Applied Molecular Biotechnology : The Next Generation of Genetic Engineering / Muhammad Sarwar Khan (Phần 1)

<span class="text_page_counter">Trang 1</span><div class="page_container" data-page="1">

Muhammad Sarwar Khan Iqrar Ahmad Khan

Debmalya Barh

CRC Press

</div><span class="text_page_counter">Trang 3</span><div class="page_container" data-page="3">

This page intentionally left blank

</div><span class="text_page_counter">Trang 4</span><div class="page_container" data-page="4">

Muhammad Sarwar KhanIqrar Ahmad Khan

</div><span class="text_page_counter">Trang 5</span><div class="page_container" data-page="5">

<small>Taylor & Francis Group</small>

<small>6000 Broken Sound Parkway NW, Suite 300</small>

<small>Boca Raton, FL 33487-2742</small>

<small>© 2016 by Taylor & Francis Group, LLC</small>

<small>CRC Press is an imprint of Taylor & Francis Group, an Informa businessNo claim to original U.S. Government works</small>

<small>Version Date: 20160302</small>

<small>International Standard Book Number-13: 978-1-4987-1483-9 (eBook - PDF)</small>

<small>This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have beenmade to publish reliable data and information, but the author and publisher cannot assume responsibility for the </small>

<small>valid-ity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyrightholders of all material reproduced in this publication and apologize to copyright holders if permission to publish in thisform has not been obtained. If any copyright material has not been acknowledged please write and let us know so we mayrectify in any future reprint.</small>

<small>Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or </small>

<small>uti-lized in any form byany electronic, mechanical, or other means, now known or hereafter invented, including </small>

<small>photocopy-ing, microfilmphotocopy-ing, and recordphotocopy-ing, or in any information storage or retrieval system, without written permission from the</small>

<small>For permission to photocopy or use material electronically from this work, please access www.copyright.com ( or contact the Copyright Clearance Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923,978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. Fororganizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged.Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only foridentification and explanation without intent to infringe</small>

<small>Visit the Taylor & Francis Web site atand the CRC Press Web site at</small>

</div><span class="text_page_counter">Trang 6</span><div class="page_container" data-page="6">

Chapter1 Emerging tools and approaches to biotechnology in the omics era... Neha Malviya, Aiman Tanveer, Sangeeta Yadav, and Dinesh Yadav Section I: Plant biotechnology

Sandhya Agarwal, Alka Grover, and SM Paul Khurana

Plant molecular biotechnology: Applications of transgenics... Muhammad Sarwar Khan, Ghulam Mustafa, Shahid Nazir, and Faiz Ahmad Joyia

The chloroplast gene-expression system 91

Yusuke Yagi and Takashi Shiina

Molecular biology of mitochondria: Genome, transcriptome,

and proteome 127 Muhammad Wagar Hameed

Plant functional genomics: Approaches and applications Mehboob-ur-Rahman, Zainab Rahmat, Maryyam Gul, and Yusuf Zafar

Whole-genome resequencing: Current status and future prospects in genomics-assisted crop improvement.

Uday Chand Jha, Debmalya Barh, Swarup K. Parida, Rintu Jha, and Narendra Pratap Singh

Molecular biotechnology of plant-microbe-insect interactions ....

Jam Nazeer Ahmad, Samina Jam Nazeer Ahmad, and Sandrine Eveillard

</div><span class="text_page_counter">Trang 7</span><div class="page_container" data-page="7">

Chapter9 Biotechnology for improved crop productivity and quality...231

Cassiana Severiano de Sousa, Maria Andréia Corréa Mendonca, Syed Shah

Hassan, Debmalya Barh, and Vasco Ariston de Carvalho Azevedo Chapter 10 Overview of methods to unveil the epigenetic code ....

Sarfraz Shafiq and Abdul Rehman Khan Section II: Animal biotechnology

Adeena Shafique, Azka Khan, Kinza Wagar, Aimen Niaz, and Alvina Gul Chapter 12 Variations in our genome: From disease to individualized cure 289

Bishwanath Chatterjee and Cecilia W. Lo

Chapter 13 Molecular biotechnology for diagnostics. ..303

Shailendra Dwivedi, Saurabh Samdariya, Gaurav Chikara, Apul Goel, Rajeev Kumar Pandey, Puneet Pareek, Sanjay KhaHri, Praveen Sharma, Sanjeev Misra, and Kamlesh Kumar Pant

Chapter 14 Techniques for cervical cancer screening and diagnosis „345 Haq Nawaz, Nosheen Rashid, Hugh J. Byrne, and Fiona M. Lyng

Chapter 15 Type 2 diabetes mellitus, obesity, and adipose tissue biology... ¡”77

Fazli Rabbi Awan and Syeda Sadia Najam

Chapter 16 Human tissue banking and its role in biomedical research ...

Shahid Mian and Ibraheem Ashankyty

Section III: Industrial and environmental biotechnology

Chapter 17 Microbial biotechnology 405 Margarita Aguilera and Jesús Manuel Aguilera~-Gémez

Chapter 18 Molecular biology of viruses: Disease perspective ..

Muhammad Mubin, Sehrish Ijaz, Sara Shakir, and Muhammad Shah Nawaz-ul-Rehman

Chapter 19 Viral biotechnology: Production perspectiVe...----.«--cc-..ceeeec...8Ỷ Kinza Wagar, Hafeez Ullah, and Alvina Gul

Chapter 20 Cell-free biosystems 465 Manju Sharma and SM Paul Khurana

Chapter 21 Magnetic nanoparticles with multifunctional water-soluble

polymers for bioapplication: 485

Muhammad Irfan Majeed, Muhammad Asif Hanif, Haq Nawaz, and Bien Tan

</div><span class="text_page_counter">Trang 8</span><div class="page_container" data-page="8">

Chapter 22 Chapter 23 Chapter 24

Chapter 25

Industrial biotechnology: Its applications in food

and chemical industrie: 517

Syed Ali Imran Bokhari, Muhammad Sarwar Khan, Nosheen Rashid, and Muhammad Irfan Majeed

Environmental biotechnology: Approaches for ecosystem

conservation 529

Vasavi Mohan, Mohammed Khaliq Mohiuddin, and Yog Raj Ahuja Marine biotechnology: Focus on anticancer drugs...

Amit Rastogi, Sameen Rugia, and Alvina Gul

Engineering genomes for biofuels 569 Niaz Ahmad, Muhammad Aamer Mehmood, Steven J. Burgess,

and Muhammad Sarwar Khan

</div><span class="text_page_counter">Trang 9</span><div class="page_container" data-page="9">

This page intentionally left blank

</div><span class="text_page_counter">Trang 10</span><div class="page_container" data-page="10">

During the last few decades several fundamental discoveries in life sciences have given rise to modern biotechnology, which is essentially based on breakthroughs in molecular

biology. It is currently one of the fastest growing areas of science and thus, this century has

rightly been termed the “Century of Biology,” hoping that such advances in life sciences will yield changes more momentous than electricity and computers.

The tremendous advances in biotechnology have also had a profound effect not only on agriculture but also on medicine and the environment. About 20% of the world’s

phar-maceuticals are produced by using biotechnological processes and it has been estimated

that about 50% of all pharmaceuticals will be produced in this manner by the year 2020. The most well-known products are insulin and many other biologicals, including inter-feron, other cytokines, and several antibiotics. With the developments in DNA sequencing, “personalized medicine” is on the anvil.

