Gene Synthesis Methods and Protocols
The de novo fabrication of custom DNA molecules is a transformative technology that significantly affects the biotechnology industry. Basic genetic engineering techniques for manipulating DNA in vitro opened an incredible field of opportunity in the life
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IN
MOLECULAR BIOLOGY™
Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes: http://www.springer.com/series/7651
Gene Synthesis Methods and Protocols
Edited by
Jean Peccoud Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
Editor Jean Peccoud, Ph.D. Virginia Bioinformatics Institute Virginia Tech Blacksburg, VA, USA [email protected]
ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61779-563-3 e-ISBN 978-1-61779-564-0 DOI 10.1007/978-1-61779-564-0 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2012930137 © Springer Science+Business Media, LLC 2012 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface The de novo fabrication of custom DNA molecules is a transformative technology that significantly affects the biotechnology industry. Basic genetic engineering techniques for manipulating DNA in vitro opened an incredible field of opportunity in the life sciences. However, genetic engineering has now moved beyond the introduction of single genes into cells to multigene cassettes, and is rapidly progressing toward whole genome engineering. In this new context, the synthesis of DNA molecules has resurged as the time and costlimiting step in genetic engineering. Today, most multigene engineering projects involve ad hoc methods of DNA assembly. A variety of PCR-based methods are in common use alongside more traditional restriction enzyme-based assembly methods. Their essential feature is the piecing together of existing DNAs that are cloned from natural sources. These techniques present a number of limitations. The use of restriction sites within natural sequences necessitates a labor intensive custom cloning strategy that is difficult to automate. As a result, molecular biologists often reach a tacit compromise between obtaining a desired sequence and the number of steps in the cloning process they are willing or able to undertake in constructing it. Theoretically, DNA fabrication methods that are rooted in chemical synthesis could transform synthesis into a generic, predictable, and scalable process allowing the generation of any user-defined DNA sequence. By liberating the process from t
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