FNR498_PCR_RZ 02.06.2006 14:55 Uhr Seite 1 C M Y CM MY CY CMY K PCR Applications Manual 3rd edition www.roche-applied-science.com Probedruck Acknowledgement We would like to thank all contributors and editors for their diligent efforts. Without their work, this project would not have been possible. Impressum Editorial Management: Degen, Hans-Joachim, Ph.D. Deufel, Annette, Ph.D. Eisel, Doris Grünewald-Janho, Stefanie Keesey, Joe, Ph.D. Art Direction: Fanz & Neumayer, Schifferstadt Layout Design and Typesetting: Active Artware GmbH, Saarbrücken © 2006 by Roche Diagnostics GmbH, Mannheim All rights reserved. No part of this booklet may be reproduced in any form without written permission of the publishers. Printed in Germany. www.roche-applied-science.com Table of Contents 1 Introduction Page 1.1 Principles of PCR and RT-PCR ........................................................................................................................... 9 1.2 The Evolution of PCR ..........................................................................................................................................11 1.3 Purpose of this PCR Applications Manual...................................................................................................15 2 General Guidelines Page 2.1 Preventing Contamination in the PCR Laboratory....................................................................................19 2.1.1 Setting Up the PCR Laboratory Space Correctly.......................................................................................20 2.1.2 Cultivating Laboratory Habits That Prevent Cross-Contamination ...................................................21 2.1.3 Using Uracil N-Glycosylase To Eliminate Carryover Contamination..................................................26 2.2 Factors To Consider When Setting Up a PCR Laboratory ......................................................................27 2.2.1 Equipment Required.............................................................................................................................................27 2.2.2 Choosing the Correct Enzymes for PCR and RT-PCR .............................................................................29 2.2.3 Other Important Reaction Components .......................................................................................................33 2.2.4 How Cycling Parameters Affect a PCR .........................................................................................................36 2.3 Typical Workflow for PCR/RT-PCR .................................................................................................................37 3 Primer Design and Template Preparation Page 3.1 Primer Design .........................................................................................................................................................41 3.1.1 Strategies for Designing Good Primers ........................................................................................................41 3.1.2 Design of PCR Primers: Helpful Web Sites that Contain Tips, Software, and Sequences.........42 3.1.3 Using the Primers in PCRs.................................................................................................................................43 3.2 Template Preparation ...........................................................................................................................................45 3.2.1 Overview ...................................................................................................................................................................45 3.2.2 Products for Manual and Automated Isolation of Nucleic Acids........................................................48 3.3 Protocols for Isolation of Typical Templates ................................................................................................54 3.3.1 Isolation of High Molecular Weight Nucleic Acids from Cultured Cells with the HIGH PURE PCR Template Preparation Kit.........................................................................................54 3.3.2 Isolation of Total RNA from Whole Blood with the HIGH PURE RNA Isolation Kit .....................57 3.3.3 Isolation of cDNA with the HIGH PURE PCR Product Purification Kit.............................................60 3 Table of Contents 4 General PCR Methods Page PCR Protocol Selection Guide...........................................................................................................................65 4.1 Basic PCR ................................................................................................................................................................ 66 4.1.1 Hot Start PCR - The new Standard................................................................................................................ 66 4.1.2 Conventional PCR..................................................................................................................................................74 4.2 High Fidelity PCR...................................................................................................................................................79 4.2.1 Reagents and Equipment Required................................................................................................................79 4.2.2 General Considerations for High Fidelity PCR........................................................................................... 80 4.2.3 Protocols for High Fidelity PCR ....................................................................................................................... 81 4.2.4 Typical Results ....................................................................................................................................................... 90 4.3 Long Template PCR...............................................................................................................................................92 4.3.1 Reagents and Equipment Required................................................................................................................92 4.3.2 General Considerations for Long Template PCR .......................................................................................93 4.3.3 Protocols for Long Template PCR................................................................................................................... 94 4.4 Amplification of Difficult Templates ............................................................................................................101 4.4.1 Reagents and Equipment Required.............................................................................................................101 4.4.2 General Considerations for Amplification of Difficult Templates .....................................................102 4.4.3 Protocols for Amplification of Difficult Templates (for Targets up to 5 kb) ..................................103 4.4.4 Typical Results ..................................................................................................................................................... 107 4.5 Guidelines for Optimizing PCR......................................................................................................................108 4.5.1 Reagents and Equipment Required.............................................................................................................108 4.5.2 General Considerations for Preventing Carryover..................................................................................109 4.5.3 Protocols for Preventing Carryover ..............................................................................................................110 4.6 Preventing Carryover......................................................................................................................................... 117 4.6.1 Choose the Appropriate Enzyme .................................................................................................................. 117 4.6.2 Use Highly Purified Templates and Primers.............................................................................................. 117 4.6.3 Design Primers Carefully ................................................................................................................................. 117 4.6.4 Use the Highest Quality Nucleotides.......................................................................................................... 118 4.6.5 Minimize Pipetting Steps with Convenient Master Reagent Mixes ................................................ 119 4.6.6 Optimize the Reaction Components............................................................................................................ 120 4.6.7 Optimize Reaction Temperatures and Times............................................................................................ 123 4 PCR Applications Manual Table of Contents 5 Basic RT-PCR Methods Page 5.1 Factors to Consider in RT-PCR...................................................................................................................... 127 5.1.1 Choosing a One-Step or a Two-Step Procedure .................................................................................... 128 5.1.2 Choosing the RT-PCR Enzymes .................................................................................................................... 128 5.1.3 Choosing the Primers for