In the case of agriculture, the gains due to high-yielding varieties as a result of the

green revolution in the 1960s have tapered off due to the increasing cost of inputs and scarcity of water in many countries. In order to make agriculture sustainable, modern bio-technology has played a significant role by developing pest-resistant and drought-tolerant crop varieties. As a result, there has been a 10-fold increase in the area under transgenic

crops in the world since 1996 when it was 1.7 million ha, which has increased to 1785 mil-lion ha in 2014. This has come about by utilizing a series of molecular biology technologies

related to genomics and new tools of bioinformatics.

During the last decade there have been several new discoveries in molecular biology that have rapidly found their way to applications. The whole new world of “omics”

com-prising genomics, proteomics, transcriptiomics, and metabolomics has revolutionized the way translational research is carried out. In addition, strides in computational, structural,

and organelle biology have opened new vistas for developing useful products.

This book, Applied Molecular Biotechnology: The Next Generation of Genetic Engineering, is timely and very much needed by our younger generation of researchers. It comprises

well-documented chapters on all aspects of new and emerging technologies related to

plants, animals, industry, and the environment. I would like to compliment the editors for compiling such a comprehensive book covering all important aspects of the technology. Tam sure it will be well received by the readers.

Professor Dr. Kauser Abdulla Malik, HI, SI, TI Distinguished National Professor Dean for Postgraduate Studies Forman Christian College (A Chartered University) Lahore

</div><span class="text_page_counter">Trang 11</span><div class="page_container" data-page="11">

This page intentionally left blank

</div><span class="text_page_counter">Trang 12</span><div class="page_container" data-page="12">

The twenty-first century is an era of technology and its applications. In the recent past, several path-breaking innovations in the field of life sciences have enabled us to resolve

a number of serious global issues using cutting-edge technologies. This book provides important revolutionary molecular biology technique-based next-generation genetic

engi-neering toward finding solutions to our needs in the fields of plant, animal, industrial, and environmental biotechnology.

The book, Applied Molecular Biotechnology: The Next Generation of Genetic Engineering, consists of 25 chapters subdivided into 3 sections. Chapter 1 by Dr. Malviya and col-leagues provides an overview of omics-based latest tools and approaches used in modern biotechnology. Section I (Plant biotechnology) begins with Chapter 2 where Dr. Agarwal’s group has demonstrated in detail how the various molecular biology technologies can be used to develop transgenic plants. In Chapter 3, Dr. Khan’s team elaborated on how these transgenic plants can fulfill our ever-growing demand for food and other plant-derived products. Drs. Yagi and Shiina have given a detailed account of the chloroplast gene expression system and its applications in Chapter 4. Chapter 5 is dedicated to organelle biotechnology. In this chapter, mitochondrial omics have been discussed by Dr. Wagar Hameed. Dr. Mehboob-ur-Rahman and colleagues discuss various approaches

and applications of plant functional genomics in the next chapter (Chapter 6). In Chapter 7, Dr. Jha et al. describe the current status and future prospects of whole genome

rese-quencing toward crop improvement. Plant-microbe and plant-insect interactions are key phenomena in plant molecular biotechnology where plant protection and productivity are concerned. A detailed account on these aspects is presented by Dr. Ahmad’s group in Chapter 8. In Chapter 9, Dr. de Sousa and colleagues have given brief, but highly useful, content in “Biotechnology for improved crop productivity and quality.” The last chapter (Chapter 10) by Drs. Shafiq and Khan provides an overview of the methods that unveil the epigenetic code.

Section II (Animal biotechnology) consists of six chapters (Chapters 11 through 16).

Chapter 11 by Dr. Shafique et al. deals with various animal models used in biomedical research. The most recent trend in medical genomics, that is, pharmacogenomics, is

dis-cussed in Chapter 12 by Drs. Chatterjee and Lo, titled “Variations in our genome: From disease to individualized cure.” Chapter 13 by Dr. Dwivedi’s group discusses how modern biotechnological approaches are used in the molecular diagnosis of various diseases such as cervical cancer, obesity, and diabetes. Chapters 14 and 15 are dedicated to these two

highly important topics. While the screening and diagnosis techniques of cervical cancer

are discussed in Chapter 14 by Dr. Nawaz and colleagues, Chapter 15 deals with the bio-logical aspects of diabetes and obesity and are documented by Drs. Awan and Najam. The last chapter (Chapter 16) under Section II deals with human tissue banking and its role in

biomedical research, by Drs. Mian and Ashankyty.

</div><span class="text_page_counter">Trang 13</span><div class="page_container" data-page="13">

xii Preface

Section II combines industrial and environmental biotechnology. The general

aspects of microbial biotechnology written by Drs. Aguilera and Aguilera-GÓmez are

included in Chapter 17. Dr. Mubin’s group discusses the molecular aspects of viral dis-eases in Chapter 18. Chapter 19 by Dr. Ullah et al. deals with the production of industrial commodities using viral biotechnology. Chapter 20 deals with an important topic “Cell free biosystems” by Drs. Sharma and Khurana. The biotechnological uses of magnetic

nanoparticles are covered by Dr. Majeed and colleagues in Chapter 21. Various

appli-cations of biotechnology in food and chemical industries are discussed in Chapter 22 by Dr. Bokhari and coauthors. In Chapter 23, Dr. Mohan's team describes how modern biotechnology can be applied to conserve our ecosystem. In this section, a special topic on marine biotechnology is also included. Dr. Rastogi and colleagues in Chapter 24 have

provided a comprehensive account on various anticancer drugs from marine resources.

The final chapter (Chapter 25) of this book deals with biofuel genomics by Dr. Khan and colleagues where genome-scale plant genetic engineering is described to develop trans-genic plants for optimum biofuel production.

Since biotechnology itself is an interdisciplinary subject, it is difficult to cover every aspect of the subject in a single book. In this book, we have tried our best to provide an overview of the latest trends of application of molecular biology techniques in plant, animal, industrial, and environmental biotechnology. We do hope this book will be a worthwhile resource to students and researchers in the field of molecular biotechnology. We welcome your suggestions to improve the next edition of the book.

Muhammad Sarwar Khan, PhD Igrar Ahmad Khan, PhD Debmalya Barh, PhD

</div><span class="text_page_counter">Trang 14</span><div class="page_container" data-page="14">

Muhammad Sarwar Khan is a highly regarded molecu-lar biologist who earned a PhD from the University of Cambridge. He was awarded the Rockefeller Foundation Fellowship under the Rice Biotechnology Program for

trans-formation at Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, which was published

in Nature Biotechnology. Dr. Khan was a founding head

of the Biotech Interdisciplinary Division at the National Institute for Biotechnology and Genetic Engineering (NIBGE), and is currently serving as the director of the Center of Agricultural Biochemistry and Biotechnology

(CABB), University of Agriculture, Faisalabad, Pakistan.

Dr. Khan has been honored with prestigious and befitting awards, including the President’s Medal for Technology, Gold Medal in Agriculture by the Pakistan Academy of Sciences, Performance Gold Medal by the Pakistan Atomic Energy Commission, and a Biotechnologist Award by the National Commission of

Biotechnology. He is a fellow of the Cambridge Commonwealth Society, the Cambridge Philosophical Society, and the Rockefeller Foundation.

