TECHNIQUES IN GENETIC ENGINEERING
THE AUTHORS ANIL KUMAR did his Masters in biochemistry with specialization in microbiology from G. B. Pant University of Agriculture & Technology, Pantnagar in 1984. He did his PhD in 1990 from University of Rajasthan, Jaipur working in the field of cancer biology and applications of hybridoma technology to characterize differentiation and tumor associated antigen(s). The study concluded that dbc-AMP induced differentiation of cultured human neuroblastoma cell line SK-N-SH is associated with the expression and suppressions of antigenic components present in the cellular organization of neuroblastoma cells. Later, he returned to his almameter in Plant Biotechnology Research Group at Pantnagar and was involved in signal
transduction research in relation to host-pathogen interaction taking Karnal bunt-wheat and Alternaria blight of Brassica as model systems. He is presently engaged on three aspects of Molecular Plant Pthology, i.e. plant disease surveillance through molecular diagnostics, pathogen indexing programme through molecular pathotyping and characterization of disease resiatance and pathogenesis through molecular signaling investigating the role of MAP Kinase and cystatin gene families as candidate genes. Dr. Anil Kumar is professor and department head since 2006. He has also been awarded the Shiksha Rattan Award for service to the cause of Biotech education.
SANJAY MOHAN GUPTA did his Masters in Biotechnology from School of Biotechnology, Devi Ahilya University, Indore in 1999. In his Mater’s dissertation, he worked on enzyme immobilization technology and developed an indigenous bed-reactor using brick dust immobilized CM-Cellulase enzyme isolated from Cowpea (Vigna sinensis L.). He did his PhD degree in biochemistry in 2007 from University of Lucknow, Lucknow and worked on genetic manipulation of banana fruit ripening. He has isolated and characterized ethylene regulated and ripening related 27 novel genes from banana pulp tissue by differential-display (DDRT-PCR) method and also studied ethylene biosynthesis, respiratory climacteric and activities of the various ripening related enzymes during ripening in banana. He is involved in research project
investigating the isolation and characterization of cold stress responsive genes from the high altitude indigenous cold tolerant plants (Hippophae salicifolia and Lepidium latifolium) for genetic transformation of the vegetable crops. He has published 35 research papers in leading national and international journals. He has more than 50 accession numbers of various genes submitted to NCBI & EST database in his credit. He is a coauthor of 02 books and 02 chapters in the field biotechnology. He has delivered various invited lectures/presentations in the national and international symposium/conferences. He is a member of the Indian Science Congress, professional society and other various scientific committees. Recently, he has been awarded with “Laboratory Scientist of the Year 2010” by DRDO.
GOHAR TAJ did her Masters in Biotechnology with specialization in study of papain kinetics under various Environmental Stresses. She also isolated and purified Ovomucoid and checked its Antitryptic Activity from Aligarh Muslim university, Aligarh in 1996. She did her Ph.D in 2002 from G. B. Pant University of Agriculture & Technology, Pantnagar in the field of signal transduction and disease resistance. During her
Ph.D research work Dr Taj developed a transgenic Brassica with introgression of osmotin gene to develop the resistance against stress through perturbation of cell signaling in Brassica juncea. Currently she is working as Assistant Professor in Department of Molecular Biology and Genetic Engineering, CBSH at G.B.P.U.A & T Pantnagar since 2003 and is incaharge of SubDIC bioinformatics.
