arun pratap singh

145 Pages·2014·12.36 MB·English

Detection of PCR-SSCP markers of the bovine lactoferrin gene and its
association with somatic cell count and lactoferrin content in milk
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 VETERINARY SCIENCE
IN
(ANIMAL GENETICS AND BREEDING)
BY
ARUN PRATAP SINGH
(B.V.Sc. & A.H.)
DAIRY PRODUCTION SECTION
NATIONAL DAIRY RESEARCH INSTITUTE (I.C.A.R.)
SOUTHERN CAMPUS, ADUGODI, BANGALORE- 560030, INDIA
2014
Regd. No.2061210
Detection of PCR-SSCP markers of the bovine lactoferrin gene and its
association with somatic cell count and lactoferrin content in milk
By
Dr. ARUN PRATAP SINGH
THESIS SUBMITIED TO THE
NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY), KARNAL
IN PARTIAL FULFILMENT OF THE REQUIREMENTS
FOR THE AWARD OF THE DEGREE OF
MASTER OF VETERINARY SCIENCE
IN
ANIMAL GENETICS & BREEDING
Approved By
n?'~
Dr. ~.P. Ramesha
Major Advisor
Members of Advisory Committee
Dr. D.N. Das, Principal Scientist fJhv
(Animal Genetics & Breeding, NORI, Bangalore)
j)V;,.U -------.
Dr. S. Isloor, Associate Professor
(Dept. of Vet. Microbiology, Veterinary College, Bangalore) - -----
Dr. Mukund A. Kataktalware, Senior Scientist
~
(Livestock Production Management, NDRI, Bangalore)
Dr. B. Surendranath, Principal Scientist ~
(Dairy Chemistry, NDRI, Bangalore)
(Joint Director's Nominee)
.......
---------------------------
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DAIRY PRODUCTION SECTION
SOUTHERN REGIONAL STATION
NATIONAL DAIRY RESEARCH INSTITUTE
ADUGODI, BAN GALORE- 560030, INDIA
11I~5I"!.~
ICAR
Dr. K. P. Ramesha
Principal Scientist
CERTIFICATE
This is to certify that the thesis entitled, "Detection of PCR-SSCP markers
of the bovine lactoferrin gene and its association with somatic cell
count and lactoferrin content in milk" submitted by ARUN PRATAP SINGH
towards the partial fulfilment of the award of the degree of MASTER OF
VETERINARY SCIENCE (ANIMAL GENETICS AND BREEDING) of the
NATIONAL DAIRY RESEARCH INSTITUTE (DEEMED UNIVERSITY), Karnal
(Haryana), India, is a bonafide research work carried out by him under my
supervision, and no part of the thesis has been submitted for any other
degree or diploma.
:::f<vX'Q
Dated: \ ~\ ch '62 \) \L \-
~?~
(Dr.K. P. Ramesha)
MAJOR ADVISOR & CHAIRMAN
DEDICATED
To my beloved parents}
who taught me to dream and set goals.
To my teachers}
who gave me the tools to achieve those
goals
To my friends}
who taught me how to live and love.
ACKNOWLEDGEMENT
I bow my head with greal reverence Iv Ihe 'Almighty God', who is a cause behind every effect
and without whose blessings this work would never have been accomplished I give all the glory,
honour and praise to the Lord, whose abundant love and grace enabled me to travel
throughout life and come to this stage.
I have great pleasure in expressing my heartfelt appreciation and gratitude to my major advisor,
Dr K. P. Ramesha, Principal Scientist and In-charge of Dairy Production Section, NDRl,
Bangalore, for his inspiring guidance, sustained encouragement, close supervision, keen
int.erest, critical appraisal, judicious planning of the project. His whole hearted cooperation,
patience and benevolence during the entire study have left certain experiences, which are worthy
ofr emembrance throughout life.
I extend my grateful acknowledgement to the members of the advisory committee, Dr. D. N. Das,
Principal Scientist, Dairy Production Section, NDRl, Bangalore, Dr. S. Isloor, Associate
Professor, Dept. of Vet. Microbiology, Veterinary College, Hebbal, Banga/ore, Dr. M. A.
Kataktalware, Scientist - Senior Scale, Dairy Production Section, NDRJ, Bangalore, and
Dr.B.Surendranath, Principal Scientist, Dairy chemistry, NDRl, Bangalore for useful advice
and suggestions during both in my course and project work.
I wish to endow my thanks to Dr. A. K. Srivastava, Director and Vice-Chancellor, NDRI
(DU), Karnal, Dr. G. R. Patil, Joint Director (Research) and Dr. Satish Kulkarni, Head,
SRS, NDRJ, Bangalore for providing the required infrastructural facilities. I also acknowledge
the scholarship provided by NDRI.
