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Protocols used in Molecular Biology Edited by Sandeep Kumar Singh Indian Scientific Education and Technology Foundation, Lucknow-226002, India Centre of Biomedical Research, SGPGI Campus, Lucknow-226014, India & Dhiraj Kumar Rameshwar College, B.R.A. Bihar University, Muzaffarpur-842001, India Protocols Used in Molecular Biology Editor: Sandeep Kumar Singh ISBN (Online): 9789811439315 ISBN (Print): 9789811439292 © 2020, Bentham eBooks imprint. Published by Bentham Science Publishers Pte. Ltd. Singapore. All Rights Reserved. BENTHAM SCIENCE PUBLISHERS LTD. End User License Agreement (for non-institutional, personal use) This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the ebook/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work. 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Ltd. 80 Robinson Road #02-00 Singapore 068898 Singapore Email: [email protected] CONTENTS FOREWORD ........................................................................................................................................... i PREFACE ................................................................................................................................................ iii LIST OF CONTRIBUTORS .................................................................................................................. iv CHAPTER 1 ISOLATION OF GENOMIC DNA FROM PLANT TISSUES ................................ 1 (cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 1 Principle .................................................................................................................................. 1 Sample Collection and Storage Conditions ............................................................................ 2 CTAB DNA Extraction Buffer: 100 ml .................................................................................. 3 T10E1 Buffer .......................................................................................................................... 4 DNA Isolation Protocol .......................................................................................................... 4 Purification .............................................................................................................................. 4 Useful Tips and Suggestions for Obtaining Optimum Results ............................................... 5 CONSENT FOR PUBLICATION ................................................................................................ 5 CONFLICT OF INTEREST ......................................................................................................... 5 ACKNOWLEDGEMENTS ........................................................................................................... 6 REFERENCES ............................................................................................................................... 6 CHAPTER 2 RNA ISOLATION PROTOCOL FROM CELLS AND TISSUES ........................... 7 (cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 7 Precautions to be Taken Before the Experiment ..................................................................... 8 I. Isolation of RNA Using TRIzol Method ............................................................................. 8 Principle .................................................................................................................................. 8 Materials Required .................................................................................................................. 9 Steps ........................................................................................................................................ 9 II. Isolation of RNA Using the CTAB Method ....................................................................... 9 Materials Required .................................................................................................................. 9 Steps ........................................................................................................................................ 10 III. Hot-Phenol RNA Extraction Method ............................................................................... 10 Materials Required .................................................................................................................. 10 Steps ........................................................................................................................................ 11 IV. QIAGEN RNeasy Kit Method .......................................................................................... 11 DNase Treatment .................................................................................................................... 12 Storage of RNA ....................................................................................................................... 12 Determining RNA Quality and Quantity ................................................................................ 12 Formaldehyde Agarose Gel Electrophoresis .......................................................................... 13 Materials Required .................................................................................................................. 13 Preparation of Formaldehyde Agarose Gel ............................................................................. 13 Preparation of RNA Samples for Electrophoresis ................................................................... 13 CONSENT FOR PUBLICATION ................................................................................................ 13 CONFLICT OF INTEREST ......................................................................................................... 14 ACKNOWLEDGEMENTS ........................................................................................................... 14 REFERENCES ............................................................................................................................... 14 CHAPTER 3 ANALYZING GENE EXPRESSION THROUGH REAL TIME PCR WHILE NEO-TISSUE REGENERATION USING DEVE- LOPED TISSUE CONSTRUCTS .................... 15 (cid:39)(cid:76)(cid:89)(cid:68)(cid:78)(cid:68)(cid:85)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:55)(cid:68)(cid:85)(cid:88)(cid:81)(cid:3)(cid:48)(cid:76)(cid:81)(cid:82)(cid:70)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:87)(cid:92)(cid:68)(cid:89)(cid:85)(cid:68)(cid:87)(cid:3)(cid:55)(cid:85)(cid:76)(cid:83)(cid:68)(cid:87)(cid:75)(cid:76)(cid:15)(cid:3)(cid:53)(cid:88)(cid:83)(cid:76)(cid:78)(cid:68)(cid:3)(cid:54)(cid:76)(cid:81)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:88)(cid:69)(cid:75)(cid:68)(cid:81)(cid:78)(cid:68)(cid:85) (cid:36)(cid:81)(cid:68)(cid:81)(cid:71)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:57)(cid:76)(cid:89)(cid:72)(cid:78)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:81)(cid:71)(cid:72)(cid:92)(cid:15)(cid:3)(cid:36)(cid:85)(cid:88)(cid:81)(cid:3)(cid:53)(cid:68)(cid:90)(cid:68)(cid:87)(cid:15)(cid:3)(cid:54)(cid:80)(cid:76)(cid:87)(cid:68)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:81)(cid:77)(cid:72)(cid:72)(cid:89) (cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:15)(cid:3)(cid:51)(cid:68)(cid:90)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:39)(cid:88)(cid:69)(cid:72)(cid:92)(cid:3)and(cid:3)(cid:51)(cid:85)(cid:68)(cid:71)(cid:72)(cid:72)(cid:83)(cid:3)(cid:54)(cid:85)(cid:76)(cid:89)(cid:68)(cid:86)(cid:87)(cid:68)(cid:89)(cid:68) INTRODUCTION .......................................................................................................................... 16 BASICS OF RT-PCR ..................................................................................................................... 17 Merits of RT-PCR ................................................................................................................... 18 PCR Cycling ........................................................................................................................... 19 RT-PCR Master Mix Components .......................................................................................... 20 Thermostable DNA Polymerase ............................................................................................. 21 Deoxyribonucleotidetriphosphates (dNTPs) ........................................................................... 21 Divalent Cation Especially Magnesium Ions .......................................................................... 21 Template ................................................................................................................................. 