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DNA SEQUENCING – METHODS AND APPLICATIONS Edited by Anjana Munshi DNA Sequencing – Methods and Applications Edited by Anjana Munshi Published by InTech Janeza Trdine 9, 51000 Rijeka, Croatia Copyright © 2012 InTech All chapters are Open Access distributed under the Creative Commons Attribution 3.0 license, which allows users to download, copy and build upon published articles even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. After this work has been published by InTech, authors have the right to republish it, in whole or part, in any publication of which they are the author, and to make other personal use of the work. Any republication, referencing or personal use of the work must explicitly identify the original source. As for readers, this license allows users to download, copy and build upon published chapters even for commercial purposes, as long as the author and publisher are properly credited, which ensures maximum dissemination and a wider impact of our publications. Notice Statements and opinions expressed in the chapters are these of the individual contributors and not necessarily those of the editors or publisher. No responsibility is accepted for the accuracy of information contained in the published chapters. The publisher assumes no responsibility for any damage or injury to persons or property arising out of the use of any materials, instructions, methods or ideas contained in the book. Publishing Process Manager Bojan Rafaj Technical Editor Teodora Smiljanic Cover Designer InTech Design Team First published April, 2012 Printed in Croatia A free online edition of this book is available at www.intechopen.com Additional hard copies can be obtained from [email protected] DNA Sequencing – Methods and Applications, Edited by Anjana Munshi p. cm. ISBN 978-953-51-0564-0 Contents Preface VII Section 1 Methods of DNA Sequencing 1 Chapter 1 DNA Representation 3 Bharti Rajendra Kumar Chapter 2 Hot Start 7-Deaza-dGTP Improves Sanger Dideoxy Sequencing Data of GC-Rich Targets 15 Sabrina Shore, Elena Hidalgo Ashrafi and Natasha Paul Section 2 Applications of DNA Sequencing 33 Chapter 3 Sequencing Technologies and Their Use in Plant Biotechnology and Breeding 35 Victor Llaca Chapter 4 DNA Sequencing and Crop Protection 61 Rosemarie Tedeschi Chapter 5 Improvement of Farm Animal Breeding by DNA Sequencing 85 G. Darshan Raj Chapter 6 The Input of DNA Sequences to Animal Systematics: Rodents as Study Cases 103 Laurent Granjon and Claudine Montgelard Chapter 7 The Application of Pooled DNA Sequencing in Disease Association Study 141 Chang-Yun Lin and Tao Wang Chapter 8 Nucleic Acid Aptamers as Molecular Tags for Omics Analyses Involving Sequencing 157 Masayasu Kuwahara and Naoki Sugimoto Preface More than a quarter of a century earlier the story of DNA sequencing began when Sanger’s studies of insulin first demonstrated the importance of sequence in biological macromolecules. Although two different DNA sequencing methods have been developed during the same period, Sanger’s dideoxy chain-termination sequencing method has became the method of choice over the Maxam–Gilbert method. The complete sequence of oX-174 was published in 1977 and then revised slightly in the following year by dideoxy method. It demonstrated that the DNA sequence could tell a fascinating story based upon the interpretation of the sequence in terms of the genetic code. Recently several next generation high throughput DNA sequencing techniques have arrived on the scene and are opening fascinating opportunities in the fields of biology and medicine. This book, “DNA Sequencing - Methods and Applications” illustrates methods of DNA sequencing and its application in plant, animal and medical sciences. This book has two distinct sections. The first one includes 2 chapters devoted to the DNA sequencing methods and the second one includes 6 chapters focusing on various applications of this technology. The content of the articles presented in the book is guided by the knowledge and experience of the contributing authors. This book is intended to serve as an important resource and review to the researchers in the field of DNA sequencing. An overview of DNA sequencing technologies right from the Sanger’s method to the next generation high throughput DNA sequencing techniques including massively parallel signature sequencing, polony sequencing, pyrosequencing, Illumina Sequencing, SOLiD sequencing etc. has been presented in chapter 1. Chapter 2 reviews how Hot Start-7-deaza-dGTP improves Sanger’s dideoxy sequencing data of GC rich template DNA. Chapter 3 demonstrates how the use of sequencing methods in combination with strategies in breeding and molecular genetic modifications has contributed