genomics protocols - michael p. starkey , ramnath elaswarapu

Methods in Molecular Biology TM VOLUME 175 Genomics Protocols Edited by Michael P Starkey Ramnath Elaswarapu HUMANA PRESS Genomics Protocols 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 183 Green Fluorescent Protein: Applications and Protocols, edited by Barry W Hicks, 2002 182 In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff Braman, 2002 181 Genomic Imprinting: Methods and Protocols, edited by Andrew Ward, 2002 180 Transgenesis Techniques, 2nd ed.: Principles and Protocols, edited by Alan R Clarke, 2002 179 Gene Probes: Principles and Protocols, edited by Marilena Aquino de Muro and Ralph Rapley, 2002 178.`Antibody Phage Display: Methods and Protocols, edited by Philippa M O’Brien and Robert Aitken, 2001 177 Two-Hybrid Systems: Methods and Protocols, edited by Paul N MacDonald, 2001 176 Steroid Receptor Methods: Protocols and Assays, edited by Benjamin A Lieberman, 2001 175 Genomics Protocols, edited by Michael P Starkey and Ramnath Elaswarapu, 2001 174 Epstein-Barr Virus Protocols, edited by Joanna B Wilson and Gerhard H W May, 2001 173 Calcium-Binding Protein Protocols, Volume 2: Methods and Techniques, edited by Hans J Vogel, 2001 172 Calcium-Binding Protein Protocols, Volume 1: Reviews and Case Histories, edited by Hans J Vogel, 2001 171 Proteoglycan Protocols, edited by Renato V Iozzo, 2001 170 DNA Arrays: Methods and Protocols, edited by Jang B Rampal, 2001 169 Neurotrophin Protocols, edited by Robert A Rush, 2001 168 Protein Structure, Stability, and Folding, edited by Kenneth P Murphy, 2001 167 DNA Sequencing Protocols, Second Edition, edited by Colin A Graham and Alison J M Hill, 2001 166 Immunotoxin Methods and Protocols, edited by Walter A Hall, 2001 165 SV40 Protocols, edited by Leda Raptis, 2001 164 Kinesin Protocols, edited by Isabelle Vernos, 2001 163 Capillary Electrophoresis of Nucleic Acids, Volume 2: Practical Applications of Capillary Electrophoresis, edited by Keith R Mitchelson and Jing Cheng, 2001 162 Capillary Electrophoresis of Nucleic Acids, Volume 1: Introduction to the Capillary Electrophoresis of Nucleic Acids, edited by Keith R Mitchelson and Jing Cheng, 2001 161 Cytoskeleton Methods and Protocols, edited by Ray H Gavin, 2001 160 Nuclease Methods and Protocols, edited by Catherine H Schein, 2001 159 Amino Acid Analysis Protocols, edited by Catherine Cooper, Nicole Packer, and Keith Williams, 2001 158 Gene Knockoout Protocols, edited by Martin J Tymms and Ismail Kola, 2001 157 Mycotoxin Protocols, edited by Mary W Trucksess and Albert E Pohland, 2001 156 Antigen Processing and Presentation Protocols, edited by Joyce C Solheim, 2001 155 Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000 154 Connexin Methods and Protocols, edited by Roberto Bruzzone and Christian Giaume, 2001 153 Neuropeptide Y Protocols, edited by Ambikaipakan Balasubramaniam, 2000 152 DNA Repair Protocols: Prokaryotic Systems, edited by Patrick Vaughan, 2000 151 Matrix Metalloproteinase Protocols, edited by Ian M Clark, 2001 150 Complement Methods and Protocols, edited by B Paul Morgan, 2000 149 The ELISA Guidebook, edited by John R Crowther, 2000 148 DNA–Protein Interactions: Principles and Protocols (2nd ed.), edited by Tom Moss, 2001 147 Affinity Chromatography: Methods and Protocols, edited by Pascal Bailon, George K Ehrlich, Wen-Jian Fung, and Wolfgang Berthold, 2000 146 Mass Spectrometry of Proteins and Peptides, edited by John R Chapman, 2000 145 Bacterial Toxins: Methods and Protocols, edited by Otto Holst, 2000 144 Calpain Methods and Protocols, edited by John S Elce, 2000 143 Protein Structure Prediction: Methods and Protocols, edited by David Webster, 2000 142 Transforming Growth Factor-Beta Protocols, edited by Philip H Howe, 2000 141 Plant Hormone Protocols, edited by Gregory A Tucker and Jeremy A Roberts, 2000 140 Chaperonin Protocols, edited by Christine Schneider, 2000 139 Extracellular Matrix Protocols, edited by Charles Streuli and Michael Grant, 2000 138 Chemokine Protocols, edited by Amanda E I Proudfoot, Timothy N C Wells, and Christine Power, 2000 137 Developmental Biology Protocols, Volume III, edited by Rocky S Tuan and Cecilia W Lo, 2000 136 Developmental