1 and A2 beta-casein

proteins is a single amino acid at position 67 in a strand of 209 amino acids. A1 beta-casein has the amino acid histidine at position 67, while A2 betacasein has a proline amino acid in the same position 67. A1 beta-casein in cow's milk is different to other mammalian beta-caseins, because of its histidine at position 67.Human milk, goat milk, sheep milk and milk of other species’ contain beta-casein which is ‘A2 like’, because they have a proline at the equivalent position in their beta-casein chains (Lonnerdal et al, 1993). The A1 beta-casein protein has been implicated as a potential etiological factor in type 1 diabetes mellitus, ischaemic heart disease and also as a modifier of behavioral symptoms associated with some neurological conditions such as autism.

12

Review of Literature

Table -8: Discovery of genetic variants of beta-casein, (.Formaggioni, ,et, al.,1999).

2.6c Beta –Casomorphin Beta-casomorphins (BCMs), the peptides originating from betacasein, form a group of peptides with a chain length of 4-11 amino acids (aa), all starting with tyrosine residue in position 60 (Kostyra et al.,2004).A single amino acid substitution at the 67th residue of the 209 amino acid chain differentiates between the A1 and A2 beta-casein variants. This difference in structure results in A1 beta-casein and the rare variants related to A1 preferentially releasing the opioid peptide beta-casomorphin-7 (BCM-7) upon digestion, which may lead to adverse physiological effects and 13

Review of Literature

constipation. The BCM-7 content of fresh and hydrolysed (digested by pepsin) bovine milk has been examined by our group lately. We have found that in hydrolysed milk with variant A1 of beta-casein, there is a 4-fold higher level of BCM-7 than in A2 milk.BCM-7 (Tyr-Pro-Phe-Pro-Gly-ProIle) was first isolated as a peptide having morphine-like activity in 1979 (Brantl et al.,1979). This bioactive peptide exhibits a strong opioid activity (Kurek et al. 1992) and has been shown to stimulate human lymphocyte T proliferation in vitro (Gill et al. 2000). It has also cytomodulatory properties (Meisel and Bockelmann et al., 1999). The sequence of BCM-7 corresponds to positions 60-66 of the bovine beta-casein aa sequence. Human BCM-7 (positions 51–57 of human beta-casein) is similar to the bovine sequence. Both peptides have 7 aa with the sequence Tyr-Pro-Phe- at the N-terminus and both have opiate properties within or close to the BCM-7 sequence. The basic difference is that in human beta-casein, polymorphism was not observed (A2 Corporation, 2006).

2.6d Beta-casomorphin 7 and its health implication in Human and Animal The bovine BCM-7 has been shown to be absorbed into the circulation of formula-fed infants (Kostyra , et al., 2004), where there appears to be variation in the BCM-7 elimination rate between babies. Furthermore, while one of these infant studies reported a negative correlation between BCM-7 exposure and aspects of infant development (Kostyra et al., 2004), the other suggested that higher blood levels of bovine BCM-7 may be a risk factor for apnoea in some infants and that bovine 14

Review of Literature

BCM-7 from milk ingested by lactating mothers may be transferred directly to the mothers’ milk ( Kostyra et al., 2005). Human digestion may break down Casomorphins into inactive dipeptides by the enzyme dipeptidyl peptidase-4 (Puschel, 1982 and Converse, 2005).This enzyme is found in the digestive tract and in some endocrine cells. There is also the potential for release of casomorphins from human milk. Human BCM7 (Tyr-Pro-PheVal-Glu-Pro-Ile) differs from the bovine form (Tyr-Pro-Phe-Pro-Gly-ProIle) at two amino acid positions. However, scientific understanding of the biochemistry and pharmacology of casomorphins is still incomplete.