TECHNIQUES IN GENETIC ENGINEERING ANIL KUMAR
Professor & Head, Department of Molecular Biology & Genetic Engineering College of Basic Sciences & Humanities G B Pant Univeristy of Agriculture & Technology, Pantnagar
SANJAY MOHAN GUPTA
Scientist, Defence Institute of Bio-Energy Research (DIBER), DRDO Govt. of India, Min. of Defence, Goraparao, Haldwani
GOHAR TAJ
Assistant Professor, Department of Molecular Biology & Genetic Engineering College of Basic Sciences & Humanities G B Pant Univeristy of Agriculture & Technology, Pantnagar
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CONTENTS FOREWORD vii PREFACE ix PART I GENE ISOLATION AND CLONING 1−57 1. EXTRACTION OF GENOMIC DNA 13−15 2. PURIFICATION OF DNA 16−17 3. QUANTITATION OF DNA 18−19 4. PLASMID DNA ISOLATION 20−25 5. RESTRICTION DIGESTION OF SOURCE DNA AND VECTOR DNA 26−31 6. LIGATION OF RESTRICTED VECTOR AND INSERT DNA 32−37 7. COMPETENT CELL PREPARATION AND TRANSFORMATION 38−42 8. TRANSFORMATION OF E. coli BY ELECTROPORATION 43−46 9. CALCULATION OF TRANSFORMATION EFFICIENCY 47−48 10. SCREENING TRANSFORMED BACTERIAL COLONIES USING X-GAL AND IPTG: αCOMPLEMENTATION 49−51 11. SCREENING BACTERIAL COLONIES BY HYBRIDIZATION 52−57 PART II CONSTRUCTION OF GENE LIBRARY 59−83 12. CONSTRUCTION OF GENOME WALKER LIBRARIES 60−66 13. CONSTRUCTION OF cDNA LIBRARIES 67−77 14. CONSTRUCTION AND SCREENING OF EUKARYOTIC LIBRARIES 78−83
EXPRESSION
PART III PURIFICATION OF EXPRESSED PROTEINS 85−95 15. ISOLATION AND ANALYSIS OF EXPRESSED PROTEIN FROM e. coli HOST CELLS USING NICKEL AFFINITY CHROMATOGRAPHY 86−93
PART IV GENE TRANSFER INTO PLANTS 95−117 16. AGROBACTERIUM MEDIATED GENETIC TRANSFORMATION OF TOBACCO AND BRASSICA 96−103 17. BIOLISTIC GENE GUN MEDIATED TRANSFORMATION 104−111 18. GENE TRANSFER INTO PROTOPLASTS THROUGH LECTROPORATION 112−117 PART V GENE TRANSFER INTO ANIMAL CELLS 119−128 19. CULTURE OF EMBRYONIC STEM CELLS 120−125 20. GENE TRANSFER INTO EMBRYONIC STEM CELL 126−128 PART VI DETECTION OF TRANSGENE AND ITS EXPRESSION ANALYSIS 129−158 21. PCR AMPLIFICATION FOR CONFIRMATION OF GENE INTEGRATION 130−134 22. DETECTION OF TRANSGENE BY SOUTHERN BLOT ANALYSIS 135−138 23. EXPRESSION PROFILING OF TRANSGENE BY NORTHERN BLOT 139−143 24. WESTERN BLOTTING 144−147 25. RNA ISOLATION AND SELECTION OF POLY (A)+ RNA BY OLIGO (dT) − CELLULOSE CHROMATOGRAPHY 148−154 26. AMPLIFICATION OF CDNA GENERATED BY REVERSE TRANSCRIPTION OF mRNA (RT-PCR METHOD) 155−158 PART VII APPENDICES 159−189 I COMMON CONVERSIONS 159−165 II GEL ELECTROPHORESIS 166 III LAMBHA MARKERS (SIZE IN KB) 163 IV ANTIBIOTICS: MODE OFACTION AND MECHANISM OF RESISTANCE 164 V KEY GENOTYPES OF e. coli 165−167 VI PREPARATION OF COMMON LABORATORY SOLUTIONS 168−169 VII MOLECULAR WEIGHT OF AMINO ACIDS 170 VIII HALF LIFE OF COMMON RADIOISOTOPES 171 IX GEL DOCUMENTATION SYSTEM 172−173 X GLOSSARY 174−186 XI FURTHER READING 187−189
FOREWORD In the last two decades, the discipline of molecular biology has seen a remarkable growth in terms of its achievements. During its early phase, the studies under this discipline were largely confined to elucidation of structure of biological macromolecules such as DNA, RNA and protein. Next, efforts were made to understand structural functional relationship, which exist among theses macromolecules in a cell. These studies eventually culminated into “central dogma of life” which states the genes of an organism are short nucleotide stretches in DNA, which are transcribed into RNA and subsequently translated into protein which has a particular function to perform. Rapid successive advancements in the field led to accumulation of the knowledge, which is now being utilized to make human life better. Molecular level studies have facilitated development of new strategies to solve recalcitrant problems of agriculture, to design new drugs for human health and to uplift socioeconomic status of people. With the completion of human genome sequence, new avenues have opened up for gene therapy of some formidable human diseases. Designer crops have become a reality and hold a significant promise in boosting up agricultural productivity. Completion of genome sequence of Arabidopsis thaliana has attracted plant biologists to solve the agricultural problems with new dimension. Infact the discipline is progressing at a fast pace and its applications are limitless. The excitements in this field have gained a rapid popularity amongst students in a very short time. As a result a number of Indian institutions and universities are running MSc and PhD programmes in Molecular Biology & Biotechnology. Many of them have also started BTech and MTech