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Organization of a Radioisotope Based Molecular Biology Laboratory

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Organization of a Radioisotope Based Molecular Biology Laboratory IAEA-TECDOC-1528 Organization of a Radioisotope Based Molecular Biology Laboratory December 2006 IAEA-TECDOC-1528 Organization of a Radioisotope Based Molecular Biology Laboratory December 2006 The originating Section of this publication in the IAEA was: Nuclear Medical Section International Atomic Energy Agency Wagramer Strasse 5 P.O. Box 100 A-1400 Vienna, Austria ORGANIZATION OF A RADIOISOTOPE BASED MOLECULAR BIOLOGY LABORATORY IAEA, VIENNA, 2006 IAEA-TECDOC-1528 ISBN 92–0–114406–7 ISSN 1011–4289 © IAEA, 2006 Printed by the IAEA in Austria December 2006 FOREWORD Polymerase chain reaction (PCR) has revolutionized the application of molecular techniques to medicine. Together with other molecular biology techniques it is being increasingly applied to human health for identifying prognostic markers and drug resistant profiles, developing diagnostic tests and genotyping systems and for treatment follow-up of certain diseases in developed countries. Developing Member States have expressed their need to also benefit from the dissemination of molecular advances. The use of radioisotopes, as a step in the detection process or for increased sensitivity and specificity is well established, making it ideally suitable for technology transfer. Many molecular based projects using isotopes for detecting and studying micro organisms, hereditary and neoplastic diseases are received for approval every year. In keeping with the IAEA’s programme, several training activities and seminars have been organized to enhance the capabilities of developing Member States to employ in vitro nuclear medicine technologies for managing their important health problems and for undertaking related basic and clinical research. The background material for this publication was collected at training activities and from feedback received from participants at research and coordination meetings. In addition, a consultants’ meeting was held in June 2004 to compile the first draft of this report. Previous IAEA TECDOCS, namely IAEA-TECDOC-748 and IAEA-TECDOC-1001, focused on molecular techniques and their application to medicine while the present publication provides information on organization of the laboratory, quality assurance and radio-safety. The technology has specific requirements of the way the laboratory is organized (e.g. for avoiding contamination and false positives in PCR) and of quality assurance in order to provide accurate information to decision makers. In addition while users of the technology accept the scientific rationale of using radio-isotopes: they often have concerns related to safety. Member States have repeatedly requested help for training when first establishing radioactive-based molecular biology laboratories in particular in radiosafety and quality assurance. The current TECDOC has been prepared in response to these requests and requirements. It represents one of the IAEA’s many efforts to contribute with the worldwide implementation of these techniques, and to be a relevant source of information to be used by molecular scientists, major national research institutions, laboratory workers and national health institutions authorities. The IAEA wishes to thank all authors and in particular to acknowledge the contribution of the following in the preparation of this publication: L. Ranford-Cartwright, T. Victor, O. Fernandes and L. García Aguirre. The IAEA officer responsible for this publication was B. Khan of the Division of Human Health. EDITORIAL NOTE The use of particular designations of countries or territories does not imply any judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and institutions or of the delimitation of their boundaries. The mention of names of specific companies or products (whether or not indicated as registered) does not imply any intention to infringe proprietary rights, nor should it be construed as an endorsement or recommendation on the part of the IAEA. CONTENTS 1. INTRODUCTION…………………………………………………………......................... 1 2. RADIOISOTOPE-BASED MOLECULAR BIOLOGY……………................................... 2 2.1. Sensitivity …………………………………………………………............................ 2 2.2. Specificity ………………………………………………………… ........................... 2 2.3. Cost-effectiveness ………………………………………………… ........................... 2 2.4. Safety issues ………………………………………………………............................ 3 3. RADIOISOTOPE-BASED METHODS…..………………………….…… ........................ 4 3.1. Polymerase chain reaction ........................................................................................... 4 3.2. Dot-blot assay .............................................................................................................. 5 3.3. Restriction fragment length polymorphism analysis……..…… ................................. 6 3.4. Single stranded conformational polymorphism ........................................................... 7 3.5. Amplified fragment length polymorphism .................................................................. 7 3.6. Mismatch cleavage assay............................................................................................. 8 3.7. Heteroduplex tracking assay ........................................................................................ 8 3.8. DNA sequencing ……………….………………………………................................ 8 3.9. Microsatellite detection ……………………………………………........................... 9 3.10. DNA “footprinting” for analysis of protein-nucleic acid interactions ............. 9 3.11. Protein truncation test..................................................................................... 10 3.12. Reporter gene assays ...................................................................................... 11 3.13. Scintillation proximity assay.......................................................................... 11 3.14. Telomerase assay ...........................................................................................11 3.15. Macroarray and microarray chip technology ................................................. 12 3.16. Stable-isotope-assisted maldi-tof mass spectrometry .................................... 13 3.17. Stable isotope coded affinity tags................................................................... 13 3.18. Solution NMR with stable isotopes................................................................ 13 4. DESIGNING A MOLECULAR BIOLOGY LABORATORY .......................................... 15 5. GENERAL WORKFLOW.................................................................................................. 16 6. DESCRIPTION OF THE AREAS ...................................................................................... 17 6.1. Area 1: Clean area …………………………………………………......................... 17 6.2. Area 2: Extraction nucleic acids from clinical specimens ......................................... 18 6.3. Area 3: Template preparation and PCR ..................................................................... 19 6.4. Area 4a: Post-amplification area — non radioactive ................................................ 19 6.5. Area 4b: The radiation area ....................................................................................... 20 7. PREVENTION OF PCR CONTAMINATION AND GOOD LABORATORY PRACTICES............................................................................. 23 8. QUALITY CONTROL ....................................................................................................... 26 9. BASIC PRINCIPLES FOR HANDLING RADIOACTIVE MATERIALS IN THE LABORATORY.............................................. 27 9.1. Information about ionizing radiation and radioactivity ............................................. 27 9.1.1. Types of radioactive decay............................................................................. 27 9.1.2. Interaction of radioactivity with matter.......................................................... 28 9.1.3. Principles of shielding……………………………………… ........................ 29 9.1.4. Effects of radioactivity on humans................................................................. 30 9.2. Detection and measurement of radioactivity ............................................................ 31 9.2.1. Survey instruments......................................................................................... 31 9.2.2. Personal monitoring devices........................................................................... 31 9.3. Establishment and management of a radioactive facility........................................... 32 9.3.1. The regulatory framework for working with ionizing and radiation dose limits .................................................................. 33 9.3.2. General principles and procedures ................................................................. 33 9.3.3. Dose limits...................................................................................................... 33 9.3.4. Government regulations on radioactive substances ....................................... 33 9.4. Basic principles for handling radioactive material in the laboratory ........................ 35 9.4.1. Design of radiation containment facility ......................................................... 37 9.4.2. Receiving radioactive material ........................................................................ 38 9.4.3. Preparing for a radioactive experiment ..........................................................
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