Dr. Khan has supervised about 80 PhD and MPhil students and researchers who are serving at national and international levels in various research institutes and universi-ties. He has published extensively in high-impact journals including Nature and Nature

Biotechnology, and is author of a number of books and book chapters. Dr. Khan has made

immense contributions in the field of chloroplast genetic engineering and is a pioneer in expressing oxygen-loving green fluorescent protein (GFP) from jellyfish in chloroplasts— plant organelles with reduced environment. He also pioneered plastid transformation in rice and sugarcane, recalcitrant plant species. As far as translational research is concerned,

Dr. Khan has developed borer-resistant transgenic sugarcane plants with no toxin residues

in the juice. His current research interests include development of edible-marker-carrying transgenics and cost-effective therapeutics and edible vaccines.

</div><span class="text_page_counter">Trang 15</span><div class="page_container" data-page="15">

xiv Editors

Iqrar Ahmad Khan has had a long career in educa-tion and agriculture earning a PhD from the University

of California, Riverside. He is currently serving as vice chancellor of the University of Agriculture, Faisalabad (UAF), Pakistan (since 2008). Dr. Khan has supervised more than 100 graduate students and researchers,

established a Center of Agricultural Biotechnology and

cofounded the Deutscher Akademischer Austauschdienst, German Academic Exchange Service (DAAD)-sponsored International Center for Decent Work and Development

(ICDD), USAID-funded Center of Advanced Studies in Agriculture and Food Security, a French Learning Center and the Chinese Confucius

Institute. He has organized numerous international conferences and established academic linkages across the continents. He has released a potato variety (PARS-70), pioneered research on breeding seedless Kinnow, and discovered new botanical varieties of wheat.

Dr. Khan initiated an internationally acclaimed program to solve a devastating problem called Witches’ Broom Disease of Lime in Oman. He is currently leading international

projects to combat citrus greening disease and mango sudden death. He has published more than 270 articles, 5 books, and several book chapters.

Dr. Khan has a diplomatic skill that has attracted international partnerships and academic linkages (Afghanistan, Australia, South Korea, China, Germany, France,

Malaysia, Indonesia, Turkey, Iran, India, Oman, Canada, UK, and USA). He has

man-aged collaborative research projects sponsored by national and international agencies. As vice chancellor, he has revamped UAF academic, research, and outreach programs. He added new academic disciplines to narrow the knowledge gaps in microbiology, biotechnology, environmental sciences, food and nutrition, climate change,

engineer-ing, rural development, and education. UAF has achieved top ranks within the national

system as well as in the Quacquarelli Symonds (QS) and National Taiwan University (NTU) rankings. Dr. Khan has a special knack for problem-solving research. He has set up an incubation center for the commercialization of knowledge. Exhibitions and business plan competitions have been made biannual features. A range of new quality

assurance mechanisms have been added and special initiatives taken to narrow the gender gap.

Dr. Khan isa fellow of Pakistan Academy of Sciences and member of several profes-sional societies and associations. In recognition of his outstanding contributions in the area of agriculture and food security he was honored with a civil award, Sitara-e-Imtiaz,

by the government of Pakistan and very recently with Ordre des Palmes Académiques

(with the grade of Officer) by the French Government for his exceptional role as an educator.

</div><span class="text_page_counter">Trang 16</span><div class="page_container" data-page="16">

Debmalya Barh, MSc, MTech, MPhil, PhD, PGDM, is the

founder of the Institute of Integrative Omics and Applied Biotechnology (IIOAB), India, a virtual global platform for multidisciplinary research and advocacy. He is a biotech-nologist and has decades of experience in integrative omics applied to translational research. Dr. Barh has written more

than 150 publications and is a globally recognized editor for

editing omics-related cutting-edge reference books for top-notch international publishers. He also serves as a reviewer for several professional international journals of global repute. Owing to his significant contributions in promoting,

R&D globally using unique strategies, in the year 2010 he was recognized by Who's Who in

the World and in 2014 he was entered in the Limca Book of Records—the Indian equivalent to the Guinness Book of World Records.

</div><span class="text_page_counter">Trang 17</span><div class="page_container" data-page="17">

This page intentionally left blank

</div><span class="text_page_counter">Trang 18</span><div class="page_container" data-page="18">

Sandhya Agarwal

Metahelix Life Sciences Ltd Bengaluru, Karnataka, India Integrated Genomic, Cellular,

Developmental and Biotechnology

University of Agriculture Faisalabad, Pakistan

Niaz Ahmad

Agricultural Biotechnology Division

National Institute for Biotechnology and Genetic Engineering

Yog Raj Ahuja

Department of Genetics and Molecular Medicine

Vasavi Medical and Research Centre

Hyderabad, Telangana, India

Ibraheem Ashankyty

The Molecular Diagnostics and Personalised Therapeutics Unit

University of Ha’il

Ha’il, Kingdom of Saudi Arabia

Fazli Rabbi Awan

Diabetes and Cardio-Metabolic

Disorders Lab

Health Biotechnology Division National Institute for Biotechnology and

Genetic Engineering Faisalabad, Pakistan

Vasco Ariston de Carvalho Azevedo Laboratory of Cellular and Molecular

Federal University of Minas Gerais Belo Horizonte, Minas Gerais, Brazil Debmalya Barh

Institute of Integrative Omics

and Applied Biotechnology (IIOAB)

Purba Medinipur, West Bengal, India

Syed Ali Imran Bokhari

Department of Bioinformatics and.

</div><span class="text_page_counter">Trang 19</span><div class="page_container" data-page="19">

Hugh J. Byrne

Focas Research Institute Dublin Institute of Technology Dublin, Ireland

Bishwanath Chatterjee

Department of Developmental Biology

University of Pittsburgh School of Medicine

Pittsburgh, Pennsylvania Gaurav Chikara

Department of Pharmacology All India Institute of Medical Sciences Jodhpur, Rajasthan, India

King George Medical University

Lucknow, Uttar Pradesh, India Cassiana Severiano de Sousa

Laboratory of Cellular and Molecular Genetics

Federal University of Minas Gerais Belo Horizonte, Minas Gerais, Brazil Shailendra Dwivedi

Department of Biochemistry

All India Institute of Medical Sciences Jodhpur, Rajasthan, India

Department of Pharmacology and

King George Medical University Lucknow, Uttar Pradesh, India Sandrine Eveillard

INRA, UMR Biologie du Fruit et Pathologie

Villenave đOrnon, France Apul Goel

Department of Urology King George Medical University

Lucknow, Uttar Pradesh, India Muhammad Wagar Hameed

Dr. Panjwani Center for Molecular Medicine and Drug Research

International Center for Chemical and

Syed Shah Hassan

Laboratory of Cellular and Molecular Genetics

Federal University of Minas Gerais Belo Horizonte, Minas Gerais, Brazil

Crop Improvement Division

Indian Institute of Pulses Research (IIPR)

Kanpur, Uttar Pradesh, India Uday Chand Jha

Crop Improvement Division

Indian Institute of Pulses Research (IIPR)

Kanpur, Uttar Pradesh, India

</div><span class="text_page_counter">Trang 20</span><div class="page_container" data-page="20">

Faiz Ahmad Joyia

Centre of Agricultural Biochemistry and

Biotechnology University of Agriculture Faisalabad, Pakistan

Abdul Rehman Khan

Department of Environmental Sciences COMSATS Institute of Information Muhammad Sarwar Khan

Centre of Agricultural Biochemistry and

King George Medical University Lucknow, Uttar Pradesh, India

Department of Developmental Biology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania

Fiona M. Lyng

DIT Centre for Radiation and Environmental Science (RESC) Focas Research Institute