Biology Protocols, Volume II, edited by Rocky S Tuan and Cecilia W Lo, 2000 135 Developmental Biology Protocols, Volume I, edited by Rocky S Tuan and Cecilia W Lo, 2000 134 T Cell Protocols: Development and Activation, edited by Kelly P Kearse, 2000 133 Gene Targeting Protocols, edited by Eric B Kmiec, 2000 132 Bioinformatics Methods and Protocols, edited by Stephen Misener and Stephen A Krawetz, 2000 131 Flavoprotein Protocols, edited by S K Chapman and G A Reid, 1999 130 Transcription Factor Protocols, edited by Martin J Tymms, 2000 129 Integrin Protocols, edited by Anthony Howlett, 1999 128 NMDA Protocols, edited by Min Li, 1999 127 Molecular Methods in Developmental Biology: Xenopus and Zebrafish, edited by Matthew Guille, 1999 126 Adrenergic Receptor Protocols, edited by Curtis A Machida, 2000 125 Glycoprotein Methods and Protocols: The Mucins, edited by Anthony P Corfield, 2000 124 Protein Kinase Protocols, edited by Alastair D Reith, 2001 123 In Situ Hybridization Protocols (2nd ed.), edited by Ian A Darby, 2000 122 Confocal Microscopy Methods and Protocols , edited by Stephen W Paddock, 1999 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™ Genomics Protocols Edited by Michael P Starkey and Ramnath Elaswarapu UK Human Genome Mapping Project Resource Centre, Cambridge, UK Humana Press Totowa, New Jersey © 2001 Humana Press Inc 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher Methods in Molecular Biology™ is a trademark of The Humana Press Inc The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work The publisher, editors, and authors are not responsible for errors or omissions or for any consequences arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents This publication is printed on acid-free paper ∞ ANSI Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials Cover design by Patricia F Cleary For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: humana@humanapr.com; or visit our Website: www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $10.00 per copy, plus US $00.25 per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923 For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc The fee code for users of the Transactional Reporting Service is: [0-89603-774-6/01 (hardcover) $10.00 + $00.25] Printed in the United States of America 10 Library of Congress Cataloging in Publication Data Genomics protocols / edited by Michael P Starkey and Ramnath Elaswarapu p ; cm.—(Methods in molecular biology ; 175) Includes bibliographical references and index ISBN 0-89603-774-6 (hardcover ; alk paper) ISBN 0-89603-708-8 (comb ; alk paper) Molecular genetics—Laboratory manuals Genomics–Laboratory manuals I Starkey, Michael P II Elaswarapu, Ramnath III Series QH440.5 G46 2001] 572.8—dc21 00-046106 Preface We must unashamedly admit that a large part of the motivation for editing Genomics Protocols was selfish The possibility of assembling in a single volume a unique and comprehensive collection of complete protocols, relevant to our work and the work of our colleagues, was too good an opportunity to miss We are pleased to report, however, that the outcome is something of use not only to those who are experienced practitioners in the genomics field, but is also valuable to the larger community of researchers who have recognized the potential of genomics research and may themselves be beginning to explore the technologies involved Some of the techniques described in Genomics Protocols are clearly not restricted to the genomics field; indeed, a prerequisite for many procedures in this discipline is that they require an extremely high throughput, beyond the scope of the average investigator However, what we have endeavored here to achieve is both to compile a collection of procedures concerned with genomescale investigations and to incorporate the key components of “bottom-up” and “top-down” approaches to gene finding The technologies described extend from those traditionally recognized as coming under the genomics umbrella, touch on proteomics (the study of the expressed protein complement of the genome), through to early therapeutic approaches utilizing the potential of genome programs via gene therapy (Chapters 27–30) Although a