I whole-heartedly thank to Dr. B. Surendranath, In-charge Education Section, and Mrs. T. R.
Thivija Kumari for their kind support in academic matters and concern shown to me
throughout my stay at SRS.
I am deeply indebted to Dr. R. S Gandhi, Dr. G. K. Sachdeva, Dr. B.R. Yadav, Dr. A. K. Gupta,
Dr. A. K. Chakravarty, Dr. Avatar Singh, Dr. L D. Gupta, Dr. Archana Verma, Dr. Dr. Vikas
Vohra, Dr. D. K. Sadana, Dr. K. N. Raja, Dr. Ganguly, Dr. Sanjeev Singh, Dr. Anad Jain, Dr.
A. Kumerashan , Dr. Latwal, Dr. Mehla, Dr. S. Jaykumar, Dr. Bandla Srinivas, Dr. M. C. A.
Devi, Dr. Subhash, Dr. Rekha Menon for their timely and unconditional help in the form of
necessary inputs and facilities throughout my M. V Sc. Programme .
.........
----------------------
I would like to thank Sri L. Krishnamoorthy, Technical Officer, Basavaraju, Akhila, Geetha
and Anantharaj for their advice, efficiency and programming skills to get my lab analysis
completed in time.
I gratefully acknowledge the valuable help and all possible cooperation extended by all
staff members of the Dairy Production and Cattle Yard Section with a special mention for
Sri Prakash, Smt Puttamma, Dr Ningaraju, Sri Punyatma, Sri Veerabhadra, Sri Ramulu,
Sri K. Murthy, Sri Puttaraju, Sri Madhesh, Sri Sandeep, Sri Kiran, Sri Chandrashekhar Sri
Ramaswami and Sri Sanjay also I express my whole hearted thanks to all Staff ofL ibrary, SRS,
NDRI, Bangalore.
I am thankful to my seniors Soumya, Rani, Divya, Mundhe Upendra, Veerendra, Mansingh,
Sakendra, Navab Singh, Jatin for their valuable suggestions and help. Sweet memories shared
with and nice ambience provided by Ashwani, Mohsin, Dhaman, Anand, Ajay, Dinesh,
Manvendra, Ramendra, Binoy, Sonali, Silo, Madhav, Snjeev, Trishul, Braj, Kiran, Sayantan,
Puneeth, Prajeesha, Asha, Preeti are unforgettable. I shall never forget the support and endless
love extended (0 me by my friends. I can 'f forget to acknowledge my juniors Akshay, Ashwin,
Sandeep, Rakesh, Swapnil, Ravi, Roshan, Sudin, Ravi, Piyush, Darshan, Christopher, Arpita,
Manoj, Naveen who gave me all the support and help throughout my work. Where emotions are
involved words cease to mean.
Words do not suffice to express my devotion and gratitude to my real soul of life my father Sri
Ram Khiladi Singh and my mother Mrs. Amita Singh, because of whom only, now I am at this
position. There everlasting concern, infinite encouragement and immense love and desire for my
better future. They always helped me in taking all the important decision in my life. My heartiest
love and gratitude to dearest dadaji and deep affection to elder sister Bhavna. It is none else
but to all in my loving family and my guide Dr. K. P. Ramesha that I dedicate this thesis. I thank
them for having faith in me and for their support.
A~*
Bangalore
2sth June, 2014 Registration No. 2061210
CONTENTS
Sr. TITLE Page
No. No.