2.6e Animal Trials An early animal study examining the possibility of a relationship between milk protein consumption and diabetes was conducted ( Elliot and Martin 1984). This study involved feeding BB rats with milk protein or an amino acid diet. Elliott and Martin concluded that cows’ milk may contain a diabetogenic agent. A further study by Elliott conducted in 1992, showed that rather than all milk being causative of Type 1 diabetes, it was possible that there was a link between digested casein, and more specifically a hexapeptide peptide derived from digested beta-casein protein, and Type 1 diabetes. The implication of a bioactive protein fragment generated by the digestion of beta casein A1 was then reported in 1995, when, in an article title referring to beta casein variants as ‘Jeckyl & Hyde’, it was further noted that this peptide bound macrophages had known immunological activity. Supplementing previous research concerning the link between the A1 variant of the beta casein protein and Type 1 diabetes was a further study presented 15

Review of Literature

by Elliott in 1997, which reported that mice fed beta-casein A1 developed high levels of Type 1 diabetes, whereas those fed beta casein A2 did not. This study further implicates BCM-7 and its associated opioid activity as it reports that the diabetogenity is attenuated by naloxone, a specific opiate blocker. BCM-7 was further implicated as the causative agent in a report published in the journal Diabetologia in 1999. A publication encompassing a range of trials involving both the BB rat and NOD (Nonobese diabetic) mouse, animal models of diabetes, was published in 2002. Its findings with regard to disease causation were inconclusive (discussed in Introduction section). However, it did consider the possibility that, although it seems unlikely that a casein variant is an exclusive promoter of diabetes, it may enhance the outcome is some cases. It is of interest that wheat proteins were exemplified as promoters of diabetes in the conclusion of this paper, as wheat gluten, like A1, is a known source of a food derived exogenous opioid.

2.7 Mutation Detection In molecular

biology and genetics, mutations are

changes

in

a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. These random sequences can be defined as sudden and

spontaneous

changes

in

the

cell.

by radiation, viruses, transposons and mutagenic

Mutations chemicals,

are as

caused well

as errors that occur during meiosis or DNA replication(Bertram et al., 2000).They can also be induced by the organism itself, by cellular processes such as hypermutation. Mutation can result in several different 16

Review of Literature

types of change in sequences. These can have no effect, alter the product of a gene, or prevent the gene from functioning properly or completely. Due to the damaging effects that mutations can have on genes, organisms have mechanisms such as DNA repair to prevent mutations. DNA polymorphisms is useful for ecological and evolutionary studies of terrestrial, marine, and microbial organisms, with applications ranging from species identification to delineation of population structure to monitoring genetic diversity.

2.7a Mutation detection techniques: Allele Specific Polymerase Chain Reaction (AS-PCR) Single nucleotide polymorphisms (SNPs) can be genotyped using Allele specific(AS) primers with the lastnucleotide at the 3' end of a primer corresponding to the site of the SNP (Ugozzoli et al., 1991). In the AS extension reaction, twosets of primers are required, one for each allele of a SNP.AS extension relies on the difference in extension efficiency of DNA polymerase between primers with matched and mismatched 3' ends. DNA polymerase extends a primer only when the 3' end is perfectly complementary to the DNA template. Thus, an AS primer is specific to one of two alleles of a SNP at the 3' end of primers and specifically amplifies one of the two alleles. Genotyping is based on determination of the primer that produces the amplicon (Sobrino et al., 2005). If a common reverse primer is used in the reaction, the reaction is called allele-specific PCR (ASPCR) (Newton et al., 2006). Typically two forward AS primers are used in AS-PCR with a shared reverse non-specific primer. Two PCR reactions are needed to detect both alleles of a SNP (Drenkard, 2000, Zhang et al., 2006). 17