number of the procedures described represent the tried and trusted, we have striven to include new variants on existing technologies in addition to exciting new approaches Where there are alternative approaches to achieving a particular goal, we have sought assistance from an expert in the field to identify the most reliable technique, one suitable for a beginner in the field Unique to the Methods in Molecular Biology series is the “Notes” section at the end of each chapter This is a veritable Aladdin’s cave of information in which an investigator describes the quirks in a procedure and the little tricks that make all the difference to a successful outcome The first section of the volume deals with the traditional positional cloning approach to gene identification and isolation The construction of a high-resolution genetic map (Chapter 1) to facilitate the mapping of monogenic traits v vi Preface and approaches to the analysis of polygenic traits (Chapter 2) are described Identification of large numbers of single-nucleotide polymorphisms (Chapter 3) will pave the way for the construction of the next generation of genetic maps Also described are such comparatively new technologies as genomic mismatch scanning (Chapter 4), for the mapping of genetic traits, and comparative genomic hybridization (Chapter 5), for the identification of gross differences between genomes Such studies are a prelude to the screening of large genomic clones, or clone contigs (Chapter 7) These transitions are made possible by the localization of genomic clones (Chapter 8) and the integration of the genetic and physical maps (Chapter 9) achieved by STS mapping Identification of cDNAs mapping to the genomic clones implicated (Chapters 12–14) is the next step toward candidate gene identification With the desire to acquire cDNAs capable of expressing authentic proteins, the emphasis in cDNA library construction is placed on a technology capable of delivering full-length cDNAs (Chapter 10) One of the consequences of genome-scale sequencing programs has been the need to annotate large stretches of anonymous sequence data, and this has been the impetus for an explosion of bioinformatics programs targeted at gene prediction (Chapter 16) The use of model organisms (Chapter 17) to expedite gene discovery, on the basis of coding sequence similarites between genes with similiar functions, is another tool accessible to the gene hunter As an alternative to genetic studies, expression profiling seeks to identify candidate genes on the basis of their differential patterns of expression, either at the level of transcription or translation A number of technologies, based on subtractive hybridization, differential display, and high throughput in situ hybridization are thus described (Chapters 18–22) Functional characterization of isolated cDNAs is the next stage in establishing the likely candidature and thus potential utility of genes isolated as targets for therapeutic intervention Predictions of protein structure and function (Chapter 23), mutagenesis (Chapter 24), or knockout studies (Chapter 25) can enable predictions of gene function The yeast two-hybrid system (Chapter 26) is described at the level of monitoring interaction between individual proteins, but also on a potential genome scale In compiling Genomics Protocols, the aim—as with all other volumes in the Methods in Molecular Biology series—has been to produce a self-contained laboratory manual useful to both experienced practitioners and beginners in the field We trust that we have been at least moderately successful We must conclude by giving a vote of thanks to all the contributing authors, and to John Walker and the staff at Humana Press for seeing this project through Michael P Starkey Ramnath Elaswarapu Contents Preface v Contributors xi Construction of Microsatellite-Based, High-Resolution Genetic Maps in the Mouse Paul A Lyons Genetic Analysis of Complex Traits Stephen P Bryant and Mathias N Chiano 11 Sequence-Based Detection of Single Nucleotide Polymorphisms Deborah A Nickerson, Natali Kolker, Scott L Taylor, and Mark J Rieder 29 Genomic Mismatch Scanning for the Mapping of Genetic Traits Farideh Mirzayans and Michael A Walter 37 