Dublin Institute of Technology D.DU. Gorakhpur University

Gorakhpur, Uttar Pradesh, India

Muhammad Aamer Mehmood Department of Bioinformatics and

GC University Faisalabad Faisalabad, Pakistan

Maria Andréia Corréa Mendonca Goiano Federal Institute Rio Verde, Goiás, Brazi

Shahid Mian

The Molecular Diagnostics and Personalised Therapeutics Unit University of Ha’il

Hai, Kingdom of Saudi Arabia Sanjeev Misra

Department of Surgical Oncology King George Medical University Lucknow, Uttar Pradesh, India

All India Institute of Medical Sciences Jodhpur, Rajasthan, India

'Vasavi Mohan

Department of Genetics and Molecular Medicine

Vasavi Medical and Research Centre

Hyderabad, Telangana, India

Mohammed Khaliq Mohiuddin Department of Genetics and Molecular

Vasavi Medical and Research Centre

Hyderabad, Telangana, India

</div><span class="text_page_counter">Trang 21</span><div class="page_container" data-page="21">

Syeda Sadia Najam

Diabetes and Cardio-Metabolic Disorders Lab

Health Biotechnology Division National Institute for Biotechnology and

Muhammad Shah Nawaz-ul-Rehman Centre of Agricultural Biochemistry and

Biotechnology University of Agriculture Faisalabad, Pakistan

Shahid Nazir

Biotechnology Research Institute Ayub Agricultural Research Institute Rajeev Kumar Pandey

Department of Dermatology and Allergic Diseases

University of Ulm Ulm, Germany

Kamlesh Kumar Pant

Department of Pharmacology and

King George Medical University Lucknow, Uttar Pradesh, India

Puneet Pareek

Department of Radiotherapy All India Institute of Medical Sciences Jodhpur, Rajasthan, India

Plant Genomics and Molecular Breeding Lab National Institute for Biotechnology

and Genetic Engineering

Faisalabad, Pakistan

Zainab Rahmat

Plant Genomics and Molecular

Breeding Lab

National Institute for Biotechnology and Genetic Engineering (NIBGE) Faisalabad, Pakistan

Nosheen Rashid

Faisalabad Institute of Research Science and Technology (FIRST)

Faisalabad, Pakistan

Amit Rastogi

Department of Molecular and Cellular

Sam Higginbottom Institute of

Agricultural, Technology and Sciences. Allahabad, Uttar Pradesh, India

</div><span class="text_page_counter">Trang 22</span><div class="page_container" data-page="22">

Contributors Saurabh Samdariya

Department of Radiotherapy

All India Institute of Medical Sciences Jodhpur, Rajasthan, India

Sarfraz Shafiq

Department of Environmental Sciences COMSATS Institute of Information

Technology Abbottabad, Pakistan

Center for Plant Biology School of Life Sciences

All India Institute of Medical Sciences

Jodhpur, Rajasthan, India Takashi Shiina

Graduate School of Life and

Environmental Science

Kyoto Prefectural University

Narendra Pratap Singh

Crop Improvement Division

Indian Institute of Pulses

D.D.U. Gorakhpur University Gorakhpur, Uttar Pradesh, India D.DU. Gorakhpur University Gorakhpur, Uttar Pradesh, India

Sangeeta Yadav

Department of Biotechnology

D.DU. Gorakhpur University

Gorakhpur, Uttar Pradesh, India

</div><span class="text_page_counter">Trang 23</span><div class="page_container" data-page="23">

This page intentionally left blank

</div><span class="text_page_counter">Trang 24</span><div class="page_container" data-page="24">

chapter one

Emerging tools and approaches to biotechnology in the omics era

Neha Malviya, Aiman Tanveer, Sangeeta Yadav, and Dinesh Yadav

One-dimensional gel electrophoresis

Polyacrylamide gel electrophoresis Agarose gel electrophoresis Isoelectric focusing

2D gel electrophoresis. Polymerase chain reaction

Sequencing techniques

Chemical degradation sequencing (Maxam and Gilbert method)..

</div><span class="text_page_counter">Trang 25</span><div class="page_container" data-page="25">

2 Applied molecular biotechnology

DNA and protein microarray ....

Nuclear magnetic resonance.

Electron Spin Resonance..

Lipid profiling for comprehensive analysis of lipid species within a cell or tissue

Technology integration for analysis and imaging of cellular data...

Bioinformatics tools

Quantitative estimation through biostatistical analysi: Application of science of omics in different fields .

Omics reflects totality and is generally referred for study of biomol-ecules influencing the structural and functional aspects of an organ-ism. Further, for convenience the science of omics are classified into several branches such as genomics, proteomics, lipidomics, tran-scriptomics, cytomics, etc. Genomics is a well-known field of omics, which includes the study of total genetic content of an organism and often encompasses several other branches such as cognitive, com-parative, functional, personal genomics, epigenomics, and metage-nomics. The study of transcriptome termed as transcriptomics is an emerging field that covers the total set of transcripts in an organism

along with the set of all ribonucleic acid (RNA) molecules. Along with the deoxyribonucleic acid and RNA, the proteins are the key players

associated with the maintenance of cellular functions. Proteomics is applied to the exploration of protein structure and functionality. The nonproteinaceous repertoires of the cell metabolites are the interme-diate products of metabolism. Its study, metabolomics is a rapidly

evolving new discipline having potential implications. Besides the

above-mentioned branches of omics, cytomics and lipidomics are also an indispensable element of the omics study. There are several basic and advanced techniques for the exploration of these branches

</div><span class="text_page_counter">Trang 26</span><div class="page_container" data-page="26">

Chapter one: Emerging tools and approaches to biotechnology in the omics era 3 and continuous efforts are being made to utilize these tools in differ-ent fields of scidiffer-entific research. Elucidation and utilization of these applications is a hallmark for global research.

Keywords: Omics, Genomics, Proteomics, Transcriptomics, Metabolomics.

Omics term is used with suffix -ome to address the study of respective field in totality such as genomics for genome, proteomics for proteome, metabolomics for metabolome,

and many more. Omics reflects the use of diverse technologies to gain an insight into

the complexity of biomolecules influencing the structure and function of organisms. The omics-driven research has led to some understanding of complex regulatory net-works that controls gene expression, protein modification, and metabolite composition. Transcriptomics, metabolomics, bioinformatics, and high-throughput DNA sequencing led to the deciphering of diverse regulatory networks in different systems resulting in enhanced understanding for its potential applications in clinical diagnosis, prognosis, and therapeutic purposes.

The relationship between different branches of omics is shown in Figure 1.1.

General classifications of omics

The outcome of innovations in sequencing technologies led to deciphering of whole genome sequences of different organisms and the branch of omics popularly known as

</div><span class="text_page_counter">Trang 27</span><div class="page_container" data-page="27">

4 Applied molecular biotechnology

genomics came into existence. The genomics reflects the study of the genome in totality

and is further classified into several subbranches as listed below.

Comparative genomics

The major principal underlying comparative genomics is that the common feature between the two organisms is encoded by the conserved DNA sequence. Conversely, divergence

between the species is defined by the sequences that encode proteins or ribonucleic acids

(RNAs). The comparative genomics provides an insight into the evolutionary aspects of organisms compared based on the sequences at whole genome level. It helps in discrimi-nating conserved sequences from divergent ones. Further, comparative genomics can also be useful to understand the variability in terms of functional DNA segments, such

as coding exons, noncoding RNAs, and also some gene regulatory regions. The genome

sequences are compared by aligning them to score the match or mismatch between them. Various softwares and algorithms have been developed for the alignment of several genome sequences simultaneously and elucidate genome evolution and function.