21 RT-PCR Primer Design .......................................................................................................... 22 Primer Designing .................................................................................................................... 22 Basics of Primer Designing .................................................................................................... 23 RT-PCR Fluorescence Detection Chemistry .......................................................................... 23 SYBR Green-based Detection ................................................................................................ 24 Mechanism of Action .............................................................................................................. 24 Advantages .............................................................................................................................. 24 Limitations .............................................................................................................................. 24 TAQMAN probe Based Detection .......................................................................................... 25 Mechanism of Action .............................................................................................................. 25 Advantages .............................................................................................................................. 26 Limitations .............................................................................................................................. 27 RT-PCR Experimental Design and Gene Expression Profiling ............................................. 27 Setting up a Reaction Mixture ................................................................................................ 28 Basic PCR Protocol and Establishment of Reaction Cycle .................................................... 29 Application of RT-PCR .......................................................................................................... 30 CHALLENGES ............................................................................................................................... 31 CONSENT FOR PUBLICATION ................................................................................................ 32 CONFLICT OF INTEREST ......................................................................................................... 32 ACKNOWLEDGEMENTS ........................................................................................................... 32 REFERENCES ............................................................................................................................... 32 CHAPTER 4 A MODIFIED WESTERN BLOT PROTOCOL FOR ENHANCED SENSITIVITY IN THE DETECTION OF A TISSUE PROTEIN ............................................................................... 35 (cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:48)(cid:68)(cid:79)(cid:79)(cid:76)(cid:78)(cid:68)(cid:85)(cid:77)(cid:88)(cid:81)(cid:68)(cid:3)(cid:53)(cid:68)(cid:82)(cid:3)(cid:42)(cid:72)(cid:71)(cid:71)(cid:68)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81) (cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:51)(cid:68)(cid:92)(cid:68)(cid:79)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77)(cid:3)(cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:53)(cid:68)(cid:78)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 35 SAMPLE PREPARATION ........................................................................................................... 36 GEL ELECTROPHORESIS ......................................................................................................... 37 BLOTTING ..................................................................................................................................... 38 WASHING, BLOCKING AND ANTIBODY INCUBATION ................................................... 39 QUANTIFICATION ...................................................................................................................... 40 TROUBLESHOOTING ................................................................................................................. 40 CONCLUSION ............................................................................................................................... 42 CONSENT FOR PUBLICATION ................................................................................................ 42 CONFLICT OF INTEREST ......................................................................................................... 42 ACKNOWLEDGEMENTS ........................................................................................................... 42 REFERENCES ............................................................................................................................... 42 CHAPTER 5 IMMUNOHISTOCHEMISTRY AS AN IMPORTANT TECHNIQUE IN EXPERIMENTAL AND CLINICAL PRACTICES ............................................................................ 44 (cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77) (cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:36)(cid:76)(cid:77)(cid:68)(cid:93)(cid:3)(cid:36)(cid:17)(cid:3)(cid:49)(cid:68)(cid:76)(cid:78)(cid:15)(cid:3)(cid:36)(cid:81)(cid:68)(cid:80)(cid:76)(cid:78)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:85)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:76)(cid:78)(cid:75)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:85)(cid:68)(cid:87)(cid:76)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 44 DIRECT TECHNIQUE ................................................................................................................. 45 NEW DIRECT TECHNIQUE (ENHANCED POLYMER ONE STEPS TRAINING METHOD) ....................................................................................................................................... 45 TWO-STEP INDIRECT TECHNIQUE ....................................................................................... 46 POLYMER CHAIN TWO STEP INDIRECT TECHNIQUE ................................................... 46 UNLABELED ANTIBODY ENZYME COMPLEX TECHNIQUES (PAP AND APAP) ...... 46 IMMUNOGOLD SILVER STAINING TECHNIQUE (IGSS) .................................................. 47 THE PRINCIPLE OF IHC ............................................................................................................ 48 TISSUE PREPARATION .............................................................................................................. 48 ANTIGEN RETRIEVAL ............................................................................................................... 50 A. Proteolytic Enzyme Digestion ........................................................................................... 50 B. Heat Mediated Antigen Retrieval Techniques ................................................................... 51 ANTIGEN-ANTIBODY INTERACTION ................................................................................... 51 AVIDIN–BIOTIN COMPLEX ...................................................................................................... 52 HAPTEN LABELLING TECHNIQUE ....................................................................................... 52 DETECTION METHODS ............................................................................................................. 52 QUANTIFICATION OF THE IMMUNOHISTOCHEMICAL STAINING ........................... 54 APPLICATION AND IMPORTANCE ........................................................................................ 54 TROUBLES AND LIMITATIONS .............................................................................................. 55 TROUBLESHOOTING OF THE EXPERIMENTAL PROBLEMS ..................................... 55 IMMUNOENZYMATIC LABELLING PROTOCOL FOR LOCALIZA- TION OF ASTROCYTES USING ANTI-GFAP ANTIBODY .................................................................... 55 FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 55 DAY 2 PROCESSING ........................................................................................................... 56 IMMUNOHISTOCHEMICAL PROTOCOL FOR LABELLING ASTRO- CYTES USING ANTI-GFAP BY IMMUNE-FLUORESCENCE METHOD ...................................................... 56 FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 56 DAY 2 PROCESSING ........................................................................................................... 