Review of Literature

One variant of AS-PCR is to use only one AS primer and two SNP-flanking primers in one PCR reaction (three-primer nested system). To enhance the specificity in the AS-PCR reaction, an additional mismatch may be deliberately introduced at the third or other position from the 3' end of each of the AS primers (Ye, 2001, Zhang et al., 2003). "a 'strong' mismatch (G/A or C/T) at the 3'-end of an allele-specific primer will likely need a 'weak' second mismatch (C/A, or G/T) and vice versa, whereas a 'medium' mismatch (A/A, C/C, G/G or T/T) at the 3'-end will likely require a 'medium' second mismatch" (Ye et al.,2001). An option is provided in the parameter setting panel for adding an additional mismatch and choosing the position of the second deliberate mismatch (the default is the third position). Two sets of AS primers, in both forward and reverse direction can be designed in BatchPrimer3. The SNP-flanking primer pair also can be designed together with AS primers or separately. The same primer selection algorithm is used to choose the AS primers with the highest scores. Effective and economical SNP genotyping method based on AS primers called tetra primer ARMSPCR (Ruiz et al., 2007). This procedure adopts principles of the tetra-primer PCR method (Ye , 1999) and the amplification refractory mutation system (ARMS) (Drenkard et al., 2000). Four primers are required to amplify a larger fragment from template DNA containing the SNP and two smaller fragments representing each of the two AS products. Primers are designed in such a way that the allelic amplicons differ in size and can be resolved by agarose gel electrophoresis. To enhance the specificity of the reaction, in addition to the first mismatch at the 3' end of AS primers, an extra mismatch

18

Review of Literature

is also deliberately introduced at the third position from the 3' end of each of the two inner AS primers.

Fig-1: Schematic illustration of primer design for the tetra-primer ARMS PCR (Ye et al., 2001). The SNP R (G/A) is presented as an example, and the other types of SNPs can be applied in the same way. Four primers, one pair of inner allelespecific (AS) primers and one pair of outer standard primers, are required in a single PCR reaction. Two AS products, one for the G allele and the other for the A allele are amplified using two pairs of primers. The former consists of a G AS primer and an outer standard primer, and other latter contains an A AS primer and an outer standard primer. A mismatch (represented by *) is deliberately introduced at the third position from the 3' end of each of the two AS primers to increase allelic specificity. Two outer standard primers 19

Review of Literature

are designed in such a way that the amplicons of two alleles differ in sizes and can be resolved by agarose gel electrophoresis.

20

Materials &Methods

3. MATERIALS AND METHODS

3.1 Chemical and equipments Chemicals used in the investigation are included in the text listed in the annexure. Composition of different solution and reagent used in the procedure is also listed in the annexure.

3.2 Experimental animals included in the study Hundred cross bred NDRI Cow (Karan Fries) were included in the study animals maintained at Cattle Yard, National Dairy Research Institute, Karnal India. The blood sample (10 ml each) were collected from the jugular vein in sterile 10ml BD Vacutainer (BD Franklin Lakes NJ USA-8362817) under aseptic conditions and transported to laboratory in a box containing ice.

3.3 Preparation of reagents, glassware and plastic ware All reagents were prepared in and washed with sterilized Mili Q water and autoclaved at 121°C for 15 min at 15 lbs pressure. Sterile disposable plasticware were used during the preparation and storage of DNA. Beakers, tubes and other glassware used for the DNA work were allowed to stand for 2 h at 37°C and rinsed several times with sterile water and then heated to 100°C for 2-3 h. Disposable gloves were worn during the preparation of materials and solutions used for the isolation and analysis of DNA.

21

Materials &Methods

3.4 Isolation of genomic DNA from Blood by Phenol: Chloroform: Isoamylalcohol (PCI) method ( De et al., 2009). About 10ml of blood sample was collected from jugular vein; and transferred and stored at 4oC before processing for DNA isolation .Plasma was separated out after centrifugation at 3000 rpm for 10 minutes. Then 10ml of R.B.C. buffer was added in 30 ml Oakridge centrifuge tube and mixed gently for 10-15min. The tubes were then incubated on ice and centrifuged at 3000rpm for 10 min at 40C.This was repeated 2-3 times until RBC s were completely removed and WBC pellet became white. Equal volume of extraction buffer was added. Then 250l of 10% SDS and 25l of 25mg/ml Proteinase-K was added to the WBC contained tube and incubated at 500C overnight. After the protein digestion equal volume of phenol was added and mixed well while inverting the tube until a uniform suspension was obtained. The tubes were then centrifuged at 12000rpm for 10 min at room temperature. The aqueous phase was collected without disturbing the interphase and equal volume of phenol: chloroform: Isoamylalcohol (25:24:1) was mixed gently for 5-10 min. and centrifuged at 12000 rpm for 10 min at room temperature. The aqueous phase was again collected in a fresh 50 ml centrifuge tube and equal volume. of Chloroform: Isoamyl alcohol (25:1) was added and gently mixed for 5 min. Centrifuging at 12000rpm for 10 min at room temperature, the aqueous phase was then transferred to a fresh 50ml centrifuge tube and 1/10thvolume. of 3M sodium acetate with 2 volumes of chilled 70% ethanol was added and mixed gently for 2 mins. A white pellet of DNA was obtained. Again tubes were centrifuged at 12000rpm for 10 min at room temperature. The pellet was 22

