Microarrays SECOND EDITION M E T H O D S I N M O L E C U L A R B I O L O G Y™ John M. Walker, SERIES EDITOR 383. Cancer Genomics and Proteomics: Methods and 360. Target Discovery and Validation Reviews Protocols,edited by Paul B. Fisher, 2007 and Protocols: Emerging Strategies for Targets 382. Microarrays, Second Edition: Volume 2, Applications and Biomarker Discovery, Volume 1, edited by and Data Analysis,edited by Jang B. Rampal, 2007 Mouldy Sioud, 2007 381. Microarrays, Second Edition: Volume 1, Synthesis 359.Quantitative Proteomics by Mass Spectrom- Methods,edited by Jang B. Rampal, 2007 etry,edited by Salvatore Sechi, 2007 380. Immunological Tolerance:Methods and Protocols, 358. Metabolomics: Methods and Protocols,edited by edited by Paul J. Fairchild, 2007 Wolfram Weckwerth, 2007 379. Glycovirology Protocols,edited by Richard J. 357. Cardiovascular Proteomics: Methods and Protocols, Sugrue, 2007 edited by Fernando Vivanco, 2006 378. Monoclonal Antibodies:Methods and Protocols, 356. High Content Screening: A Powerful Approach edited by Maher Albitar, 2007 to Systems Cell Biology and Drug Discovery, edited by D. Lansing Taylor, Jeffrey Haskins, 377.Microarray Data Analysis:Methods and and Ken Guiliano, 2007 Applications,edited by Michael J. Korenberg, 2007 355. Plant Proteomics:Methods and Protocols,edited 376. Linkage Disequilibrium and Association by Hervé Thiellement, Michel Zivy, Catherine Mapping:Analysis and Application, edited by Damerval, and Valerie Mechin, 2006 Andrew R. Collins, 2007 354. Plant–Pathogen Interactions:Methods and 375. In Vitro Transcription and Translation Protocols: Protocols,edited by Pamela C. Ronald, 2006 Second Edition, edited by Guido Grandi, 2007 353. DNA Analysis by Nonradioactive Probes: Methods 374.Quantum Dots:Methods and Protocols, edited and Protocols,edited by Elena Hilario and John. F. by Charles Z. Hotz and Marcel Bruchez, 2007 MacKay, 2006 373.Pyrosequencing® Protocols, edited by Sharon 352. Protein Engineering Protocols,edited by Kristian Marsh, 2007 Müller and Katja Arndt, 2006 372.Mitochondrial Genomics and Proteomics 351. C. elegans: Methods and Applications,edited by Protocols, edited by Dario Leister and Johannes Kevin Strange, 2006 Herrmann, 2007 350. Protein Folding Protocols,edited by Yawen Bai 371. Biological Aging:Methods and Protocols, edited by and Ruth Nussinov 2007 Trygve O. Tollefsbol, 2007 349. YAC Protocols, Second Edition,edited by Alasdair 370. Adhesion Protein Protocols,Second Edition, edited MacKenzie, 2006 by Amanda S. Coutts, 2007 348. Nuclear Transfer Protocols: Cell Reprogramming 369. Electron Microscopy:Methods and Protocols, and Transgenesis,edited by Paul J. Verma and Alan Second Edition, edited by John Kuo, 2007 Trounson, 2006 368. Cryopreservation and Freeze-Drying Protocols, 347. Glycobiology Protocols,edited by Inka Second Edition, edited by John G. Day and Glyn Brockhausen-Schutzbach, 2006 Stacey, 2007 346. Dictyostelium discoideum Protocols,edited by 367. Mass Spectrometry Data Analysis in Proteomics, Ludwig Eichinger and Francisco Rivero, 2006 edited by Rune Matthiesen, 2007 345. Diagnostic Bacteriology Protocols, Second Edi- 366. Cardiac Gene Expression: Methods and Protocols, tion,edited by Louise O'Connor, 2006 edited by Jun Zhang and Gregg Rokosh, 2007 344.Agrobacterium Protocols, Second Edition: 365. Protein Phosphatase Protocols: edited by Greg Volume 2, edited by Kan Wang, 2006 Moorhead, 2007 343.Agrobacterium Protocols, Second Edition: 364. Macromolecular Crystallography Protocols: Volume 1, edited by Kan Wang, 2006 Volume 2, Structure Determination,edited by Sylvie 342. MicroRNA Protocols,edited by Shao-Yao Ying, 2006 Doublié, 2007 341.Cell–Cell Interactions: Methods and Protocols, 363. Macromolecular Crystallography Protocols: edited by Sean P. 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Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 ISSN 1064-3745 E-ISBN 978-1-59745-303-5 Library of Congress Control Number: 2007924175 Dedication To my parents and gurus. v sdfsdf Preface To meet the emerging needs of genomics, proteomics, and the other omics, microarrays have become unique and important tools for high-throughput analysis of biomolecules. Microarray technology provides a highly sensitive and precise technique for obtaining information from biological samples. It can simultaneously handle a large number of analytes that may be processed rapidly. Scientists are applying microarray technology to understand gene expression, to analyze mutations and single-nucleotide polymorphisms, to sequence genes, and to study antibody–antigen interactions, aptamers, carbohydrates, and cell functions, among many other research subjects. The objective of Microarrays is to enable the researcher to design and fabricate arrays and binding studies with biological analytes. An additional goal is to provide the reader with a broader description of microarray technology tools and their potential applications. In this edition, Microarrays is divided in two parts: Volume 1 deals with methods for preparation of microarrays, and Volume 2 with applications and data analysis. Various methods and applications of microarrays are described and accompanied