57 CONSENT FOR PUBLICATION ................................................................................................ 57 CONFLICT OF INTEREST ......................................................................................................... 57 ACKNOWLEDGEMENTS ........................................................................................................... 57 REFERENCES ............................................................................................................................... 57 CHAPTER 6 PROTOCOLS FOR THE DETECTION AND PROTEOME ANALYSIS OF THE YELLOW MOSAIC VIRUS INFECTED SOYABEAN LEAVES .................................................... 60 (cid:37)(cid:68)(cid:83)(cid:68)(cid:87)(cid:79)(cid:68)(cid:3)(cid:46)(cid:72)(cid:86)(cid:68)(cid:89)(cid:68)(cid:3)(cid:51)(cid:68)(cid:89)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:68)(cid:83)(cid:68)(cid:87)(cid:75)(cid:85)(cid:88)(cid:71)(cid:88)(cid:3)(cid:46)(cid:68)(cid:81)(cid:68)(cid:78)(cid:68)(cid:79)(cid:68) INTRODUCTION .......................................................................................................................... 60 METHOD ........................................................................................................................................ 61 Protein Extraction and Virus Detection .................................................................................. 61 Iso-electric Focusing ............................................................................................................... 62 SDS-PAGE/ 2nd Dimension ................................................................................................... 62 Colloidal Coomassie Staining ................................................................................................. 63 Image Analysis ........................................................................................................................ 64 CONSENT FOR PUBLICATION ................................................................................................ 64 CONFLICT OF INTEREST ......................................................................................................... 64 ACKNOWLEDGEMENTS ........................................................................................................... 64 ABBREVIATIONS ......................................................................................................................... 64 REFERENCES ............................................................................................................................... 64 CHAPTER 7 2D-DIGE A POWERFUL TOOL FOR PROTEOME ANALYSIS .......................... 67 (cid:54)(cid:88)(cid:71)(cid:75)(cid:76)(cid:85)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:75)(cid:72)(cid:78)(cid:75)(cid:68)(cid:85)(cid:15)(cid:3)(cid:45)(cid:68)(cid:76)(cid:3)(cid:42)(cid:82)(cid:71)(cid:75)(cid:72)(cid:77)(cid:68)(cid:3)and(cid:3)(cid:39)(cid:76)(cid:81)(cid:72)(cid:86)(cid:75)(cid:3)(cid:53)(cid:68)(cid:77)(cid:3)(cid:48)(cid:82)(cid:71)(cid:76) INTRODUCTION .......................................................................................................................... 68 Methodology ........................................................................................................................... 68 Steps of the Experiment .......................................................................................................... 70 ADVANTAGES OF DIGE ............................................................................................................. 71 Applications of DIGE ............................................................................................................. 72 CONCLUSION ............................................................................................................................... 72 CONSENT FOR PUBLICATION ................................................................................................ 72 CONFLICT OF INTEREST ......................................................................................................... 72 ACKNOWLEDGEMENTS ........................................................................................................... 72 REFERENCES ............................................................................................................................... 72 CHAPTER 8 MOLECULAR TECHNIQUES FOR GENOTYPING .............................................. 74 (cid:54)(cid:75)(cid:68)(cid:79)(cid:76)(cid:81)(cid:76)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:82)(cid:80)(cid:68)(cid:79)(cid:76)(cid:3)(cid:54)(cid:68)(cid:81)(cid:92)(cid:68)(cid:79)(cid:15)(cid:3)(cid:54)(cid:88)(cid:85)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:3)and(cid:3)(cid:48)(cid:68)(cid:71)(cid:68)(cid:81)(cid:3)(cid:47)(cid:68)(cid:79)(cid:3)(cid:37)(cid:85)(cid:68)(cid:75)(cid:80)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:87)(cid:87) 74 INTRODUCTION .......................................................................................................................... 74 1. Saliva Collection and Storage ............................................................................................. 75 2. Preparation for Cell Lysis ................................................................................................... 76 3. RNA Removal ..................................................................................................................... 76 4. Protein and Lipid Removal ................................................................................................. 76 5. Isolation and Purification of Genomic DNA ...................................................................... 76 6. Rehydration of g DNA ........................................................................................................ 77 Several Protocols Have Been Developed to Isolate DNA from Saliva ................................... 77 Sample Collection ................................................................................................................... 77 DNA Extraction ...................................................................................................................... 78 DNA Evaluation ...................................................................................................................... 78 Methods for DNA Extraction from Whole Blood .................................................................. 79 Two-step Lysis ........................................................................................................................ 79 One-step Lysis ........................................................................................................................ 79 Isolating Genomic DNA Using Magnetic Beads .................................................................... 79 GENOMIC DNA ISOLATION OF DNA FROM BLOOD ........................................................ 80 Reagents Required .................................................................................................................. 80 Procedure ................................................................................................................................ 80 VISUALIZATION OF DNA THROUGH AGAROSE GEL ELECTRO- PHORESIS .......... 81 Materials Required .................................................................................................................. 81 METHOD ............................................................................................................................... 81 Range of separation of linear DNA of different get concentrations ....................................... 81 Reagents .................................................................................................................................. 83 6x Gel-loading Buffer .................................................................................................... 83 DNA Staining Solution (10mg/ml) ......................................................................................... 83 EDTA (0.5M pH 8) ................................................................................................................. 83 TAE (50X) .............................................................................................................................. 83 TBE (5X) ................................................................................................................................ 83 TPE (10X) ............................................................................................................................... 