Materials &Methods

wash twice with500ul of 70% ethanol and the tubes was put for air drying the pellet which was eventually dissolved in 100-200ul of 1XTE buffer and stored in the -20 o C for future use.

3.5 Quality check and Quantification of genomic DNA: 3.5a Spectrophotometric quantification of DNA Spectrophotometric quantification of isolated DNA was done by using (Nano-drop) spectrophotometer. The optical density of the diluted DNA sample was taken at 260/280 nm using DNA storage solution (Mili-Q water) as blank. The conversion factor for DNA was taken as µg/µl per OD

260/280

units (Sambrook et al., 2001). Ratio of OD at 260 nm and 280 nm was 1.92.0; which indicated that the DNA was pure.

3.5b Quality checked by Agarose gel electrophoresis of DNA The integrity of the isolated DNA was examined electrophoretically by running the DNA in 1% agarose gel. The 1% gel of high quality molecular biology grade agarose (Sigma Chem. Co., USA) was prepared by dissolving the agarose in 1X TAE buffer (pH 8.0) followed by heating in a microwave oven. Ethidium bromide stock solution was added directly to molten agarose solution at the rate of 0.5 g/ml of gel volume before casting the gel. The surface of the gel casting tray and comb were leveled before pouring the gel. After complete setting of the gel, the comb was removed carefully and the gel casting tray was placed in the electrophoresis tanks containing 1X TAE (pH 8.0) buffer. Then3l of the DNA sample was mixed with 1l of 6X loading buffer and then loaded slowly into the wells of the 23

Materials &Methods

submarine gel using a micropipette. Electrophoresis was carried out at 100V. for half an hour. After completion of electrophoresis, the gel was examined under UV transilluminator /Gel documentation system. (Sambrook et al., 2001). 3.6 Primer designing for Tetra-primer ARMS PCR This procedure adopts principles of the tetra-primer PCR method and the Amplification Refractory Mutation System (ARMS). Four primers are required to amplify a larger fragment from template DNA containing the SNP and two smaller fragments representing each of the two Allele specific (AS) products. Primers were designed in such a way that the allelic amplicons differ in size and can be resolved by agarose gel electrophoresis. The gene sequences of Beta-casein (CSN2) variant A2 gene, exon VII (CSN2

Bos

indicus

Accession

no.

BC111172.1,EF123100.1,Bos

AY366420.1,

Bos

taurus-

grunniens-HQ902899.1,JN655525.1,

JN655525.1,EU310401,JN051276.1,JN051275.1 for Bos indicus species) were retrieved from GenBank (www.ncbi.nlm.nih.gov).The downloaded nucleic acid sequences were aligned using multiple alignment software ClustalW. Exon sequences for the above mentioned genes were also retrieved from the Ensemble genome browser. Tetra Primers for ARM-PCR were designed from the inter-exonic regions of respective genes by using the Primer3 software and were got custom synthesized from Sigma Pvt. Ltd. Bangalore.

24

Materials &Methods

Fig. -2: Schematic illustration of primer design for the Tetra-primer ARMS PCR.