Functional genomics

Functional genomics is applied to test and extend hypotheses that emerge from the analy-sis of sequence data. Elucidating the functions of identified genes of the sequenced genome of an organism is the sole purpose of functional genomics. While sequencing projects

yield preliminary results, functional genomics focuses on the functional aspects such as

regulation of gene expression, functions and interaction of different genes, etc. Functional genomics means genome-wide analysis through high-throughput methods. Hence it pro-vides an overview of the biological information encoded by the organism’s genome. The encyclopedia of DNA elements (ENCODE) project is a much-anticipated project, which aims to recognize all the functional elements of genomic DNA both in coding and non-coding regions.

Metagenomics is emerging as an important discipline to access the biocatalytic potential of unculturable microorganisms. Despite very rich microbial diversity of the range of a mil-lion species per 1 g of soil, very few microorganisms can be cultured under in vitro condi-tions. With the advances made in the field of metagenomics, DNA can be extracted from environmental samples from which genomic library can be prepared. This library can be further explored by screening the clones for biological activity to identify clones

possess-ing desired characteristics. A number of biocatalysts such as laccase, xylanase,

endogluca-nase, exoglucaendogluca-nase, and lipase have been recently identified from metagenomic libraries. Epigenomics

Epigenetics refers to the external modification of DNA. It alters the physical structure of

DNA without altering the DNA sequence. The DNA methylations, that is, the addition of a methyl group, or a “chemical cap,” to part of the DNA molecule and histone modification

are examples of epigenetic changes. Epigenetic changes can be carried over to the follow-ing generation if the modifications occur in sperm or egg cells. But most of these epigenetic changes get corrected during reprogramming of fertilized eggs. Cellular differentiation is also an example of epigenetic change in eukaryotic biology. Epigenetic mechanisms are

influenced by many other factors such as prenatal development and in childhood,

envi-ronmental influence, drugs, aging, diet, etc.

</div><span class="text_page_counter">Trang 28</span><div class="page_container" data-page="28">

Chapter one: Emerging tools and approaches to biotechnology in the omics era Personal genomics

The completion of human genome project has provided valuable information regarding variations of the human genome. Single nucleotide polymorphism (SNP), copy number

variation, and complex structural variations can be typed with the help of sequencing

data. An ambitious personal genome project (PGP) has been initiated to truly understand the genesis of most complex human traits—from deadly diseases to the talents and other features that makes every individual unique. PGP is widely supported by the nonprofit

PersonalGenomes.org, which works to publicize genomic technology and knowledge at a

global level. This might be useful for disease management and understanding of human health. It also deals with the ethical, legal, and social issues (ELSI) related to personal

Cognitive genomics

The brain is an important organ of an organism that helps to deal with the complex,

infor-mation-rich environment. The blueprint for the brain is contained in the genetic material, that is, DNA of an organism. Brain development and cognitive or behavioral variability among individuals is a complicated process that results from genetic attribute of the per-son as well as their interactions with the environment. In cognitive genomics, cognitive

function of the genes and also the noncoding sequences of an organism's genome related

to health and activity of the brain are being studied. Genomic locations, allele frequencies, and precise DNA variations are analyzed in cognitive genomics. Cognitive genomics have immense potential for investigating the genetic reasons for neurodegenerative and mental disorders such as Down syndrome, Autism, and Alzheimer’s disease.

Till date several genome sequencing projects have been completed and efforts are now being made to decipher the functional roles of different identified genes, their role in different cellular processes, genes regulation, genes and gene product interaction, and expression level of genes in various cell types. Transcription being the primary step in gene regulation processes, the information about the transcript levels is a prerequisite for understanding gene regulatory networks. The functional elucidation of the identified genes in totality is a subject matter of transcriptomics. It deals with the study of the com-plete set of RNAs/transcriptomes encoded by the genome of a cell or organism at a specific

time and under a specific set of conditions. The techniques that are frequently used for

genome-wide analysis for gene expression are complementary DNA (cDNA) microarrays and protein microarrays, CDNA-amplified fragment length polymorphism (AFLP), and serial analysis of gene expression (SAGE).

Proteomics is a comprehensive study of proteins in totality identified in a cell, organ, or organism at a particular time. The complexity of diverse physiological processes and biological structures hinders the applicability of proteomics, though the advent of recent proteomic techniques enables large-scale, high-throughput analyses, identification, and functional study of the proteome. For convenience, the proteomics can further be studied in different subbranches such as structural genomics, immunoproteomics, proteogenomics,

nutriproteomics, etc.

</div><span class="text_page_counter">Trang 29</span><div class="page_container" data-page="29">

6 Applied molecular biotechnology

Structural genomics

It aims to decipher the 3D structure of all proteins encoded by a particular genome using either experimental tools or in silico tools or sometimes both. In structural genomics, the

structure of the total number of identified protein of particular genome is determined

while in traditional structural prediction, structure of only one particular protein is deter-mined. Through the availability of full genome sequences of number of organisms, struc-tural prediction can be done by using both the experimental and modeling approaches, as well as previously known protein structures. The sequence-structure-function relation-ship provides an opportunity to analyze the putative functions of the identified proteins of an organism under purview of structural genomics.

Immunoproteomics is the study of proteins solely associated with immune response with

the aid of diverse techniques and approaches. Immunoproteomics encompasses a

rap-idly growing collection of techniques for identifying and measuring antigenic peptides or proteins. The approaches include gel- and array-based, mass spectrometry, DNA-, and bioinformatics-based techniques. Immunoproteomics is purposely used for understanding of disease, its progression, vaccine preparation, and biomarkers.

Proteogenomics is the study that uses proteomic information, mainly derived from mass spectrometry, to improve gene annotations. It is a field of junction of the genomics and proteomics. Previously, the genomics and proteomics studies were done independently. In genomics studies, large-scale annotation was done for identification of genes and its corresponding protein sequences, after sequencing of the genome. The proteomics aims to elucidate the protein expression observed in different tissues under specific conditions along with an insight into various posttranslational modifications. In proteogenomics

there is amalgamation of both genomics as well as proteomics for elucidating the gene

The study of proteins of nutritional values of an organism can be referred as

nutri-proteomics. It can be defined as the interaction of nutrients with the proteins by

study-ing the effect of nutrients on protein synthesis, interaction of nutrients with proteins, and modulation of protein-protein interaction through nutrients.

The diverse chemical reactions leading to growth and development of an organism is often referred to as metabolism and includes both anabolic and catabolic reactions.

It is a branch of omics associated with study of metabolites of an organism in totality.

This advancing field of science has much importance in pharmacological studies, func-tional genomics, toxicology, drug discovery, nutrition, cancer, and diabetes. As we know metabolites are the end result of all regulatory complex processes present in the cells hence metabolic changes represent reporters of alterations in the body in response to a drug or a disease.

</div><span class="text_page_counter">Trang 30</span><div class="page_container" data-page="30">

Chapter one: Emerging tools and approaches to biotechnology in the omics era vụ Metabonomics

Metabonomics reflects the quantitative estimation of the metabolite of a particular organ-ism and also includes the study of factors both exogenous and endogenous influencing

the change in metabolite concentration. The exogenous factors such as environmental

factors, xenobiotics, and endogenous factors such as physiology and development are predominantly considered in metabonomics. Like genomics, transcriptomics, and pro-teomics, metabonomics has immense potential in the discovery and development of new

Lipidomics is the study of network of lipids and their interacting protein partners in

organs, cells, and organelles. The lipidomic analysis is mainly done by mass spectrometry, commonly preceded by separation by liquid chromatography or gas chromatography.