83 POLYACRYLAMIDE GEL ELECTROPHORESIS ................................................................. 83 Materials ................................................................................................................................. 83 Buffers and Solutions .................................................................................................... 83 METHOD ........................................................................................................................................ 84 POLYMERASE CHAIN REACTION ......................................................................................... 86 Materials Required .................................................................................................................. 86 Method .................................................................................................................................... 86 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ................................... 87 Reagents Required .................................................................................................................. 87 Method .................................................................................................................................... 87 CONSENT FOR PUBLICATION ................................................................................................ 87 CONFLICT OF INTEREST ......................................................................................................... 87 ACKNOWLEDGEMENTS ........................................................................................................... 88 REFERENCES ............................................................................................................................... 88 CHAPTER 9 SODIUM BISULFITE CONVERSION OF HUMAN GENOME FOR DNA METHYLATION STUDIES .................................................................................................................. 89 (cid:36)(cid:68)(cid:86)(cid:87)(cid:75)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:75)(cid:85)(cid:68)(cid:3)and(cid:3)(cid:52)(cid:68)(cid:71)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:86)(cid:75)(cid:68) INTRODUCTION .......................................................................................................................... 90 Mapping Whole Genome DNA Methylation Using Sodium Bisulfite Conversion of DNA Followed by Next-generation Sequencing .............................................................................. 92 Sodium Bisulfite Conversion of DNA ............................................................................ 92 Bisulfite Conversion Protocol ................................................................................................. 93 CONCLUDING REMARKS ......................................................................................................... 95 CONSENT FOR PUBLICATION ................................................................................................ 95 CONFLICT OF INTEREST ......................................................................................................... 96 ACKNOWLEDGEMENTS ........................................................................................................... 96 REFERENCES ............................................................................................................................... 96 CHAPTER 10 CHROMATIN IMMUNOPRECIPITATION (CHIP) ............................................. 97 (cid:46)(cid:68)(cid:89)(cid:92)(cid:68)(cid:81)(cid:77)(cid:68)(cid:79)(cid:76)(cid:3)(cid:54)(cid:75)(cid:68)(cid:85)(cid:80)(cid:68)(cid:15)(cid:3)(cid:54)(cid:88)(cid:69)(cid:68)(cid:86)(cid:75)(cid:3)(cid:38)(cid:75)(cid:68)(cid:81)(cid:71)(cid:85)(cid:68)(cid:3)(cid:54)(cid:82)(cid:81)(cid:78)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:54)(cid:75)(cid:68)(cid:78)(cid:88)(cid:81)(cid:87)(cid:68)(cid:79)(cid:68)(cid:3)(cid:48)(cid:68)(cid:75)(cid:76)(cid:79)(cid:78)(cid:68)(cid:85) OVERVIEW OF CHIP ASSAY .................................................................................................... 98 GENERAL DESCRIPTION OF CHIP PROTOCOL ................................................................ 99 1. Cross-Linking ..................................................................................................................... 99 2. Cell Lysis ............................................................................................................................ 100 3. Shearing of DNA ................................................................................................................ 100 4. Immunoprecipitation ........................................................................................................... 100 5. Reversal of Cross-links and Characterization of DNA ....................................................... 101 6. Analysis of DNA ................................................................................................................. 101 Requirements .......................................................................................................................... 101 Troubleshooting ...................................................................................................................... 105 Limitation ................................................................................................................................ 105 Variation ................................................................................................................................. 106 APPLICATION .............................................................................................................................. 109 CONSENT FOR PUBLICATION ................................................................................................ 110 CONFLICT OF INTEREST ......................................................................................................... 110 ACKNOWLEDGEMENTS ........................................................................................................... 110 REFERENCES ............................................................................................................................... 110 CHAPTER 11 OSTEOSARCOMA CELL CULTURE AND MAINTENANCE TO DETECT THE APOPTOTIC EFFECT OF SOME PROMISING COMPOUNDS BY POTENT MARKERS VIZ. DNA FRAGMENTATION AND CASPASE-3 ACTIVATION ................................................. 114 (cid:36)(cid:86)(cid:76)(cid:73)(cid:3)(cid:45)(cid:68)(cid:73)(cid:85)(cid:76)(cid:15)(cid:3)(cid:45)(cid:88)(cid:75)(cid:76)(cid:3)(cid:53)(cid:68)(cid:76)(cid:86)(cid:15)(cid:3)(cid:54)(cid:88)(cid:71)(cid:75)(cid:76)(cid:85)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:48)(cid:71)(cid:3)(cid:36)(cid:85)(cid:86)(cid:75)(cid:68)(cid:71) INTRODUCTION .......................................................................................................................... 114 Osteosarcoma Cells Culture and Maintenance ....................................................................... 115 DNA Fragmentation ................................................................................................................ 115 Detection of Apoptosis via Caspase-3 Activity in Cancer Cells ............................................ 117

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Protocols used in Molecular Biology PDF

186 Pages·2020·5.221 MB·English
by  Sandeep Singh, Dhiraj Divakar
#Biology #Molecular