programmes in biotechnology. Successes of these courses depend on exposure of students to basic molecular biological techniques, which form backbone for advanced research in this exciting area. Acquiring the minimal skills to perform basic molecular biology procedures can be obtained by several means. These include through laboratory coursework, advanced degrees, spending dedicated period of time in laboratory specializing in molecular biology procedures, training courses, etc. Perhaps the most effective are either on-hand training or laboratory experience that leads to an advanced degree. With the same intention, this manual of Techniques in Genetic Engineering has been designed by department of molecular biology and genetic enginnering to cater to the needs of all students of molecular biology and biotechnology as well as related disciplines. I welcome and gratefully acknowledge the efforts of authors for preparation of this manual, which will help
students in learning some of the rudimentary reactions and principles of molecular biology, which are crucial for the analysis of macromolecules involved in the flow of genetic information, i.e. central dogma as proposed by Watson. I am confident that this manual will enable the students not only to build the concepts but also solve thesquest to practice molecular biological techniques. With best wishes K. DHARMALINGAM Senior Professor School of Biotechnology Madurai Kamaraj University, Madurai
PREFACE The practical aspect along with the theoretical background would help in better understanding of the various techniques of genetic engineering. This manual tries to address and compile some of the routinely used protocols in molecular biology and genetic engineering tools for easy access. Its aim is not only to bring together the protocols, but also to explain the basics behind the methodologies followed. The purpose is to provide the students with a view of genetic engineering, especially those who have just ventured into this field and need a head start. The protocols have been written as a handy guide that can be carried as a pocket guide for easy reference. They are easy to follow with each step explained in layman terms. Even though the field of genetic engineering is too exhaustive to be covered in an introductory text like this, yet an effort has been made to list some of the protocols that could serve as a foundation for starting any biotechnological investigation. In addition, in order to keep high academic standard and quality teaching, it is mandatory to have on hands training of students in the various tools and techniques used for exploration of living system. With this intention, we have attempted to write this book so as to provide complete set of practical exercises covering all the essential basic techniques involved in molecular biology. The book has been written in the form of an experimental manual which, we hope, will be useful for various graduate and postgraduate students offering the course for their major and minor in molecular biology and biotechnology. Students belonging to BSc, BTech MSc and MTech programmes taking the experimental course in molecular biology will find it useful in learning the techniques by performing experiments given in the manual. We have experience in teaching the course “Techniques in Genetic Engineering” for the past one decade and based on our experience; the manual has been prepared. The book includes all the basic paradigms and principles used in nucleic acid based analysis and consists of total 26 exercises. Each practical exercise starts with the brief background for understanding the purpose of performing the experiment, followed by reagents required, detailed procedure for the experiment and observations to be recorded. Each lab exercise is presented in an organized and a systematic manner so that the students understand the essential concepts while performing the experiments. The project of preparing the manual was initiated at the behest of our dynamic and visionary leader Dr Rajendra Dobhal, Project Director, State Biotechnology Programme, Haldi who showed us the path to achieve academic excellence in the field
of science, specially the emerging field of biotechnology. Due to his guidance, persuasion and motivation from time-to-time, we have been able to bring this manual in its present shape and for the first time in the hands of our students. We hope that the manual will help students not only to understand the concepts of genetic engineering but also solve their quest to practice the techniques.
ANIL KUMAR, SANJAY MOHAN GUPTA, GOHAR TAJ