Detection of Chromosomal Abnormalities by Comparative Genomic Hybridization Mario A J A Hermsen, Marjan M Weiss, Gerrit A Meijer, and Jan P A Baak 47 Construction of a Bacterial Artificial Chromosome Library Sangdun Choi and Ung-Jin Kim 57 Contiguation of Bacterial Clones Sean J Humphray, Susan J Knaggs, and Ioannis Ragoussis 69 Mapping of Genomic Clones by Fluorescence In Situ Hybridization Margaret A Leversha 109 Map Integration: From a Genetic Map to a Physical Gene Map and Ultimately to the Sequence Map Panagiotis Deloukas 129 10 Construction of Full-Length–Enriched cDNA Libraries: The Oligo-Capping Method Yutaka Suzuki and Sumio Sugano 143 vii viii Contents 11 Construction of Transcript Maps by Somatic Cell/Radiation Hybrid Mapping: The Human Gene Map Panagiotis Deloukas 155 12 Preparation and Screening of High-Density cDNA Arrays with Genomic Clones Günther Zehetner, Maria Pack, and Katja Schäfer 169 13 Direct Selection of cDNAs by Genomic Clones Daniela Toniolo 189 14 Exon Trapping: Application of a Large-Insert Multiple-Exon-Trapping System Martin C Wapenaar and Johan T Den Dunnen 201 15 Sequencing Bacterial Artificial Chromosomes David E Harris and Lee Murphy 217 16 Finding Genes in Genomic Nucleotide Sequences by Using Bioinformatics Yvonne J K Edwards and Simon M Brocklehurst 235 17 Gene Identification Using the Pufferfish, Fugu rubripes, by Sequence Scanning Greg Elgar 249 18 Isolation of Differentially Expressed Genes Through Subtractive Suppression Hybridization Oliver Dorian von Stein 263 19 Isolation of Differentially Expressed Genes by Representational Difference Analysis Christine Wallrapp and Thomas M Gress 279 20 Expression Profiling and the Isolation of Differentially Expressed Genes by Indexing-Based Differential Display Michael P Starkey 295 21 Expression Profiling by Systematic High-Throughput In Situ Hybridization to Whole-Mount Embryos Nicolas Pollet and Christof Niehrs 309 22 Expression Monitoring Using cDNA Microarrays: A General Protocol Xing Jian Lou, Mark Schena, Frank T Horrigan, Richard M Lawn, and Ronald W Davis 323 23 Prediction of Protein Structure and Function by Using Bioinformatics Yvonne J K Edwards and Amanda Cottage 341 Contents ix 24 Identification of Novel Genes by Gene Trap Mutagenesis Anne K Voss and Tim Thomas 377 25 Determination of Gene Function by Homologous Recombination Using Embryonic Stem Cells and Knockout Mice Ahmed Mansouri 397 26 Genomic Analysis Utilizing the Yeast Two-Hybrid System Ilya G Serebriiskii, Garabet G Toby, Russell L FInley, Jr., and Erica A Golemis 415 27 Methods for Adeno-Associated Virus–Mediated Gene Transfer into Muscle Terry J Amiss and Richard Jude Samulski 455 28 Retroviral-Mediated Gene Transduction Donald S Anson 471 29 Gene Therapy Approaches to Sensitization of Human Prostate Carcinoma to Cisplatin by Adenoviral Expression of p53 and by Antisense Jun Kinase Oligonucleotide Methods Ruth Gjerset, Ali Haghighi, Svetlana Lebedeva, and Dan Mercola 495 30 Ribozyme Gene Therapy Leonidas A Phylactou 521 Index 531 524 Phylactou Fig Hammerhead ribozymes can be cloned into vectors containing prokaryotic promoters and synthesized by in vitro transcription oligonucleotide strands can include a degenerate base so that both active and inactive molecules can be present in the ligation reaction (7) 3.2.1 Annealing and Digestion of Ribozyme Oligonucleotides Mix pmol of both ribozyme strands in 10 mM Tris-HCl; 10 mM MgCl2; 10 mM NaCl; and 50 mM dithiothreitol (DTT), pH 7.9 Incubate at 95°C for min, then at 65°C for 10 min, followed by gradual cooling to room temperature Concentrate the annealed oligonucleotides by ethanol precipitation Perform sequential digests of the double-stranded oligonucleotides using appropriate restriction enzymes, e.g., HindIII and EcoRI (for pGEM-4Z) Following digestion, extract the restriction endonucleases with phenol/chloroform and precipitate the digested double-stranded oligonucleotides with ethanol Sequentially digest µg of pGEM-4Z with HindIII and EcoRI (as described in steps and 5) 3.2.2 Construction of Hammerhead Ribozymes Perform an overnight ligation at 6°C of 0.1 pmol of double-digested doublestranded ribozyme oligonucleotides and the linearized vector by using 