3.7 Optimization of Annealing Temperature of T-ARMS PCR Optimization of the primer pair was carried out by performing touchdown-PCR, to check their specificity and for determining the optimum annealing temperature. It is a method used to increase specificity without compromising amplicon yield. The principle is to initiate synthesis at a very high annealing temperature which permits only perfectly matched primertemplate hybrid to form the annealing temperature was dropped in a stepwise fashion with the progression of each cycle. Once copy of target sequence accumulates over the first few cycles, the high temperature annealing becomes much less critical for specificity, it is the previous product which form major template. The benefit of decreasing the annealing temperature is to increase the probability of stable primer-target interaction. Touchdown-PCR was carried out by selecting different temperatures. The

25

Materials &Methods

components of the PCR mastermix was prepared as per Table -9 and the PCR cycling conditions are listed in Table-10: The PCR mastermix was prepared in a 0.5ml tube by mixing nuclease free water,10XDream Taq buffer, dNTPs, primers and DreamTaq DNA Polymerase. Sufficient master mix for the number of reactions plus one extra for a no-template control was prepared. The master mix was aliquoted into individual 0.2ml PCR tubes. Then 1.5l of genomic DNA was added to each aliquot of mastermix usinga clean micropipette. The PCR reagents were mixed gently and spun down.The tubes were then placed onto the PCR machine.TARMS touchdown PCR program was run after which it was stored at -4oC.

Table 9: Composition of the PCR mixture Component

Stock Con.

Volume/Tube (l)

Nuclease-free water

Working con.

15.8l

Buffer

10x

2.5l

1x

dNTPs

10mM in each 1ml

0.5l

0.2mM

100pm

1l

10pm

100pm

1l

10pm

100pm

1l

10pm

100pm

1l

10pm

Enzyme

5U/l

0.2l

1U/25l

Template

300-737 ng/l

2l

70ng/25l

Primer Outer forward Outer reverse Inner forward Inner reverse

Total

25l 26

Materials &Methods

Table -10: Touchdown PCR cycling Parameters Step Temperature, 0CTimeCycle number Initial Denaturation

95

3 min

Denaturation

95

30sec

Annealing

59-54

30sec

Extension

72

20 sec

Denaturation

94

30sec

Annealing

54

30sec

Extension

72

20sec

Final Extension

72

Δ t = -10c/ cycle

Χ5

Χ 30

4 min

3.8 Agarose gel electrophoresis of PCR Product. The 3% agarose gel of high quality molecular biology grade agarose (Sigma Chem. Co., USA) was prepared by dissolving the agarose in 1X TAE buffer (pH 8.0) followed by heating in a microwave oven. Ethidium bromide stock solution was added directly to molten agarose solution at the rate of 0.5 g/ml of gel volume before casting the gel. The surface of the gel casting tray was leveled before pouring the gel. After complete setting of the gel, the comb was removed carefully and the gel casting tray was placed in the electrophoresis tanks containing 1X TAE (pH 8.0) buffer. The amplified DNA samples were mixed with 5 l of tracking dye and then loaded slowly into the wellsof the submarine gel using micropipette. Electrophoresis was carried out at 150 V for half an hour. After completion of electrophoresis, the gel was examined under UV transilluminator /Gel documentation system ( BioRad, Molecular Imager, GelDoc TM XR, Imaging System). 27

Results & Discussion

4. RESULTs AND Discussion

4.1 DNA Isolation Genomic DNA was isolated from peripheral blood leukocyte of Karan Fries crossbred cattle by standard Phenol chloroform methodology (Sambrook et al., 2001). as mentioned in Material & Method. A total of100 DNA samples were included in the present study from Karan Fries cattle as given in table below. The concentration of the DNA isolated is provided in Table-12.

4.2 Quality and quantity of genomic DNA Quality and quantity of isolated genomic DNA was evaluated by horizontal Agarose gel electrophoresis (0.9 % of agarose gel) and measuring absorbance at 260 and 280 nm in spectrophotometer. Isolated DNA showed distinct band of genomic DNA under UV transilluminator indicating good quality of isolated DNA Fig-3.The ratio of optical density at two wavelength (OD260/OD 280) indicate quality of isolated DNA. OD ratio between1.7 to 1.9 indicate good quality of DNA.OD260/OD 280 ratio if less than 1.7 indicate protein contamination and more than 2.0 indicate RNA contamination. Most of DNA sample of the present study were found within range 1.7 to 1.9.The concentration DNA varied from 3000to 737ng/ul. The working solution contained approximately 100ng of DNA .These diluted DNA samples were used subsequently as template DNA for PCR amplification.