Cytomics is the study of cytomes or the molecular single-cell phenotypes study resulting

from genotype.

Pharmaconomics is the study of a complex genetic basis of interpatient variability in

response to drug therapy. This subbranch of omics is an interesting field for

pharmaceuti-cal industries, clinicians, academicians, and patients as well. Using pharmacogenomics, the biopharmaceutical industries can improve the drug developmental process more rap-idly and safely.

This includes

1. Nutritional genomics aims to study the relationship between human genome,

nutri-tion, and health.

2. Toxicogenomics aims to study gene and protein activity in response to toxic

Psychogenomics aims to understand the biological substrate of normal behavior and also understand the diseased conditions to unravel the behavioral abnormalities at genomic scale.

4. Stem cell genomics aims to understand human biology and disease states, which further progress toward clinical translation.

Basic tools and techniques used in science of omics

Electrophoretic techniques

Electrophoresis is the movement of molecules under the influence of uniform electric field. The separation of DNA, RNA, and proteins can be easily analyzed by gel electrophoresis

</div><span class="text_page_counter">Trang 31</span><div class="page_container" data-page="31">

8 Applied molecular biotechnology

sos-ract |

Figure 1.2 Types of gel electrophoresis.

of the specific weight of the target to be analyzed. When the target molecule is protein or small nucleic acids, polyacrylamide gel is used. If the target molecule is larger nucleic acids, then agarose gel is preferred. Broadly, there are two types of gel electrophoresis designated as one dimensional and two dimensional (2D) (Figure 1.2).

One-dimensional gel electrophoresis Polyacrylamide gel electrophoresis

The uniform pore size provided by the polyacrylamide gel is utilized for separating pro-teins/DNA. The concentrations of acrylamide and bisacrylamide control the pore size of the gel.

Sodium dodecyl sulfate—polyacrylamide gel electrophoresis It is the most widely used

electrophoretic technique, which separates proteins primarily by mass. NATIVE PAGE It separates protein according to their charge/mass ratio.

Pulse field gel electrophoresis DA greater than ~40 kb length cannot be easily

sepa-rated by applying constant electrical field. This problem is solved by pulsed field gel elec-trophoresis (PFGE) in which electric field is switched periodically between two different directions.

Agarose gel electrophoresis

It is frequently used for qualitative and quantitative estimation of nucleic acid, that is, DNA and RNA.

Isoelectric focusing

It separates the proteins in a pH gradient based on their isoelectric point (pl).

</div><span class="text_page_counter">Trang 32</span><div class="page_container" data-page="32">

Chapter one: Emerging tools and approaches to biotechnology in the omics era 9

2D gel electrophoresis

It is the combination of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS— PAGE)/isoelectric focusing (IEF): first dimension is generally IEF. Second dimension is generally SDS-PAGE. Proteins having same molecular weight or same pl are also resolved.

Polymerase chain reaction

It can be simply defined as in vitro amplification of DNA in a sequential manner resulting in thousands to millions of its copies. This technique was developed by Kary Mullis in

1983. Polymerase chain reaction (PCR) consists of repetitive cycles of heating and cooling

associated with events of DNA denaturation, annealing, and extension carried out at par-ticular temperature for certain duration, which need extensive optimization depending on the template DNA and primers synthesized. The PCR utilizes the ability of DNA poly-merase to synthesize new strand of DNA complementary to the target region. In general, DNA polymerase adds a nucleotide only when 3-OH group preexists and hence it needs a primer to which it can add the first nucleotide. During the process, the DNA amplified serves as a template for subsequent amplification resulting in multiple copies at end. In this way, the DNA template is exponentially amplified. At last, after several cycles of PCR reaction, billions of copies of specific sequences (amplicons) are accumulated.

The following components are required for PCR reaction:

1. DNA template: It is the DNA having target sequence. Application of high tempera-ture is required for denaturation. The quantity and quality of the template DNA is an important consideration for PCR amplification.

2. Thermostable DNA polymerase: Taq DNA polymerase is the most commonly used

enzyme though Pfu DNA polymerase is often used because of its higher fidelity when copying DNA. The processivity and fidelity of the enzyme is an important consideration, which ultimately determines the strategy for subsequent cloning by specific cloning vectors.

Primers: A pair of synthetic oligonucleotides is a prerequisite to prime DNA

synthe-sis and is an important component of the PCR reaction. The efficiency and specificity of the amplification is greatly influenced by the primers designed. The availabil-ity of softwares such as PRIMER-3, DNAStar, etc., in recent years has significantly contributed for proper designing of the primers. Some of the important

consider-ations for primer designing are setting of GC content in the range of 40%-60%,

pro-vision for equal distribution of four bases, avoiding polypurine or polypyrimidine tracts or dinucleotide repeats, maintaining the length of primers in the range of 18-25 mer, avoiding complementarity between the forward and reverse primers, etc.

In general, higher concentration of primers favors mispriming leading to nonspecific amplification.

4. Deoxynucleoside triphosphates (dNTPs): These are building blocks of new DNA strand. The standard PCR contains equimolar concentration of dATP, dGTP, dCTP, and dTTP and the concentration is in the range of 200-250 UM for each dNTP. Buffer solution: It is generally provided with 10x concentration and comprises

Tris-based buffer and salt like KCI and provides a suitable environment for optimum

activity of DNA polymerase.

Divalent cations: Mg” is used commonly, but sometimes Mn?† is used in the PCR buf-fer. Itis an important cofactor for thermostable DNA polymerases. The concentration =

” e

</div><span class="text_page_counter">Trang 33</span><div class="page_container" data-page="33">

10 Applied molecular biotechnology of the Mg* is standardized for PCR amplification though 1.5 mM is the optimum concentration in most of the PCR reaction set up. The excess of Mg” reduces the enzyme fidelity and leads to nonspecific amplification.

Besides these ingredients, in many cases specific chemicals are also added, which are referred to as PCR enhancer and additives such as betaine, DMSO, formamide, BSA, etc., which often increases the specificity of PCR amplification resulting in enhanced yield and

also minimizes the undesired products.

The basic steps involved in PCR amplification are (i) Denaturation: DNA melting by

disrupting the hydrogen bonds between complementary bases often carried out at 94-98°C for 30 s. (ii) Annealing: Assists binding of primers to the template DNA and the anneal-ing temperature needs optimization based on the Tm value of the primers synthesized.

Generally, during standardization of annealing temperature for a particular set of reac-tion, the annealing temperature is kept at about 3-5°C below the Tm of the primers. (iii)

Extension: Binding of primers to the template DNA during annealing step is followed by synthesis of new cDNA strand with the aid of dNTPs and DNA polymerase in 5’ to 3’ direction. Since Taq DNA polymerase has optimum activity at 72°C, the extension is car-ried out at this temperature, though variation can be done based on the complexity of the

template DNA and primers used. Time of extension is variable and is optimized based on

the expected size of DNA fragment to be amplified and type of DNA polymerase used. To ensure that any remaining single-stranded DNA is fully extended, the final extension step is carried out at 70-74°C for 5-15 min.