1 INTRODUCTION 1
2 REVIEW OF LITERATURE 4
2.1 Deoni 4
2.2 Murrah 5
2.3 Structure of Lactoferrin 6
2.4 Concentration of Lactoferrin Protein 8
2.5 Lactoferrin metabolism 9
2.6 Biological functions of lactoferrin 9
2.7 Lactoferrin Gene 16
2.8 Lactoferrin gene polymorphism 18
2.9 DNA isolation 23
2.10 Polymerase Chain Reaction (PCR) 23
2.11 Agarose gel electrophoresis 27
2.12 Identification of new Polymorph isms 27
2.13 Single Strand Conformational Polymorphism (SSCP) 28
2.14 Direct Sequencing 30
2.15 Sequence data analysis 31
3 MATERIALS AND METHODS 34
3.1 Experimental animals 34
3.2 Blood collection 34
3.3 Isolation of DNA by High Salt method 34
3.4 Determination of purity and yield of DNA 35
3.5 Dilution of samples (Preparation of template DNA) for PCR 36
3.6 Primer designing 36
3.7 Standardization of PCR conditions 36
3.8 Polymerase Chain Reaction - Single Strand Confonnation 41
Pol ymorphism(PCR -S SCP)
3.9 Poly Acrylamide Gel Electrophorcsis (PAGE) 42
3.10 Custom Sequencing 45
3.11 Genotype frequencies 46
3.12 Quantitative determination of Bovine lactoferrin (LTF/LF) 46
concentration in milk by using Bovine lactoferrin (L TF/LF) ELISA Kit
3.13 Estimation of somatic cell count 49
3.14 Statistical analysis 50
4 RESULTS AND DISCUSSION 51
4.1 DNA extraction, yield and purity 51
4.2 Standardization of Polymerase Chain Reaction (PCR) for SSCP 51
4.3 Single Strand Conformation Polymorphism (PCR-SSCP) analysis 57
4.4 SequencinK and analysis 67
4.5 Association of SSCP variants on Somatic Cell Count (SCC) and 88
lactoferrin (L TF) content in milk
5 SUMMARY AND CONCLUSIONS 96
BIBLIOGRAPHY
•
APPENDIX
LIST OF TABLES
S.No Title Page No
2.1 Single nucleotide polymorphisms repotied in the coding region of bovine 19
lactoferrin gene
2.2 Association of lactoferrin gene with somatic cell count 20
2.3 Frequency of different SSCP variants found in coding region of bubaline 21
lactoferrin gene loci
2.4 Frequency of different SSCP variants identified in coding region of 22
lactoferrin gene loci in Sahiwal cattle
3.1 Details of primer sequences (5' to 3' sequences) used for amplification 37
3.2 Composition of reaction mixture for PCR for amplification 38
3.3 Thermo cycling conditions for PCR amplification of exons 39
3.4 The conditions of electrophoresis for SSCP analysis 42
3.5 Components in the preparation of PAGE gel 43
4.1 Details of annealing temperature and product size of different Exons 52
4.2 Details of optimized conditions for SSCP analysis 58
4.3 Frequency of SSCP variants in exon2, 3, 6, 8, 9, 14 and 15 oflactoferrin 66
gene in Deoni cattle
4.4 Frequency of SSCP variants in exon4, 5, 10, 13 and 16 oflactoferrin gene 67
in Murrah buffaloes
4.5 Summary of Single nucleotide polymorphisms observed in lactoferrin 69
gene in Deoni cattle
4.6 Summary of Single nucleotide polymorphisms observed in lactoferrin 70
gene in Deoni cattle Murrah buffaloes
4.7 Somatic cell count and lactoferin content in milk of Deoni cattle and 89
Murrah buffaloes
4.8 Effect of SSCP variants and non-genetic factors on Somatic cell count and 91
lactoferrin content in milk
4.9 Least squares means of Somatic cell count (log) in Deoni cattle 91
4.10 Least squares means of lactoferrin (log) in Deoni cattle 92
4.11 Least squares means of Somatic cell count (log) in Murrah buffalo 93
4.12 Least squares means of lactoferrin(log) in Murrah buffalo 94
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LIST OF FIGURES
Figure Title Page
No. No.
2.1 Deoni cow maintained at Cattle Yard, NDRI Southern Campus 4
2.2 Murrah buffalo maintained at commercial farms at Dharwad, Karnataka 5
2.3 Structure of Lactoferrin 6
2.4 Genomic organization of bovine lactoferrin gene 17
4.1 Quality checking of DNA on 0.8% agarose gel 51
4.2 Resolution of PCR amplified product of exon 1 on 1.5% agarose gel 53
4.3 Resolution of PCR amplified product of exon 2 on 1.5% agarose gel 53
4.4 Resolution ofPCR amplified product of exon 3 on 1.5% agarose gel 53
4.5 Resolution of PCR amplified product of exon 4 on 1.5% agarose gel 53
4.6 Resolution ofPCR amplified product of exon 5 on 1.5% agarose gel 54
4.7 Resolution ofPCR amplified product of exon 6 on 1.5% agarose gel 54
4.8 Resolution of PCR amplified product of exon 7 on 1.5% agarose gel 54