28

Results & Discussion

Table-11: Quantity and Quality of isolated DNA using Nanodrop Animal No:

260/280 ratio

DNA conc. (ng/ul)

Animal No:

260/280 ratio

DNA yield

Animal No:

260/280 ratio

DNA yield

7108 6911 6489 6712 6767 7301 6577 7305 7343 7066 7081 7315 6879 8933 8517 7355 6949 7088 8517 7489 6911 7216 7135 7348 6989 6079 7206 7218 720 6879

1.9 2 1.9 1.9 1.9 2.03 2.08 1.89 1.9 2 2 2 1.9 1.97 1.8 2 1.9 2 1.9 1.9 1.9 2.03 2.2 1.89 1.9 2 2 2 1.9 2

3087 1163 2736 2989 2867 2731 4737 3216 2646 3376 1163 2736 2989 2867 4024 3343 1163 2736 2989 2867 2731 4737 3216 2646 3376 1163 2736 2989 2867 3218

7206 7276 7289 7343 7215 7826 8669 8242 6806 7070 739 7165 724 6929 6329 8195 1062 6732 7355 7808 6978 6625 7772 7109 7099 6949 7216 7870 6922 6379

1.97 1.8 1.9 2 1.9 1.9 1.9 2.03 2.08 1.89 1.9 2 2 2 1.9 1.97 2 1.9 2 1.9 1.9 1.9 2.03 2.08 1.89 1.9 2 2 2 1.9

4024 3343 2989 2867 2731 4737 3216 2646 3376 1163 2736 2989 2867 524 3343 4737 887 1163 2736 2989 2867 2731 4737 3216 2646 3376 1163 2736 2989 2646

6821 6566 6821 7085 7057 6489 7323 6996 6989 7364 9922 6979 7323 6722 7023 5217 7155 6821 7162 7066 6989 6089 7162 6879 6922 7109 7230 7241 6577

1.9 1.97 1.8 2 1.9 1.9 1.9 2.03 2.08 1.89 1.9 2 2 2 1.9 2.02 1.89 2.08 2.2. 2.2 2 2 2 1.9 1.9 1.89 1.9 2 2

2867 4024 3343 2731 737 3216 2646 3376 1163 2736 2989 2735 4737 3218 2646 1163 2736 2989 3343 2731 1117 2216 2646 3376 1163 2736 2989 2735 3137

29

Results & Discussion

Agarose gel electrophoresis

Fig-3: Agarose gel electrophoresis (1%) of genomic DNA isolated from peripheral blood leukocyte photograph were taken at different interval of gel electrophoresis in 1xTAE buffer (A) after 30min. (B) after 60 min. (C) after 90 min.

30

Results & Discussion

A Fig-4A: Agarose gel electrophoresis in (1%) of isolated genomic DNA in 1xTAE buffer. Lane 1: DNA molecular weight marker in bp (base pair). Lane 2-14: Genomic DNA (3ul) from different Karan Fries animal as indicate their number above the well.

31

Results & Discussion

B

C Fig-4 B, C: Agarose gel electrophoresis in (1%) of isolated genomic DNA in 1xTAE buffer. Lane 1: DNA molecular weight marker in bp (base pair), Lane 2-9: Genomic DNA (3ul) from different Karan Fries animal as indicate their number above the well.

32

Results & Discussion

D

E Fig- 4 D, E: Agarose gel electrophoresis in (1%) of isolated genomic DNA in 1xTAE buffer. Lane 1: DNA molecular weight marker in bp (base pair), Lane 2-14: Genomic DNA (3ul) from different Karan Fries animal as indicate their number above the well. 33

Results & Discussion

4.3 PCR amplification of beta-casein 7th exon for differentiation of A1&A2 Tetra Primer ARMS was designed and Touchdown PCR optimized to amplification of the A1 and A2 casein 7th exon the procedure described in the section 4.5. T

C/T

C Outer Product T-Specific Allele C-specific Allele

Fig-5: Schematic diagram showing allele specific PCR amplified product which could be obtained after PCR.