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Protocols used in Molecular Biology Edited by Sandeep Kumar Singh Indian Scientific Education and Technology Foundation, Lucknow-226002, India Centre of Biomedical Research, SGPGI Campus, Lucknow-226014, India & Dhiraj Kumar Rameshwar College, B.R.A. Bihar University, Muzaffarpur-842001, India Protocols Used in Molecular Biology Editor: Sandeep Kumar Singh ISBN (Online): 9789811439315 ISBN (Print): 9789811439292 © 2020, Bentham eBooks imprint. Published by Bentham Science Publishers Pte. Ltd. Singapore. All Rights Reserved. BENTHAM SCIENCE PUBLISHERS LTD. End User License Agreement (for non-institutional, personal use) This is an agreement between you and Bentham Science Publishers Ltd. Please read this License Agreement carefully before using the ebook/echapter/ejournal (“Work”). Your use of the Work constitutes your agreement to the terms and conditions set forth in this License Agreement. If you do not agree to these terms and conditions then you should not use the Work. 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Any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims) will be governed by and construed in accordance with the laws of Singapore. Each party agrees that the courts of the state of Singapore shall have exclusive jurisdiction to settle any dispute or claim arising out of or in connection with this License Agreement or the Work (including non-contractual disputes or claims). 2. Your rights under this License Agreement will automatically terminate without notice and without the need for a court order if at any point you breach any terms of this License Agreement. In no event will any delay or failure by Bentham Science Publishers in enforcing your compliance with this License Agreement constitute a waiver of any of its rights. 3. You acknowledge that you have read this License Agreement, and agree to be bound by its terms and conditions. To the extent that any other terms and conditions presented on any website of Bentham Science Publishers conflict with, or are inconsistent with, the terms and conditions set out in this License Agreement, you acknowledge that the terms and conditions set out in this License Agreement shall prevail. Bentham Science Publishers Pte. Ltd. 80 Robinson Road #02-00 Singapore 068898 Singapore Email: [email protected] CONTENTS FOREWORD ........................................................................................................................................... i PREFACE ................................................................................................................................................ iii LIST OF CONTRIBUTORS .................................................................................................................. iv CHAPTER 1 ISOLATION OF GENOMIC DNA FROM PLANT TISSUES ................................ 1 (cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 1 Principle .................................................................................................................................. 1 Sample Collection and Storage Conditions ............................................................................ 2 CTAB DNA Extraction Buffer: 100 ml .................................................................................. 3 T10E1 Buffer .......................................................................................................................... 4 DNA Isolation Protocol .......................................................................................................... 4 Purification .............................................................................................................................. 4 Useful Tips and Suggestions for Obtaining Optimum Results ............................................... 5 CONSENT FOR PUBLICATION ................................................................................................ 5 CONFLICT OF INTEREST ......................................................................................................... 5 ACKNOWLEDGEMENTS ........................................................................................................... 6 REFERENCES ............................................................................................................................... 6 CHAPTER 2 RNA ISOLATION PROTOCOL FROM CELLS AND TISSUES ........................... 7 (cid:51)(cid:68)(cid:79)(cid:79)(cid:68)(cid:89)(cid:76)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 7 Precautions to be Taken Before the Experiment ..................................................................... 8 I. Isolation of RNA Using TRIzol Method ............................................................................. 8 Principle .................................................................................................................................. 8 Materials Required .................................................................................................................. 9 Steps ........................................................................................................................................ 9 II. Isolation of RNA Using the CTAB Method ....................................................................... 9 Materials Required .................................................................................................................. 9 Steps ........................................................................................................................................ 10 III. Hot-Phenol RNA Extraction Method ............................................................................... 10 Materials Required .................................................................................................................. 10 Steps ........................................................................................................................................ 11 IV. QIAGEN RNeasy Kit Method .......................................................................................... 11 DNase Treatment .................................................................................................................... 12 Storage of RNA ....................................................................................................................... 12 Determining RNA Quality and Quantity ................................................................................ 12 Formaldehyde Agarose Gel Electrophoresis .......................................................................... 13 Materials Required .................................................................................................................. 13 Preparation of Formaldehyde Agarose Gel ............................................................................. 13 Preparation of RNA Samples for Electrophoresis ................................................................... 13 CONSENT FOR PUBLICATION ................................................................................................ 13 CONFLICT OF INTEREST ......................................................................................................... 14 ACKNOWLEDGEMENTS ........................................................................................................... 14 REFERENCES ............................................................................................................................... 14 CHAPTER 3 ANALYZING GENE EXPRESSION THROUGH REAL TIME PCR WHILE NEO-TISSUE REGENERATION USING DEVE- LOPED TISSUE CONSTRUCTS .................... 15 (cid:39)(cid:76)(cid:89)(cid:68)(cid:78)(cid:68)(cid:85)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:55)(cid:68)(cid:85)(cid:88)(cid:81)(cid:3)(cid:48)(cid:76)(cid:81)(cid:82)(cid:70)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:87)(cid:92)(cid:68)(cid:89)(cid:85)(cid:68)(cid:87)(cid:3)(cid:55)(cid:85)(cid:76)(cid:83)(cid:68)(cid:87)(cid:75)(cid:76)(cid:15)(cid:3)(cid:53)(cid:88)(cid:83)(cid:76)(cid:78)(cid:68)(cid:3)(cid:54)(cid:76)(cid:81)(cid:75)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:88)(cid:69)(cid:75)(cid:68)(cid:81)(cid:78)(cid:68)(cid:85) (cid:36)(cid:81)(cid:68)(cid:81)(cid:71)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:57)(cid:76)(cid:89)(cid:72)(cid:78)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:81)(cid:71)(cid:72)(cid:92)(cid:15)(cid:3)(cid:36)(cid:85)(cid:88)(cid:81)(cid:3)(cid:53)(cid:68)(cid:90)(cid:68)(cid:87)(cid:15)(cid:3)(cid:54)(cid:80)(cid:76)(cid:87)(cid:68)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:81)(cid:77)(cid:72)(cid:72)(cid:89) (cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:15)(cid:3)(cid:51)(cid:68)(cid:90)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:39)(cid:88)(cid:69)(cid:72)(cid:92)(cid:3)and(cid:3)(cid:51)(cid:85)(cid:68)(cid:71)(cid:72)(cid:72)(cid:83)(cid:3)(cid:54)(cid:85)(cid:76)(cid:89)(cid:68)(cid:86)(cid:87)(cid:68)(cid:89)(cid:68) INTRODUCTION .......................................................................................................................... 16 BASICS OF RT-PCR ..................................................................................................................... 17 Merits of RT-PCR ................................................................................................................... 18 PCR Cycling ........................................................................................................................... 19 RT-PCR Master Mix Components .......................................................................................... 20 Thermostable DNA Polymerase ............................................................................................. 