3–5 M excess of the former in a total volume of 10 µL Transform competent E coli cells with µL of the ligation mix, and select recombinant clones Extract plasmid DNA from individual colonies, and identify active and inactive ribozyme clones by restriction digestion and dideoxy sequencing Linearize the ribozyme-containing constructs by restriction digestion Choose an appropriate restriction endonuclease (i.e., EcoRI for a ribozyme cloned in the appropriate orientation between the HindIII and EcoRI sites of pGEM-4Z) so that the construct is linearized at the end of the cloned ribozyme sequence (see Note 2) Ribozyme Gene Therapy 525 3.2.3 Ribozyme Synthesis by In Vitro Transcription Mix 33 nmol of linearized plasmid with all four ribonucleotide triphosphates (ATP, CTP, UTP, and GTP), at a final concentration of mM, in the presence of mM MgCl2; mM spermidine; 50 mM Tris-HCl, pH 7.5; mM DTT; 25 U of ribonuclease inhibitor; and 20 U of T7 RNA polymerase (for a ribozyme cloned in the appropriate orientation between the HindIII and EcoRI sites of pGEM-4Z) in a total volume of 50 µL Add all the reagents (see Note 3) at room temperature to avoid precipitation of DNA Incubate the reaction at 37°C for 2–4 h and stop the reaction by adding of U of RNase-free DNase I and further incubating at 37°C for 15 Remove enzymes by phenol/chloroform extraction and recover the newly synthesized transcript by ethanol precipitation Check the quality and amount of ribozymes by reading their absorbance at 260 nm and or by denaturing (7 M urea) polyacrylamide gel electrophoresis (PAGE) alongside markers of known concentration (see Note 4) 3.3 Production of RNA Target A labeled target RNA can be synthesized by in vitro transcription As template, a PCR product can be used that contains the T7 promoter upstream of the target cDNA The T7 promoter can be incorporated into the PCR fragment by a second PCR reaction The first PCR product is generated from cDNA synthesized from total RNA extracted from cells or tissue expressing the target gene (Fig 3) 3.3.1 Target Synthesis by Direct Template Production The method used to synthesize labeled RNA target in our laboratory uses a PCR product as the template for in vitro transcription The PCR product used for transcription is the result of two rounds of amplification reaction (7) (Fig 3) During the first, the target cDNA, containing the ribozyme binding site, is amplified, and in the second round of amplification, a T7 promoter is added to the 5' end of the PCR product Target RNA is then synthesized by in vitro transcription, using T7 RNA polymerase Alternatively, target RNA can be constructed and synthesized by cloning the first PCR product followed by linearization of the target cDNA-containing construct and in vitro transcription (see Note 5) Extract total RNA from cells or tissue, expressing the target gene, using TRIZOL according to the manufacturer’s instructions Carry out a reverse transcription with either an oligo(dT) primer or a primer specific for the RNA of interest Set up a PCR amplification of the newly synthesized cDNA using upstream and downstream primers designed to amplify the part of the target cDNA, that con- 526 Phylactou Fig Construction and synthesis of labeled target RNA used for in vitro ribozyme testing Template for in vitro transcription can be in the form of either linearized plasmid or a PCR product Target can be cloned into the vector of choice in the form of a PCR-amplified cDNA, derived from total RNA extracted from target-expressing cells Alternatively, a T7 promoter can be added to the PCR product by a second PCR amplification, thus creating a template for target synthesis by in vitro transcription tains the ribozyme cleavage site Ensure that the PCR reaction has been successful by checking a small sample by agarose or PAGE Use ng of the first-round PCR product as a template for the second round of amplification Employ the same downstream primer as in the first round of PCR, but the upstream primer should be a universal primer composed of the T7 promoter (see Note 6) Ensure that the PCR reaction has been successful by checking a small sample by agarose or PAGE 3.3.2 In Vitro Target RNA Synthesis RNA targets are synthesized