PCR-based DNA fingerprinting technique is quite popular in forensic sciences owing to the fact that even extremely small amounts of sample (DNA) can be amplified. PCR is often used in molecular diagnosis of many diseases. PCR-based molecular markers are routinely used for diversity analysis, it is often used in sequencing techniques, for muta-genesis studies, for expression studies, etc.

In recent years several alterations in the basic PCR has been attempted for diverse

applications. The variants of PCR and its diverse application are shown in Table 1.1 and Figure 1.3.

Sequencing techniques

It is the process of deciphering the arrangement of A, C, G, and T nucleotide for a particu-lar DNA molecule and came into existence in 1970 popuparticu-larly known as Sanger (or dideoxy) method and the Maxam-Gilbert (chemical cleavage) method. The technological innova-tions in sequencing methods was attempted for enhancing the speed, accuracy, automa-tion, read length, cost-effectiveness, etc., over the years and these developments have been

marked as first-, second-, third-, and fourth-generation sequencing technologies as shown

in Table 1.2. The second-, third-, and fourth-generation sequencing are also referred as next generation sequencing (NGS) methods.

Chemical degradation sequencing (Maxam and Gilbert method)

It uses specific chemicals for the cleavage of nucleotides and requires double-stranded template DNA radioactively labeled at one 5’ end of the DNA. The method involves modification of a base chemically and then the DNA strand is cleaved by reaction that specially cleaved the DNA at the point where base is modified. Four reactions desig-nated as G, A+G, C, and C+T is set up by degrading with specific chemicals followed by

</div><span class="text_page_counter">Trang 34</span><div class="page_container" data-page="34">

Chapter one: Emerging tools and approaches to biotechnology in the omics era 11

Figure 1.3 Some variants of PCR.

its analysis on polyacrylamide gel. The read length of approximately 400 bp is possible with this method. The use of toxic chemicals and difficulty in automation limits its use.

Enzymatic or chain termination method (Sanger’s method)

This method involves enzymatic polymerization of DNA fragments complementary to the single-stranded template DNA in a reaction setup comprising ingredients such as dNTP, specific primer, and a modified nucleoside called terminator of dideoxynucleoside triphos-phate (ddNTP) involved with chain termination. A set of four different tubes containing the appropriate amount of one of the four terminators (ddNTPs) is needed for the sequenc-ing. Here, P-labeled primer is preferably used for generating different fragments having, the same 5’ end and these fragments are finally resolved by denaturing polyacrylamide gel electrophoresis. It requires single-stranded DNA as a template and has several

advan-tages such as possibility for automation, increased sequence read length, no toxic

chemi-cals required, etc. The automated DNA sequencing is an automation of Sanger’s method of sequencing which has greatly enhanced the accuracy, speed, and read length. This was followed by further advancement in sequencing technologies as described in Table 1.2.

DNA and protein microarray

Microarray is a 2D array ona solid substrate that detects large amounts of biological mate-rial using multiplexed, parallel processing, and detection methods. Most commonly used is DNA microarray though protein, peptide, and carbohydrate microarrays are also

gain-ing importance.

</div><span class="text_page_counter">Trang 35</span><div class="page_container" data-page="35">

Applied molecular biotechnology

Table 1.1 Variants of PCR and its applications

Type of PCR Description Application

Multiplex PCR Utilizes more than one set of primers meant Molecular diagnosis with for simultaneous amplification of different __ provision for analyzing regions of the DNA. different markers

simultaneously confirming the existence of targeted diseases.

Nested PCR It involves two sets of primers designed in Enhances specificity of PCR such a manner that one of the primers amplification.

amplifies the shorter internal fragment of the larger fragment.

Reverse It is associated with the amplification of Generally used for expression

transcriptase RNA sequences by first converting into studies as an alternative of PCR (RT-PCR) double-stranded cDNA using reverse northern hybridization.

<small>transcriptase enzyme.</small>

Semiquantitative __ It is basically used for quantification of PCR _Used for determining the PCR products during the exponential phase relative amount of cDNA ina

prior to saturation stage and involves both housekeeping and target genes. The simultaneous amplification and

quantification of a targeted cDNA molecules.

It is associated with the preferential amplification of one strand owing to the unequal concentration of primers in the

<small>reaction setup.</small>

It is basically a condition to avoid adding all the components of PCR reaction simultaneously resulting in accumulation of nonspecific PCR amplicons. The best way is to add the critical component of PCR reactions, that is, DNA polymerases only when the temperature reaches 90-95°C. This is basically a method of

standardization of annealing temperature with primers designed from protein sequences, that is, degenerate primers. The optimization for perfect annealing temperature can be achieved by first increasing the temperature from 3°C to 5°C for few cycles above the theoretically calculated Tm of the primers, followed by reducing the temperature unless the exact annealing temperature as witnessed by the yield of the reaction.

Tt is a method used to allow amplification of unknown region.

given sample.

For assessing the copy number of genes.

Helpful to generate single-stranded DNA, which can be used for sequencing and hybridization probing. This enhances the possibility of

getting the expected DNA fragment in PCR. The accumulation of nonspecific products is also minimized. The sole purpose is to optimize

the annealing temperature for better yield.

Itis used in chromosome walking, for cloning unknown regions of genomic sequences. (Continued)

</div><span class="text_page_counter">Trang 36</span><div class="page_container" data-page="36">

Chapter one: Emerging tools and approaches to biotechnology in the omics era 13

Table 1.1 (Continued) Variants of PCR and its applications

Type of PCR Description Application

Long PCR It relies on the use of a mixture of two Used in physical mapping and thermostable polymerases, one having 3-5’ directcloning from genome. exonucleases (proofreading activity) while

other lacking like use of Taq and Pfu DNA polymerases. Amplification of fragments in the range of 10-25 kb is possible by long PCR. Comparatively longer extension time, additives such as glycerol and DMSO are preferred for long PCR.

In situ PCR The PCR amplification is merged with It can be used for detecting histological localization with the aid of in cellular DNA, cDNA situ hybridization technique. Several associated with diseased alterations like increased concentration of conditions.

Mp”, increased amount of DNA polymerases is done in in situ PCR. Tissue preparation is an important consideration in this PCR.

High-fidelity Use of DNA polymerase with proofreading The sole purpose is to

PCR activity such as Pfu, Tli, etc. minimize the chances of mutations resulting from the use of DNA polymerases lacking proofreading activity. It is preferred for PCR-based cloning and expression studies.

Differential Amplify and display many cDNAs derived The purpose is to display all

display PCR from mRNA of a given cell or tissue type. mRNA of a cell. It can be used Uses two different types of

oligonucleotides, (i) anchored antisense primers which are 10-14 mer designed complementary to poly(A) tail of mRNA and last two nucleotides of transcribed sequences, and (ii) arbitrary primer of 10 mer and often long arbitrary sense primers of 25-28 mers are also used.

to detect differential expression of mRNAs that are expressed in low abundance.

DNA microarray

DNA microarray or DNA chip or biochip comprises millions of DNA spots onto a solid support for comprehensive genome analysis. The parallelism, miniaturization, speed, multiplexing, automation, and combinatorial synthesis are typical features of DNA micro-array. Each DNA spot contains 10”? moles of a specific DNA sequence, called as probe

needed to hybridize cDNA. This hybridization is sensed and quantified by chemically

labeled or fluorescent targets to determine relative abundance of nucleic acid sequences in the target. The following steps are associated with DNA microarray:

® Printing or deposition of high-density nucleic acid samples (CDNA or oligonucle-otides) onto very precise area of the support system referred to as fabrication of chips followed by immobilization to the substrate.