21 Deoxyribonucleotidetriphosphates (dNTPs) ........................................................................... 21 Divalent Cation Especially Magnesium Ions .......................................................................... 21 Template ................................................................................................................................. 21 RT-PCR Primer Design .......................................................................................................... 22 Primer Designing .................................................................................................................... 22 Basics of Primer Designing .................................................................................................... 23 RT-PCR Fluorescence Detection Chemistry .......................................................................... 23 SYBR Green-based Detection ................................................................................................ 24 Mechanism of Action .............................................................................................................. 24 Advantages .............................................................................................................................. 24 Limitations .............................................................................................................................. 24 TAQMAN probe Based Detection .......................................................................................... 25 Mechanism of Action .............................................................................................................. 25 Advantages .............................................................................................................................. 26 Limitations .............................................................................................................................. 27 RT-PCR Experimental Design and Gene Expression Profiling ............................................. 27 Setting up a Reaction Mixture ................................................................................................ 28 Basic PCR Protocol and Establishment of Reaction Cycle .................................................... 29 Application of RT-PCR .......................................................................................................... 30 CHALLENGES ............................................................................................................................... 31 CONSENT FOR PUBLICATION ................................................................................................ 32 CONFLICT OF INTEREST ......................................................................................................... 32 ACKNOWLEDGEMENTS ........................................................................................................... 32 REFERENCES ............................................................................................................................... 32 CHAPTER 4 A MODIFIED WESTERN BLOT PROTOCOL FOR ENHANCED SENSITIVITY IN THE DETECTION OF A TISSUE PROTEIN ............................................................................... 35 (cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:48)(cid:68)(cid:79)(cid:79)(cid:76)(cid:78)(cid:68)(cid:85)(cid:77)(cid:88)(cid:81)(cid:68)(cid:3)(cid:53)(cid:68)(cid:82)(cid:3)(cid:42)(cid:72)(cid:71)(cid:71)(cid:68)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81) (cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:51)(cid:68)(cid:92)(cid:68)(cid:79)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77)(cid:3)(cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:53)(cid:68)(cid:78)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 35 SAMPLE PREPARATION ........................................................................................................... 36 GEL ELECTROPHORESIS ......................................................................................................... 37 BLOTTING ..................................................................................................................................... 38 WASHING, BLOCKING AND ANTIBODY INCUBATION ................................................... 39 QUANTIFICATION ...................................................................................................................... 40 TROUBLESHOOTING ................................................................................................................. 40 CONCLUSION ............................................................................................................................... 42 CONSENT FOR PUBLICATION ................................................................................................ 42 CONFLICT OF INTEREST ......................................................................................................... 42 ACKNOWLEDGEMENTS ........................................................................................................... 42 REFERENCES ............................................................................................................................... 42 CHAPTER 5 IMMUNOHISTOCHEMISTRY AS AN IMPORTANT TECHNIQUE IN EXPERIMENTAL AND CLINICAL PRACTICES ............................................................................ 44 (cid:43)(cid:68)(cid:85)(cid:72)(cid:85)(cid:68)(cid:80)(cid:3)(cid:37)(cid:76)(cid:85)(cid:79)(cid:68)(cid:15)(cid:3)(cid:54)(cid:68)(cid:70)(cid:75)(cid:70)(cid:75)(cid:76)(cid:71)(cid:68)(cid:3)(cid:49)(cid:68)(cid:81)(cid:71)(cid:3)(cid:53)(cid:68)(cid:76)(cid:15)(cid:3)(cid:54)(cid:68)(cid:88)(cid:80)(cid:76)(cid:87)(cid:85)(cid:68)(cid:3)(cid:54)(cid:72)(cid:81)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75)(cid:15)(cid:3)(cid:58)(cid:68)(cid:79)(cid:76)(cid:68)(cid:3)(cid:61)(cid:68)(cid:75)(cid:85)(cid:68)(cid:15)(cid:3)(cid:49)(cid:72)(cid:72)(cid:85)(cid:68)(cid:77) (cid:55)(cid:76)(cid:90)(cid:68)(cid:85)(cid:76)(cid:15)(cid:3)(cid:36)(cid:76)(cid:77)(cid:68)(cid:93)(cid:3)(cid:36)(cid:17)(cid:3)(cid:49)(cid:68)(cid:76)(cid:78)(cid:15)(cid:3)(cid:36)(cid:81)(cid:68)(cid:80)(cid:76)(cid:78)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:85)(cid:68)(cid:15)(cid:3)(cid:54)(cid:75)(cid:76)(cid:78)(cid:75)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:85)(cid:68)(cid:87)(cid:76)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:92)(cid:68)(cid:3)(cid:51)(cid:85)(cid:68)(cid:87)(cid:68)(cid:83)(cid:3)(cid:54)(cid:76)(cid:81)(cid:74)(cid:75) INTRODUCTION .......................................................................................................................... 44 DIRECT TECHNIQUE ................................................................................................................. 45 NEW DIRECT TECHNIQUE (ENHANCED POLYMER ONE STEPS TRAINING METHOD) ....................................................................................................................................... 45 TWO-STEP INDIRECT TECHNIQUE ....................................................................................... 46 POLYMER CHAIN TWO STEP INDIRECT TECHNIQUE ................................................... 46 UNLABELED ANTIBODY ENZYME COMPLEX TECHNIQUES (PAP AND APAP) ...... 46 IMMUNOGOLD SILVER STAINING TECHNIQUE (IGSS) .................................................. 47 THE PRINCIPLE OF IHC ............................................................................................................ 48 TISSUE PREPARATION .............................................................................................................. 48 ANTIGEN RETRIEVAL ............................................................................................................... 50 A. Proteolytic Enzyme Digestion ........................................................................................... 50 B. Heat Mediated Antigen Retrieval Techniques ................................................................... 51 ANTIGEN-ANTIBODY INTERACTION ................................................................................... 51 AVIDIN–BIOTIN COMPLEX ...................................................................................................... 52 HAPTEN LABELLING TECHNIQUE ....................................................................................... 52 DETECTION METHODS ............................................................................................................. 