34

Results & Discussion

Fig-6. Agarose gel electrophoresis (2%) of allele specific PCR product these main graph are applied in lane 2-5:-Lane 1=Marker DNA as in, Lane 2 and 5=A2A2 genotype specific PCR product, Lane 3=A1A2 genotype specific PCR product, Lane 4= A1A1 genotype specific PCR product.

4.4 PCR amplification of beta casein variants Primers were designed for amplification of bovine beta casein variants after analyzing the mutation point in the bovine beta casein gene .The important primer parameter like GC percent, Tm value, secondary structure and self-complementarity, were taken into consideration while designing the primer optimization of the concentration of PCR component and PCR cycling parameters were described in material method section .Touchdown PCR with 30 cycle at final annealing temperature of 55 0C. This optimum primer concentration and cycling parameter result in efficient amplification 35

Results & Discussion

of desired fragment size of beta casein variants by PCR. The amplified PCR product for A1 allele (“A” allele) was 199bp and for A2 allele (“C” allele) i.e. wild type was 111bp shown in Fig- 5, based on the position of primer location and amplified specific bands.

A

B FIG-6A, B: Agarose gel electrophoresis (2%) of allele specific PCR product these main graph A , B are showing A1, A2 genotypes. 36

Results & Discussion

a C

D FIG-6 C, D: Agarose gel electrophoresis (2%) of allele specific PCR product these main graphs C, D are showing A1, A2 genotypes 37

Results & Discussion

E

F FIG-6 E,F: Agarose gel electrophoresis (2%) of allele specific PCR product these main graph E , F are showing A1, A2 genotypes

38

Results & Discussion

4.4 Gene and Genotypic frequency In Karan Fries beta casein gene, three genotype were revealed namely, CC, AC, and AA (Fig -6A to Fig-6E). At this particular SNP location two nucleotide present namely A and C. The genotype and allele frequency for both these allele were calculated and presented in Table13.The homogenous genotype CC was found to be the predominant genotype in Karan fries breed .Overall frequencies of CC,AC,AA genotypes were 0.73,0.19, and 0.08 respectively. In Karan Fries cattle the wild type allele i.e. “C” nucleotide is prevalent. Out of 100 animal tested, 73animals (73%) showed A2A2 genotype containing “C” nucleotide (wild type allele; A2 allele). Nineteen animals were having A1A2 genotype containing “C” and “A” nucleotide (Both A1 and A2 allele respectively) in the same animals. While eight animals (8%) showed “A” nucleotide). The Table -13shows the inheritance pattern of variant allele (A2 allele containing “A” nucleotide) was present in the both heterozygous and homozygous conditions. Only eight animals were found homozygous for A1A1genotype containing only “A” allele (A1 allele).

39

Results & Discussion

Table-12: A1, A2 genotypes and their frequencies in Indian Cross breed cattle (Karan Fries) in NDRI Genotype and allele frequency of bovine beta casein variants in Karan fries cattle. A1,A2

Variant

Genotype

genotypes

Number

Frequencies

A1A1

8

0.08

A2A2

73

0.73

A1A2

19

Allele frequencies

A2

82.5

A1

17.5

0.19

From the above result we can say that frequency of undesirable “A” nucleotide is 0.175 which is supposed to be responsible for production of A1 milk .This frequency of undesirable “A” nucleotide allele may be attributed to the fact that Karan fries breed is evolved from European Holstein breed with higher A1A1 genotype .Thus, we can say that Indian cattle breed are still on the safer side that allele responsible for A2 milk production is comparatively high of “C” nucleotide containing wild type allele .