</div><span class="text_page_counter">Trang 37</span><div class="page_container" data-page="37">

Applied molecular biotechnology

“Surunsuod aury “9soư

0} anp Yy8ry SỊ ayer 10119 ay,

saseq 00z tt sSo[ pur ‘tadeayp

219IsbJ “ySry SỊ ayes AvmMI0y

Surouanbas VNI 10J SAAO[[e OS[E

pur (sapnoa[anu jo ađÁ) aưo jo

Supeai jo Á2eimsop paoueyug

Aq payayap are yoty ‘paonpoad are suor

“SIsayuÁs paseq-asetouiÁJod yNa Áq sapyoaponu jo

uopeiodioaut Surmp ‘Sutsuanbes yNq [eondoouen,

2[n2a[ott ø[8us se 0} paxtoga1 Á[uOurtuo2 st

asIoAar 8uTA|oAut pouiauu 8ur2uenbas SỊ I]

“onbrtr5ø1 sip jo sưonttuun[ 1ofeut

axe aw uns 8uo[ pue t)8uô[ peas 10s 2L 'p3sn

Suraq st ya uoIs[nuio os[e 2191 'soqoxd ø|qeAea[

y8nomy ưone[ saA[oAuT (ITQS) uoq22ap

pur ưone8I[ Áq sapnoatnuo8iio jo Supuanbag'P2ZII0n st 101EU†U119) ð|dIS12A21 ,£ 6 Suruteyu0D

pue saxoydozony ano; jo auo Aq pa[2qe[ toe2

‘sapnoajanu jo sad} anoy uoyovax 8u[2uanbas ayy tị

'(uopeatitidure a8prq) aovpins pros uo

payrdure st aouanbas 1281 1osn[2 ønbru22) sry uy

‘qy8y 8ưneaaua3 Aq ase1aran[ Aq paysayruew st WNC.

yuadseu ay] Out apHoapnu Jo ưore1odO2uT ayy,

2t] areredas 0) spoyaur 2n21otdo1i2Ja Ut pasp) «

Siu9I801]'VNC Jo 195 paysau oteouo8

</div><span class="text_page_counter">Trang 38</span><div class="page_container" data-page="38">

Emerging tools and approaches to biotechnology in the omics era

tu ST UREN “ABojouypay aaodoueu

E2S 2uIOU98 afoyr JOJ HOISIAOadi YIM sot8ojoutoz 8uyouonbas ưoneaaus8-patti

“8utpuanbas jo paads 8H, 1o 8uppuanbas a[n2o[otu a[8us ayy sautqtuo2 I]

Surauanbas uopieiaua8-(ano,[

AQUULUT 2A1139/J3-1SO2 UT UNI apyoaponu ayy autua‡ap 0} pasn st uore8trT

aad paouanbas aq ue> s[[¿qoưeu __ 'sJjeqoueu pa[[83-os ayy ou†so2uanbas VNC [[ews

Na Sueur sjaaneredwo> jo uoneoyydure asi 8unJ[ox v sasn pouaur ayy

ïN SỊ ayer 10119 pue yy8uay pear 19AAO[ UỊ synsar

Ajjesoua8 yy ‘sind qy8x 1o uorsstwa ue <q pappe 2pRoa[anu ayy jo 8utaoi[uot auM-[ear 10J uoIs(Aoxd

6 SỊ 0101] pue WNC are[duuay Jo uoniespridure sproae

peardq 0001 3I'S2pm8aAeM apour-o1az jo saysodord ayy uo paseg

</div><span class="text_page_counter">Trang 39</span><div class="page_container" data-page="39">

16 Applied molecular biotechnology ¢ Hybridization of resulting microarray with a fluorescently labeled probe.

* Detection of fluorescent markers using high-resolution laser scanner.

* Gene expression pattern is analyzed based on signal emitted from each spot using digital imaging software.

® The expression pattern of two different samples can be compared.

The microarray fabrication can be performed either by in situ synthesis of nucleic acids or by exogenous deposition of prepared materials on solid substrates. Prefabricated micro-arrays can also be purchased from companies such as Affymetrix, Research Genetics, CLONTECH, Incyte Genomics, Operon Technologies, Genometrix Inc., etc.

Two commonly observed variants of DNA microarray is oligonucleotide and CDNA microarray. The commonly used methods of chip fabrication include contact printing, photolithography, pin and ring, piezoelectric printing, and bubble jet technology.

Protein microarray

Small quantities of diverse purified proteins are assorted on a solid support. The purity of proteins, possessing its native conformation and its concentration are important conside-rations for protein microarray. Typically, fluorescently labeled probes are used for signal generation and identification.

Other than protein and DNA microarrays several different versions of microarrays are emerging as shown in Figure 1.4.

Figure 1.4 Types of microarrays.

</div><span class="text_page_counter">Trang 40</span><div class="page_container" data-page="40">

Chapter one: Emerging tools and approaches to biotechnology in the omics era 7

. Peptide microarray: It is a collection of small peptides attached onto solid support either glass or plastic. The peptide microarray is generally used for assessing the

protein interactions, elucidating the binding and functionality with the target

. Tissue microarray: In this a number of tissues, typically from different organs, are thrown together in the same block and tissue distributions of a particular antigen/

protein are assessed.

. Cellular microarray: Living-cell microarray allows the assessment of cellular response to different external stimuli.

4. Chemical compound microarray: Used for the study of the interaction between chemical compounds and biological targets.

Antibody microarray: Used for protein expression profiling and comparative analysis

between different samples.

Carbohydrate microarray: Used for measurements of glycan—protein interactions and disease diagnosis.

7. Phenotype microarray: Used for genotype-phenotype characterization and determin-ing most favorable conditions for different cellular activities.

N °

Blotting techniques for the study of DNA, RNA, and proteins

Blotting techniques are techniques for identification of targeted nucleic acid or protein by

immobilization onto specific support either nylon or nitrocellulose followed by detection with probes. Nucleic acids blotting techniques include blotting of nucleic acids from gels (southern hybridization, northern hybridization), dot/slot blotting, and colony /plaque blotting. This technique consists of four major steps namely, (i) resolution of protein and nucleic acid samples by electrophoretic means, (ii) transfer and immobilization on solid support (by capillary blotting, vacuum blotting, electrophoretic transfer), (iii) binding of analytical probe, and (iv) visualization of bound probe to target molecule usually by auto-radiography. The fixation of the nucleic acid sample to the membrane can be achieved by several methods such as ultraviolet cross-linking, oven baking, alkali fixation, and

micro-wave fixation. The blotting techniques designated as southern, northern, and western

blotting are routinely used molecular biology technique. Southern blotting identifies DNA fragments that bind with the probes, through hybridization complementary fragments of target DNA. Northern blotting identifies messenger RNA (mRNA) after hybridization to their corresponding DNA sequences, whereas, western blotting identifies particular

pro-teins using specific antibodies as probes.

Spectroscopic techniques

The main principle underlying spectroscopic techniques is that each and every atom and molecule absorbs and emits light at certain wavelengths. It involves the interaction

between electromagnetic radiation and matter, which can be an atom, molecule, ion, or solids. This may result in absorption, emission, or scattering. Since each chemical element has its own characteristic spectrum, this nature of interaction is used to analyze matter and to interpret its physical properties. The spectroscopic technique is meant for

determi-nation of concentration or amount of a given species. The different forms of spectroscopy

are discussed in the following sections.

</div>