by in vitro transcription Our standard protocol uses [α-32P UTP] to incorporate labeled nucleotide The method used is similar to that used for synthesizing ribozyme Ribozyme Gene Therapy 527 Mix 33 nmol of the second PCR product (Subheading 3.31., step 4) with ATP, CTP, and GTP at a final concentration of 0.5 mM; 50 µCi of [α-32P UTP] (800 Ci/mmol); mM MgCl2; mM spermidine; 50 mM Tris-HCl, pH 7.5; mM DTT; 12.5 U of ribonuclease inhibitor; and 10 U of T7 RNA polymerase in a total volume of 20 µL Add all reagents at room temperature to avoid precipitation of DNA Incubate the reaction at 37°C for 60 min, and halt by adding U of RNase-free DNase I and further incubating at 37°C for 15 Remove all the enzymes by phenol/chloroform extraction, and then recover the newly synthesized transcript by ethanol precipitation Determine the specific activity of the labeled target by precipitation with trichloroacetic acid followed by liquid scintillation counting (see Note 7) 3.4 In Vitro Ribozyme Cleavage Assay In vitro synthesized ribozymes can then be tested for their ability to cleave the target RNA prior to cell culture experiments This can be done by incubating the ribozyme with the labeled mini-target under optimum conditions (see Note 8) (8) Mix the hammerhead ribozyme (active or inactive) with the labeled target RNA in the presence of 50 mM Tris-HCl, pH 7.5, and 20 mM MgCl2 in a total volume of 20 µL Incubate the samples at either 37°C (physiologic temperature) or at 50°C (cleavage being more efficient at the higher temperature Halt the reaction by adding of 20 mM EDTA Separate the labeled cleavage products from the target RNA by denaturing (7 M urea) PAGE and detect by autoradiography (Fig 4) Notes To avoid the time and expense of cloning the ribozyme sequence adjacent to a prokaryotic promoter, an alternative approach is to produce hammerhead ribozymes directly off a synthetic DNA template (7) The template can contain the ribozyme sequence and the promoter The annealing of the oligonucleotides is performed as in Subheading 3.2.1., followed by in vitro transcription as described in Subheading 3.2.3 Restriction enzymes that create a 3' overhang should be avoided since the transcription will be inefficient Particular care should be taken to prepare ribonuclease-free solutions since contamination will result in degradation of both target and ribozyme All commercially available reagents used for RNA work are prepared with ribonuclease-free solutions Solutions made in the laboratory that will be used for RNA work should be prepared with diethylpyrocarbonate (DEPC) treated water (0.5% [v/v] DEPC in dH2O) Mix vigorously and leave at room temperature overnight Autoclave to break down the DEPC to CO2 and H2O 528 Phylactou Fig Example of hammerhead ribozyme-mediated cleavage of a target RNA Labeled target RNA has been incubated with a hammerhead ribozyme at different time intervals (lanes and 3), or with its catalytically inactive version (lane 4) at 37°C Lane shows incubation of the labeled target in the absence of ribozyme Labeled target and cleavage products have been detected by denaturing PAGE A combination of the two methods is advised, because neither of them alone can provide a very accurate calculation of the ribozyme concentration An alternative way to synthesize labeled target RNA is by cloning the first PCR product described in Subheading 3.3.1 If the PCR primers include restriction sites at their 5' ends, the PCR product can be cloned as described in Subheading 3.2.2 The target-containing vector can be linearized by restriction digest as described in Subheading 3.2.2 In vitro transcription is then used to produce the labeled target RNA in the same way as described in Subheading 3.3.2 The T7 RNA polymerase promoter sequence 5'-CTCACTATAGCC is incorporated at the 5' end of the universal upstream primer (5'-AATTTAATACGACT CACTATAG-3') used in the second PCR amplification The conditions for both PCR reactions are standard, with an appropriate annealing temperature that depends on the GC content of the PCR primers complementary to the target cDNA sequence We usually find that the radioactive ribonucleotide is not limiting during transcription when an RNA target of