USER GUIDE DNA Fragment Analysis by Capillary Electrophoresis Publication Number 4474504 Revision B For Research Use Only. Not intended for use in diagnostic procedures. For Research Use Only. Not intended for use in diagnostic procedures. The information in this guide is subject to change without notice. DISCLAIMER LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. TRADEMARKS All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. AmpErase, AmpliTaq, AmpliTaq Gold, and TaqMan are registered trademarks of Roche Molecular Systems, Inc. AFLP is a registered trademark of Keygene N.V. Millipore is a registered trademark of Merck KGaA. © 2014 Thermo Fisher Scientific Inc. All rights reserved. Contents About This Guide . 13 Revision history . 13 Purpose . 13 Prerequisites . 13 Structure of this guide . 14 ■ CHAPTER 1 Introduction to Fragment Analysis . 15 Fragment analysis versus sequencing…what is the difference? . 15 Fragment analysis . 15 Sequencing . 16 What can I do with fragment analysis? . 16 Types of applications . 16 Applications described in this guide . 17 What is capillary electrophoresis? . 18 Fragment analysis workflow . 19 ■ CHAPTER 2 Experimental Design . 21 Experimental design considerations . 21 DNA polymerase enzymes . 22 Overview . 22 Derivatives of Tth DNA polymerase . 24 Enzyme characteristics . 24 Fluorescent labeling methods . 25 Singleplexing versus multiplexing . 26 Singleplexing . 26 Multiplexing . 26 Multiplexing (pooling) strategies . 27 Multiplex design software . 28 Multiplexing guidelines . 28 Primer design guidelines . 29 Primer design criteria . 29 Primer design software . 29 Factors affecting Tm and primer annealing . 30 Effects of template secondary structure . 31 Selective amplification . 31 Preferential amplification . 31 DNA Fragment Analysis by Capillary Electrophoresis 3 Contents Non-specific amplification . 32 Minimizing binding to other primers . 32 Post-amplification manipulations . 33 Addition of 3' A nucleotide by Taq polymerase . 33 Dyes . 36 Dyes and chemical forms . 36 Multicomponent analysis with fluorescent dyes . 36 Factors that affect dye signal . 37 Emission and absorption (excitation) wavelengths and relative intensities . 38 Points to consider when selecting dyes for custom primers . 39 Example: selecting dyes . 39 Dye sets . 41 Dye sets and matrix standards . 41 Creating a custom dye set . 41 Size standards . 42 Functions of a size standard . 42 Size-standard peak intensity . 42 Selecting a GeneScan™ size standard . 43 Peaks not used for sizing . 43 Preparing a size standard . 43 GeneScan™ 120 LIZ® Size Standard . 44 GeneScan™ 500 LIZ® Size Standard . 44 GeneScan™ 600 LIZ® and GeneScan™ 600 LIZ® v2.0 Size Standards . 45 GeneScan™ 1200 LIZ® Size Standard . 47 GeneScan™ 350 ROX™ Size Standard . ..