52 QUANTIFICATION OF THE IMMUNOHISTOCHEMICAL STAINING ........................... 54 APPLICATION AND IMPORTANCE ........................................................................................ 54 TROUBLES AND LIMITATIONS .............................................................................................. 55 TROUBLESHOOTING OF THE EXPERIMENTAL PROBLEMS ..................................... 55 IMMUNOENZYMATIC LABELLING PROTOCOL FOR LOCALIZA- TION OF ASTROCYTES USING ANTI-GFAP ANTIBODY .................................................................... 55 FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 55 DAY 2 PROCESSING ........................................................................................................... 56 IMMUNOHISTOCHEMICAL PROTOCOL FOR LABELLING ASTRO- CYTES USING ANTI-GFAP BY IMMUNE-FLUORESCENCE METHOD ...................................................... 56 FOR CRYOSTAT SECTIONS DAY 1 PROCESSING ........................................................ 56 DAY 2 PROCESSING ........................................................................................................... 57 CONSENT FOR PUBLICATION ................................................................................................ 57 CONFLICT OF INTEREST ......................................................................................................... 57 ACKNOWLEDGEMENTS ........................................................................................................... 57 REFERENCES ............................................................................................................................... 57 CHAPTER 6 PROTOCOLS FOR THE DETECTION AND PROTEOME ANALYSIS OF THE YELLOW MOSAIC VIRUS INFECTED SOYABEAN LEAVES .................................................... 60 (cid:37)(cid:68)(cid:83)(cid:68)(cid:87)(cid:79)(cid:68)(cid:3)(cid:46)(cid:72)(cid:86)(cid:68)(cid:89)(cid:68)(cid:3)(cid:51)(cid:68)(cid:89)(cid:68)(cid:81)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:54)(cid:88)(cid:85)(cid:68)(cid:83)(cid:68)(cid:87)(cid:75)(cid:85)(cid:88)(cid:71)(cid:88)(cid:3)(cid:46)(cid:68)(cid:81)(cid:68)(cid:78)(cid:68)(cid:79)(cid:68) INTRODUCTION .......................................................................................................................... 60 METHOD ........................................................................................................................................ 61 Protein Extraction and Virus Detection .................................................................................. 61 Iso-electric Focusing ............................................................................................................... 62 SDS-PAGE/ 2nd Dimension ................................................................................................... 62 Colloidal Coomassie Staining ................................................................................................. 63 Image Analysis ........................................................................................................................ 64 CONSENT FOR PUBLICATION ................................................................................................ 64 CONFLICT OF INTEREST ......................................................................................................... 64 ACKNOWLEDGEMENTS ........................................................................................................... 64 ABBREVIATIONS ......................................................................................................................... 64 REFERENCES ............................................................................................................................... 64 CHAPTER 7 2D-DIGE A POWERFUL TOOL FOR PROTEOME ANALYSIS .......................... 67 (cid:54)(cid:88)(cid:71)(cid:75)(cid:76)(cid:85)(cid:3)(cid:46)(cid:17)(cid:3)(cid:54)(cid:75)(cid:72)(cid:78)(cid:75)(cid:68)(cid:85)(cid:15)(cid:3)(cid:45)(cid:68)(cid:76)(cid:3)(cid:42)(cid:82)(cid:71)(cid:75)(cid:72)(cid:77)(cid:68)(cid:3)and(cid:3)(cid:39)(cid:76)(cid:81)(cid:72)(cid:86)(cid:75)(cid:3)(cid:53)(cid:68)(cid:77)(cid:3)(cid:48)(cid:82)(cid:71)(cid:76) INTRODUCTION .......................................................................................................................... 68 Methodology ........................................................................................................................... 68 Steps of the Experiment .......................................................................................................... 70 ADVANTAGES OF DIGE ............................................................................................................. 71 Applications of DIGE ............................................................................................................. 72 CONCLUSION ............................................................................................................................... 72 CONSENT FOR PUBLICATION ................................................................................................ 72 CONFLICT OF INTEREST ......................................................................................................... 72 ACKNOWLEDGEMENTS ........................................................................................................... 72 REFERENCES ............................................................................................................................... 72 CHAPTER 8 MOLECULAR TECHNIQUES FOR GENOTYPING .............................................. 74 (cid:54)(cid:75)(cid:68)(cid:79)(cid:76)(cid:81)(cid:76)(cid:3)(cid:42)(cid:88)(cid:83)(cid:87)(cid:68)(cid:15)(cid:3)(cid:54)(cid:82)(cid:80)(cid:68)(cid:79)(cid:76)(cid:3)(cid:54)(cid:68)(cid:81)(cid:92)(cid:68)(cid:79)(cid:15)(cid:3)(cid:54)(cid:88)(cid:85)(cid:72)(cid:86)(cid:75)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)(cid:60)(cid:68)(cid:71)(cid:68)(cid:89)(cid:3)and(cid:3)(cid:48)(cid:68)(cid:71)(cid:68)(cid:81)(cid:3)(cid:47)(cid:68)(cid:79)(cid:3)(cid:37)(cid:85)(cid:68)(cid:75)(cid:80)(cid:68)(cid:3)(cid:37)(cid:75)(cid:68)(cid:87)(cid:87) 74 INTRODUCTION .......................................................................................................................... 74 1. Saliva Collection and Storage ............................................................................................. 75 2. Preparation for Cell Lysis ................................................................................................... 76 3. RNA Removal ..................................................................................................................... 76 4. Protein and Lipid Removal ................................................................................................. 76 5. Isolation and Purification of Genomic DNA ...................................................................... 76 6. Rehydration of g DNA ........................................................................................................ 77 Several Protocols Have Been Developed to Isolate DNA from Saliva ................................... 77 Sample Collection ................................................................................................................... 77 DNA Extraction ...................................................................................................................... 78 DNA Evaluation ...................................................................................................................... 78 Methods for DNA Extraction from Whole Blood .................................................................. 79 Two-step Lysis ........................................................................................................................ 79 One-step Lysis ........................................................................................................................ 79 Isolating Genomic DNA Using Magnetic Beads .................................................................... 79 GENOMIC DNA ISOLATION OF DNA FROM BLOOD ........................................................ 80 Reagents Required .................................................................................................................. 80 Procedure ................................................................................................................................ 80 VISUALIZATION OF DNA THROUGH AGAROSE GEL ELECTRO- PHORESIS .......... 81 Materials Required .................................................................................................................. 