4.9 Resolution of PCR amplified product of exon 8 on 1.5% agarose gel 54
4.10 Resolution of PCR amplified product of exon 9 on 1.5% agarose gel 55
4.11 Resolution of PCR amplified product of exon lOon 1.5% agarose gel 55
4.12 Resolution of PCR amplified product of exon lIon 1.5% agarose gel 55
4.13 Resolution of PCR amplified product of exon 12 on 1.5% agarose gel 55
4.14 Resolution of PCR amplified product of exon 13 on 1.5% agarose gel 56
4.15 Resolution ofPCR amplified product of exon 14 on 1.5% agarose gel 56
4.16 Resolution of PCR amplified product of exon 15 on 1.5% agarose gel 56
4.17 Resolution of PCR amplified product of exon 16 on 1.5% agarose gel 56
4.18 Resolution of PCR amplified product of exon 17 on 1.5% agarose gel 56
4.19 PCR-SSCP patterns of exon 1 of lactoferrin gene in Deoni cattle 58
4.20 PCR-SSCP patterns of exon 4 of lactoferrin gene in Deoni cattle 58
4.21 PCR-SSCP patterns of exon 5 of lactoferrin gene in Deoni cattle 58
4.22 PCR-SSCP patterns of exon 7 of lactoferrin gene in Deoni cattle 58
4.23 PCR-SSCP patterns of exon 10 of lactoferrin gene in Deoni cattle 59
4.24 PCR-SSCP patterns of exon II of lactoferrin gene in Deoni cattle 59
4.25 PCR-SSCP patterns of exon 12 of lactoferrin gene in Deoni cattle 59
4.26 PCR-SSCP patterns of exon 13 oflactoferrin gene in Deoni cattle 59
4.27 PCR-SSCP patterns of exon 16 of lactoferrin gene in Deoni cattle 59
4.28 PCR-SSCP patterns of exon 17 oflactoferrin gene in Deoni cattle 59
4.29 PCR-SSCP patterns of exon I oflactoferrin gene in Murrah buffaloes 60
4.30 PCR-SSCP patterns of exon 2 of lactoferrin gene in Murrah buffaloes 60
4.31 PCR-SSCP patterns of exon 3 of lactoferrin gene in Murrah buffaloes 60
4.32 PCR-SSCP patterns of exon 6 of lactoferrin gene in Murrah buffaloes 60
4.33 PCR-SSCP patterns of exon 7 of lactofelTin gene in Murrah buffaloes 60
4.34 PCR-SSCP patterns of exon 8 of lactoferrin gene in Murrah buffaloes 60
4.35 PCR-SSCP patterns of exon 9 of lactoferrin gene in Murrah buffaloes 61
4.36 PCR-SSCP patterns of exon 11 of lactofemn gene in Murrah buffaloes 61
4.37 PCR-SSCP patterns of exon 12 of lactofemn gene in Murrah buffaloes 61
4.38 PCR-SSCP patterns of exon 14 of lactoferrin gene in Murrah buffaloes 61
4.39 PCR-SSCP patterns of exon 15 of lactofemn gene in Murrah buffaloes 61
4.40 PCR-SSCP patterns of exon 17 of lactoferrin gene in Murrah buffaloes 61
4.41 PCR-SSCP patterns of exon 2 oflactoferrin gene in-Deoni cattle 62
4.42 PCR-SSCP patterns of exon 3 of lactofemn gene in Deoni cattle 63
4.43 PCR-SSCP patterns of exon 6 of lactoferrin gene in Deoni cattle 63
4.44 PCR-SSCP patterns of exon 8 of lactoferrin gene in Deoni cattle 63
4.45 PCR-SSCP patterns of exon 9 of lactoferrin gene in Deoni cattle 63
4.46 PCR-SSCP patterns of exon 14 of lactofemn gene in Deoni cattle 64
4.47 PCR-SSCP patterns of exon 15 of lactofemn gene in Deoni cattle 64
4.48 PCR-SSCP patterns of exon 4 of lactoferrin gene in Murrah buffaloes 65
4.49 PCR-SSCP patterns of exon 5 of lactoferrin gene in Murrah buffaloes 65
4.50 PCR-SSCP patterns of exon 10 of lactoferrin gene in Murrah buffaloes 65
4.51 PCR-SSCP patterns of exon 13 of lactoferrin gene in Murrah buffaloes 65
4.52 PCR-SSCP patterns of exon 16 of lactofemn gene in Murrah buffaloes 66
4.53 Clustal W multiple alignment sequence of exon 2 in Deoni cattle 72
4.54 Sanger Trace Figures of SSCP variant sites of exon 2 in Deoni cattle 72
4.55 Clustal W multiple alignment sequence of exon 3 in Deoni cattle 73
4.56 Sanger Trace Figures of SSCP variant sites of exon 3 in Deoni cattle 73
4.57 Clustal W multiple alignment sequence of exon 6 in Deoni cattle 74
4.58 Sanger Trace Figures of SSCP variant sites of exon 6 in Deoni cattle 74
4.59 Clustal W multiple alignment sequence of exon 8 in Deoni cattle 75
4.60 Sanger Trace Figures of SSCP variant sites of exon 8 in Deoni cattle 76
4.61 Clustal W multiple alignment sequence of exon 9 in Deoni cattle 77
4.62 Sanger Trace Figures of SSCP variant sites of exon 9 in Deoni cattle 78
4.63 Clustal W multiple alignment sequence of exon 14 in Deoni cattle 79
4.64 Sanger Trace Figures of SSCP variant sites of exon 14 in Deoni cattle 79
4.65 Clustal W multiple alignment sequence of exon 15 in Deoni cattle 80