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MZO-02 Vardhman Mahaveer Open University, Kota Cell, Molecular Biology and Biotechnology MZO-02 Vardhman Mahaveer Open University, Kota Cell, Molecular Biology and Biotechnology Course Development Committee Chair Person Prof. Vinay Kumar Pathak Vice-Chancellor Vardhman Mahaveer Open University, Kota Coordinator and Members Convener SANDEEP HOODA Department of Zoology School of Science & Technology Vardhman Mahaveer Open University, Kota Members  Prof. L.R.Gurjar  Dr. Anuradha Dubey Director (Academic) Deputy Director Vardhman Mahaveer Open School of Science & Technology University, Kota Vardhman Mahaveer Open University, Kota  Dr. Arvind Pareek  Prof. K.K. Sharma Director (Regional Centre) MDSU,Ajmer Vardhman Mahaveer Open University, Kota  Prof. Maheep Bhatnagar  Prof. S.C. Joshi MLSU, Udaipur University of Rajasthan, Jaipur  Dr. Anuradha Singh  Dr. M.M.Ranga Department of Zoology Department of Zoology Govt. College, Kota Govt. College, Ajmer Editing and Course Writing Editor SANDEEP HOODA Assistant Professor & Convener of Zoology School of Science & Technology Vardhman Mahaveer Open University, Kota Writing Writer Name Unit Writer Name Unit No. No. Dr. Subhash Chandra 1 Sandeep Hooda 2 Director (Regional Centre) Department of Zoology Vardhman Mahaveer Open S c h o o l o f S c i e n c e & Technology University, Kota Vardhman Mahaveer Open University, Kota Dr. Digvijay Singh 3, 4 Dr. Anima Sharma 5,8,9,10, Lecture in Zoology Dept. of Biotechnology, JECRC 13 AssistantDirector, Bikaner University, Jaipur Directorate of College Education, Rajasthan Dr. Arvind Pareek 6, 16 Dr. Farah Sye 7 Director (Regional Centre) Dept. of Zoology, Vardhman Mahaveer Open University of Rajasthan, Jaipur University, Kota Dr. Meera Shrivastva Dept. of Zoology Govt. Dungar College, Bikaner Dr. Ruchi Seth 11 Dr. Hardik Pathak 12, 15 Dept. of Biotechnology, D e p t. of Biotechnology, JECRC University, Jaipur JECRC University Jaipur Dr. Abhisekh Vashistha 14 Dept. of Microbiology, Maharaja Ganga Singh University, Bikaner Academic and Administrative Management Prof. Vinay Kumar Pathak Prof. L.R. Gurjar Vice-Chancellor Director (Academic) Vardhman Mahaveer Open University, Kota Vardhman Mahaveer Open University, Kota Prof. Karan Singh Dr. Subodh Kumar Director (MP&D) Additional Director (MP&D) Vardhman Mahaveer Open University, Kota Vardhman Mahaveer Open University, Kota ISBN : All Right reserved. No part of this Book may be reproduced in any form by mimeograph or any other means without permission in writing from V.M. Open University, Kota. Printed and Published on behalf of the Registrar, V.M. Open University, Kota. Printed by : MZO-02 Vardhman Mahaveer Open University, Kota Index Unit No. Unit Name Page No. Unit - 1 Microscopy 1 Unit - 2 Cytological techniques: Staining techniques ,isolation and 11 fractionation of cell , Cell Culture,. Centrifugation and Ultracentrifugation, Electrophoresis, Chromatography, Cytometry Unit - 3 Plasma membrane and intracellular compartments 38 Unit - 4 Vesicular Traffic Organelles 60 Unit - 5 Energy transducers and other organelles 75 Unit - 6 Cell Signalling 86 Unit - 7 Signaling pathways in malignant transformation of cells, 102 cell transformation, role of oncogenes, siRNA and miRNA basics, regulation of transcription and translation of proteins by miRNA Unit - 8 Chromosomes 125 Unit - 9 Genome expression analysis: FISH, GISH, M-FISH, 141 Minichromosomes and Giant chromosomes Gene expression; Process of gene expression: Translation, Transcription; Gene control regions; Gene expression analysis techniques; Giant chromosomes Minichromosomes Unit - 10 Cell cycle, Mitosis and Meiosis 162 Unit - 11 Biotechnology basics: Genetic engineering, culture media, 179 culture methods, restriction enzymes, cloning vectors, somatic hybridization Unit - 12 Recombinant DNA Technology 232 Unit - 13 Bioreactors and downstream processing 257 Unit - 14 Biotechnology in Medicine 272 Unit - 15 Molecular mapping of Genome 316 Unit - 16 Gene Regulation 344 MZO-02 Vardhman Mahaveer Open University, Kota Preface The present book entitled “Cell, Molecular Biology and Biotechnology” has been designed so as to cover the unit-wise syllabus of MZ-02 