81 METHOD ............................................................................................................................... 81 Range of separation of linear DNA of different get concentrations ....................................... 81 Reagents .................................................................................................................................. 83 6x Gel-loading Buffer .................................................................................................... 83 DNA Staining Solution (10mg/ml) ......................................................................................... 83 EDTA (0.5M pH 8) ................................................................................................................. 83 TAE (50X) .............................................................................................................................. 83 TBE (5X) ................................................................................................................................ 83 TPE (10X) ............................................................................................................................... 83 POLYACRYLAMIDE GEL ELECTROPHORESIS ................................................................. 83 Materials ................................................................................................................................. 83 Buffers and Solutions .................................................................................................... 83 METHOD ........................................................................................................................................ 84 POLYMERASE CHAIN REACTION ......................................................................................... 86 Materials Required .................................................................................................................. 86 Method .................................................................................................................................... 86 RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) ................................... 87 Reagents Required .................................................................................................................. 87 Method .................................................................................................................................... 87 CONSENT FOR PUBLICATION ................................................................................................ 87 CONFLICT OF INTEREST ......................................................................................................... 87 ACKNOWLEDGEMENTS ........................................................................................................... 88 REFERENCES ............................................................................................................................... 88 CHAPTER 9 SODIUM BISULFITE CONVERSION OF HUMAN GENOME FOR DNA METHYLATION STUDIES .................................................................................................................. 89 (cid:36)(cid:68)(cid:86)(cid:87)(cid:75)(cid:68)(cid:3)(cid:48)(cid:76)(cid:86)(cid:75)(cid:85)(cid:68)(cid:3)and(cid:3)(cid:52)(cid:68)(cid:71)(cid:68)(cid:85)(cid:3)(cid:51)(cid:68)(cid:86)(cid:75)(cid:68) INTRODUCTION .......................................................................................................................... 90 Mapping Whole Genome DNA Methylation Using Sodium Bisulfite Conversion of DNA Followed by Next-generation Sequencing .............................................................................. 92 Sodium Bisulfite Conversion of DNA ............................................................................ 92 Bisulfite Conversion Protocol ................................................................................................. 93 CONCLUDING REMARKS ......................................................................................................... 95 CONSENT FOR PUBLICATION ................................................................................................ 95 CONFLICT OF INTEREST ......................................................................................................... 96 ACKNOWLEDGEMENTS ........................................................................................................... 96 REFERENCES ............................................................................................................................... 96 CHAPTER 10 CHROMATIN IMMUNOPRECIPITATION (CHIP) ............................................. 97 (cid:46)(cid:68)(cid:89)(cid:92)(cid:68)(cid:81)(cid:77)(cid:68)(cid:79)(cid:76)(cid:3)(cid:54)(cid:75)(cid:68)(cid:85)(cid:80)(cid:68)(cid:15)(cid:3)(cid:54)(cid:88)(cid:69)(cid:68)(cid:86)(cid:75)(cid:3)(cid:38)(cid:75)(cid:68)(cid:81)(cid:71)(cid:85)(cid:68)(cid:3)(cid:54)(cid:82)(cid:81)(cid:78)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:54)(cid:75)(cid:68)(cid:78)(cid:88)(cid:81)(cid:87)(cid:68)(cid:79)(cid:68)(cid:3)(cid:48)(cid:68)(cid:75)(cid:76)(cid:79)(cid:78)(cid:68)(cid:85) OVERVIEW OF CHIP ASSAY .................................................................................................... 98 GENERAL DESCRIPTION OF CHIP PROTOCOL ................................................................ 99 1. Cross-Linking ..................................................................................................................... 99 2. Cell Lysis ............................................................................................................................ 100 3. Shearing of DNA ................................................................................................................ 100 4. Immunoprecipitation ........................................................................................................... 100 5. Reversal of Cross-links and Characterization of DNA ....................................................... 101 6. Analysis of DNA ................................................................................................................. 101 Requirements .......................................................................................................................... 101 Troubleshooting ...................................................................................................................... 105 Limitation ................................................................................................................................ 105 Variation ................................................................................................................................. 106 APPLICATION .............................................................................................................................. 109 CONSENT FOR PUBLICATION ................................................................................................ 110 CONFLICT OF INTEREST ......................................................................................................... 110 ACKNOWLEDGEMENTS ........................................................................................................... 110 REFERENCES ............................................................................................................................... 110 CHAPTER 11 OSTEOSARCOMA CELL CULTURE AND MAINTENANCE TO DETECT THE APOPTOTIC EFFECT OF SOME PROMISING COMPOUNDS BY POTENT MARKERS VIZ. DNA FRAGMENTATION AND CASPASE-3 ACTIVATION ................................................. 114 (cid:36)(cid:86)(cid:76)(cid:73)(cid:3)(cid:45)(cid:68)(cid:73)(cid:85)(cid:76)(cid:15)(cid:3)(cid:45)(cid:88)(cid:75)(cid:76)(cid:3)(cid:53)(cid:68)(cid:76)(cid:86)(cid:15)(cid:3)(cid:54)(cid:88)(cid:71)(cid:75)(cid:76)(cid:85)(cid:3)(cid:46)(cid:88)(cid:80)(cid:68)(cid:85)(cid:3)and(cid:3)(cid:48)(cid:71)(cid:3)(cid:36)(cid:85)(cid:86)(cid:75)(cid:68)(cid:71) INTRODUCTION .......................................................................................................................... 114 Osteosarcoma Cells Culture and Maintenance ....................................................................... 115 DNA Fragmentation ................................................................................................................ 115 Detection of Apoptosis via Caspase-3 Activity in Cancer Cells ............................................ 117

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