40

Summary & Conclusion

5. Summary and conclusion

Bovine beta-casein contains 209 amino acids. Beta casein A1 and A2 variants differ only at position 67, which is histidine in A1 or proline in A2 milk. A bioactive seven-amino-acid peptide, b-casomorphin-7 (BCM-7) can be released from digestion of A1 beta-casein containing milk. Hundred Karan Fries cross bred cattle DNA were genotyped for the A1 and A2 type of beta casein gene. A T-ARMS PCR (Tetra primer Amplification Refractory Mutation System PCR) was used to detect the A1 and A2 beta casein type by this allele specific PCR. Out of 100 animals tested, 73 animals (73%) showed A2A2 genotype containing “C” nucleotide (wild type allele; A2 allele). Nineteen animals had A1A2 genotype containing “C” and “A” nucleotide (both A1 and A2 allele respectively) in the same animals. Eight animals (8%) showed “A” nucleotide (variant allele, A2 allele). The allelic frequencies for “A” and “C” nucleotide containing alleles were 0.175 and 0.825 respectively. In Karan Fries cattle the wild type allele i.e., “C” nucleotide is prevalent.

41

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Corporation.2006.

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Sciences", 41, 527-537. Brantl V, Teschemacher H, Henschen A, Lottspeich F,1979. Novel opioid peptides derived from casein (b-casomorphins). I. Isolation from bovine caseinpeptone. Hoppe Seylers Z Physiol Chem 360:1211–1216. Braunitzer G., Chen R., Schrank B., Stangl A. ,1972 , Automatische sequenz analyse -lactoglobulin AB). "Hoppe-Seyler's Zeitung für Phisiologie und Chemie", 353, 832-834. Brew K., Castellino F.J., Vanaman T.C., Hill R.L.,1970 , The complete aminoacid sequence

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Brignon G., Ribadeau-Dumas B., Mercier J.C., Pélissier J.P., Das B.C.,1977. Complete

aminoacid

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APPENDIX COMPOSITION OF DIFFERENT BUFFERS & REAGENTS USED IN DNA ISOLATION FROM BLOOD AND GENE CLONING 1.RBC Lysis Buffer (1X)(500 ml) Reagents 1 M NH4Cl 1M KHCO3 0.5 M EDTA(pH-8.0) Autoclaved distilled Water

Final working 155 mM 10 mM 0.1mM

Volume concentration 77.5 ml 5 ml 100 μl Up to 500 ml

2. DNA Extraction Buffer (1X) (100ml) Reagents 1M NaCl 0.5 M EDTA 1M Tris-HCl(pH-8.0) Autoclaved distilled Water

Final working 75 mM 2 mM 10 mM

Volume concentration 7.5ml 0.4 ml 1 ml Up to 100 ml

3. Proteinase K (20 mg/ml) Reagent Proteinase K TE buffer

Amount 200 mg 10ml

Store at -20°C.

APPENDIX

4. 10% (w/v) Sodium Dodecyl Sulphate (SDS) , pH-7.0 Reagent SDS Autoclave distilled water Heat to 60°C to assist dissociation

Amount 10 g 100 ml

5. 50X Tris Acetate EDTA (TAE) (500 ml) Reagent 2M Tris Base Acetic Acid 0.05 M EDTA Autoclave water Final working concentration-1XTAE

Amount 121 g 28.55 ml 50 ml/18.6 g Upto 500 ml

6. Saturated Phenol/Chloroform/Isoamyl alcohol (25:24:1) (50 ml). Reagent Saturated Phenol(pH-8.0) Chloroform Isoamyl alcohol

Amount 25 ml 24 ml 1 ml

7. Chloroform/Isoamyl alcohol (24:1) (25 ml). Reagent Chloroform Isoamyl alcohol

Amount 24 ml 1 ml

APPENDIX

8. 70% Ethanol (100 ml) Reagent Ethanol Autoclaved distilled water

Amount 70 ml 30 ml

9. 3M Sodium acetate Reagent Sodium acetate Autoclaved distilled water Adjust to pH 5.2 with CH3COOH

Amount 246 g 1000ml

10. TE (10:1) Buffer (pH-8.0) Reagent Tris-HCl Na2EDTA

Amount 10 mM 1mM

11. Ethidium bromide Reagent Ethidiumbromide Autoclaved distilled water

Amount 10mg 1ml

APPENDIX