to our knowledge of plant genetics and remarkable increase in agricultural productivity. Chapter 4 provides information on applications of DNA sequencing in crop protection. This chapter highlights the perspectives for new sustainable and environmental friendly strategies for controlling pests and diseases of crop plants. VIII Preface Chapter 5 has discussed the application of DNA sequencing in improving the breeding strategies of farm animals. The development of molecular markers using DNA sequencing serves as an underlying tool, for geneticists and breeders to create desirable farm animals. Chapter 6 aims at showing how DNA sequencing technology has reboosted rodent systematics leading to a much better supported classification of this order. The molecular data generated by DNA sequencing has played an important role in rodent systematics over the last decades indicating the importance of this kind of information in evolutionary biology as a whole. Chapter 7 has discussed the application of pooled DNA sequencing in disease association studies. It is a cost effective strategy for genome-wide association studies (GWAS) and successfully identifies hundreds of variants associated with complex traits. Some strategies of pooling design including PI- deconvolution shifted- transversal design, multiplex scheme and overlapping pools to recover linkage disequilibrium information have also been introduced. Statistical methods for the detection of variants and case-control association studies accounting for high levels of sequencing errors have been discussed. Chapter 8 focuses on the development of nucleic acid-aptamers and the outlook for related technologies. Aptamers can be readily amplified by PCR and decoded by sequencing and it is possible to apply them as molecular tags to quantitative bimolecular analysis and single cell analysis. The scientific usefulness of DNA sequencing continues to be proven, and the number of sequenced and catalogued genomes has grown more than five times from where it was at the middle of the decade. Anjana Munshi Department of Molecular Biology, Institute of Genetics and Hospital for Genetic Diseases, Hyderabad, India Section 1 Methods of DNA Sequencing 1 DNA Representation Bharti Rajendra Kumar B.T. Kumaon Institute of Technology, Dwarahat,Almora, Uttarakhand, India 1. Introduction The term DNA sequencing refers to methods for determining the order of the nucleotides bases adenine,guanine,cytosine and thymine in a molecule of DNA. The first DNA sequence were obtained by academic researchers,using laboratories methods based on 2- dimensional chromatography in the early 1970s. By the development of dye based sequencing method with automated analysis,DNA sequencing has become easier and faster. The knowledge of DNA sequences of genes and other parts of the genome of organisms has become indispensable for basic research studying biological processes, as well as in applied fields such as diagnostic or forensic research. DNA is the information store that ultimately dictates the structure of every gene product, delineates every part of the organisms. The order of the bases along DNA contains the complete set of instructions that make up the genetic inheritance. The rapid speed of sequencing attained with modern DNA sequencing technology has been instrumental in the sequencing of the human genome, in the human genome project. Fig. 1. DNA Sequence Trace DNA can be sequenced by a chemical procedure that breaks a terminally labelled DNA molecule partially at each repetition of a base. The length of the labelled fragments then identify the position of that base. We describe reactions that cleave DNA preferentially at guanines,at adenines,at cytosine and thymines equally, and at cytosine alone. When the product of these four reactions are resolved by size,by electrophoresis on a polyacrylamide gel, the DNA sequences can be read from the pattern of radioactive bands. The technique 4 DNA Sequencing – Methods and Applications will permit sequencing of atleast 100 bases from the point of labelling. The purine specific reagent is dimethyl sulphate; and the pyrimidine specific reagent is hydrazine. In 1973 , Gilbert and Maxam reported the sequence of 24 base pairs using a method known as wandering- spot analysis. The chain termination method developed by Sanger and coworkers in 1975 owing to its relative easy and reliability. In 1975 the first complete DNA genome to be sequenced is that of bacteriophage X174. By knowing the DNA sequence, the cause of the various diseases can be known. We can determine the sequence responsible for various disease and can be treated with the help of Gene therapy. DNA sequencing is very significant in research and forensic
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