course for M.Sc. Zoology (Previous) students of Vardhman Mahaveer Open University, Kota. The basic principles and theory have been explained in simple, concise and lucid manner. Adequate examples, diagrammes, photographs and self-learning exercises have also been included to enable the students to grasp the subject easily. The unit writers have consulted various standard books and internet on the subject and they are thankful to the authors of these reference books. ---------------- Unit-1 Microscopy Structure of the Unit 1.0 Objectives 1.1 Introduction 1.1.1 Refractive index 1.1.2 The Lenses 1.2 Light Microscopy 1.3 Electron Microscopy 1.4 Atomic Force Microscopy 1.5 Summary 1.6 Self-Learning Exercise 1.7 References 1.0 Objectives After going through this unit you will be able to understand  How small organisms and small sections of animal and plants may be enlarged using lenses.  The mechanisms of Microscopes to resolve the objects.  That different microscopes are used to study different small objects as we are not able observe them by necked eyes. 1.1 Introduction This is the most essential lesson for a student before you start reading about Instrumentation. Different scientists have contributed a lot to develop Microscopes. Antony van Leeuwenhoek (1632-1723) was the first person to observe and describe micro-organisms accurately. The optical microscope was first used systematically by Robert Hooke in 1864 to study polished sections of opaque materials, notably metals and alloys, and, he was able to reveal distinct phases in a microstructure. Notably, the optical microscope remains the fundamental tool for phase identification. It magnifies an image by sending a beam of light through the object. The condenser lens 1 focuses the light on the sample and the objective lenses (10X to 100-2000X) magnify the beam, which contains the image, to the projector lens so the image can be viewed by the observer. In compound microscopes there is combined effect of two or more than two lenses where as generally one lens is used to enlarge the object in dissecting microscopes. We shall discuss compound microscopes in detail here. The lenses are used in Microscopes and there is the bending of light when passing through lenses. As you know light is refracted (bent) when passing from one medium to another. 1.1.1 Refractive index It is a measure of how greatly a substance slows the velocity of light. One important part is that; direction and magnitude of bending is determined by the refractive indexes of the two media forming the interface 1.1.2 The Lenses The Focus light rays at a specific place called the focal point and the distance between center of lens and focal point is the focal length. One another important aspect is that the strength of lens is related to focal length. Thus; if the short focal length, there will be more magnification. 1.2 The Light Microscope Many types of light microscopes have been discovered and they are called as per their light background as well as their functioning. They have different properties also (Table 1). The light microscopes are of following types: 1. Bright-field microscope 2. Dark –field microscope 3. Phase-contrast microscope 4. Fluorescence microscopes These are compound microscopes as image is formed by action of two or more than lenses. 1. The Bright –Field Microscope This microscope produces a dark image against a brighter background. There are several objective lenses are present in the microscope. Total magnification is the product of the magnifications of the ocular lens and the objective lens. 2 Microscope Resolution is the ability of a lens to separate or distinguish small objects that are close together. Wavelength of light used is a major factor in the resolution. As we have discussed earlier that if we use shorter wavelength, there will be greater resolution. Table 1: The properties of microscope objectives Objective Property Scannin Low High oil g power power Immersion Magnification 4x 10x 40-45x 90-100x Numerical aperture 0.10 0.25 0.55-0.65 1.25-1.4 Approximate focal length(f) 40mm 16mm 4mm 1.8-2.0mm Working distance 17- 4-8mm 0.5-.7mm 0.1mm 20mm Approximate resolving 2.3um 0.9um 0.35um 0.18um Power with light of 450nm (blue light) 3