by exemplary protocols. Volume 2 also covers topics related to bioinformatics, an important aspect of microarray technologies because of the enormous amount of data coming out of microarray experiments. Together, the two volumes provide useful information to the novice and expert alike. From this point onward, I will discuss the contents of Volume I: Synthesis Methods. For readers just entering the array technology field, as well as those who are well versed, the history of microarray technology from its conception is covered in the first chapter. Surface activation chemistries and various types of matrices involved in the synthesis of microarrays are summarized in Chapters 2 and 3. As the major objective of this volume is to provide detailed synthesis methods for constructing microarrays, so the emphasis of the remaining chapters is on methods and protocols. I tried to include various types of protocols. Some may look very similar, but in fact each protocol has a unique utility based on the research problem or individual interests. Chapter 4 details array optimization processes based on numerous factors, for example, the printing quality, spot morphology, and quantification of hybridized target. Chapter 5 presents array-based comparative genomic hybridization (array CGH) and includes procedures for making bacterial artificial chromosome vii viii Preface DNA arrays. Chapter 6 describes the 60-mer oligonucleotide probes immobilized on coated glass slides to study the effect of target concentration, retention, signal linearity, and properties of fluophores in quantitative gene expression measurements. The array production method using premodified DNA can be directly applied to construction of oligo or cDNA arrays. Such arrays can be used for detection of chromosomal abnormalities in complex genomes. Chapter 7 highlights the use of unique bifunctional reagents, NTMTA and NTPAC for building glass and plastic biochips. Chapter 8 explains the use of sensitive reagents for the determination of the functional group density in the microarray system by spectrophotometric methods. Chapter 9 illustrates the synthesis of high-density arrays using a digital microarray synthesis platform. The use of long optimized oligonucleotide probes (150-mers) for high and specific signal intensity for the measurement of gene expression is described in Chapter 10. In addition to sequence and probe length, the importance of other parameters, such as the surface of the glass slide, linkers/spacers, and the conditions for hybridization are also highlighted in Chapter 10. Chapter 11 deals with in situ synthesized oligoarrays using the Southern Array Maker (SAM) synthesizer and standard phosphoramidite chemistry. The array probes, including cystic fibrosis, were synthesized onto the flat surface of aminated polypropylene. The printing and use of the synthetic oligonucleotide probes for the detection of multiplex ligation-dependent probe amplification products is explained in Chapter 12. The synthesis and the use of grafted pyrrole oligonucleotide probes are demonstrated in Chapter 13. Chapter 14 deals with the optimization of hybridization conditions for in situ synthesized oligoarrays on plastic. Chapters 15 and 16 are devoted for the synthesis of peptide arrays. Creation of protein microarrays in microplate is described in Chapter 17. Arrays of the captured monoclonal antibodies corresponding to specific interleukins are printed down onto the bottom of the wells. A Biomek® 2000 workstation equipped with a high-density replicating tool is used for printing the low-density arrays. For higher density arrays, a microarrayer system (BioDot, Inc.) is employed. Printing the protein arrays onto specially polymer-coated glass slide while maintaining the activity and structure of the protein is described in Chapter 18. Chapter 19 focuses on the printing of cell microarrays for the functional exploration of genomes. Chapter 20 is related to suspension arrays. The oligo- coated microparticles are hybridized with the target molecule. The protocol for quantification of oligohybridization complex is analyzed by a europium (111) detection system. Glyco-bead array for calculating the sugar-binding lectins is described in Chapter 21. As we all are aware that array technology is moving forward from micro to nano, Chapter 22 describes this emerging technology. Preface ix The chapter highlights the utility of nanoarrays, particularly the analysis of nanoarrays by using label-free nucleic acids and proteins and others. I believe this volume, Synthesis Methods, will provide valuable information to scientists at all levels, from the novice to those intimately familiar with array technology. I would like to thank all the contributing authors for providing manuscripts. My thanks are also due to colleagues for their help in completing this work. I thank John Walker for editorial guidance and the staff of Humana Press for making it possible to include large body of available microarray technologies in this volume. Finally, my thanks to my family, especially to my sweet wife Sushma Rampal, for providing all sorts of incentives to complete this project successfully. Jang B. Rampal