Automated DNA Sequencing Chemistry Guide ©Copyright 1998, The Perkin-Elmer Corporation This product is for research purposes only. ABI PRISM, MicroAmp, and Perkin-Elmer are registered trademarks of The Perkin-Elmer Corporation. ABI, ABI PRISM, Applied Biosystems, BigDye, CATALYST, PE, PE Applied Biosystems, POP, POP-4, POP-6, and Primer Express are trademarks of The Perkin-Elmer Corporation. AmpliTaq, AmpliTaq Gold, and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. Centricon is a registered trademark of W. R. Grace and Co. Centri-Sep is a trademark of Princeton Separations, Inc. Long Ranger is a trademark of The FMC Corporation. Macintosh and Power Macintosh are registered trademarks of Apple Computer, Inc. pGEM is a registered trademark of Promega Corporation. Contents 1 Introduction. 1-1 New DNA Sequencing Chemistry Guide . 1-1 Introduction to Automated DNA Sequencing . 1-2 ABI PRISM Sequencing Chemistries . 1-5 PE Applied Biosystems DNA Sequencing Instruments . 1-7 Data Collection and Analysis Settings . 1-12 2 ABI PRISM DNA Sequencing Chemistries . 2-1 Overview . 2-1 Dye Terminator Cycle Sequencing Kits . 2-2 Dye Primer Cycle Sequencing Kits . 2-8 Dye Spectra . 2-12 Chemistry/Instrument/Filter Set Compatibilities . 2-13 Dye/Base Relationships for Sequencing Chemistries . 2-14 Choosing a Sequencing Chemistry. 2-15 3 Performing DNA Sequencing Reactions . 3-1 Overview . 3-1 DNA Template Preparation . 3-2 Sequencing PCR Templates . 3-10 DNA Template Quality. 3-15 DNA Template Quantity. 3-17 Primer Design and Quantitation . 3-18 Reagent and Equipment Considerations. 3-20 Preparing Cycle Sequencing Reactions . 3-21 Cycle Sequencing . 3-27 Preparing Extension Products for Electrophoresis . 3-33 Removing Unincorporated Dye Terminators . 3-34 Preparing Dye Primer Reaction Products for Electrophoresis . 3-46 Preparing and Loading Samples for Gel Electrophoresis. 3-50 Preparing and Loading Samples for Capillary Electrophoresis . 3-53 4 Optimizing Gel Electrophoresis . 4-1 Introduction . 4-1 Reagents . 4-2 i Avoiding Problems with Sequencing Gels . 4-4 5 Optimizing Capillary Electrophoresis . 5-1 Introduction . 5-1 Capillary Electrophoresis Consumables . 5-2 Optimizing Electrokinetic Injection . 5-4 Optimizing Electrophoresis Conditions. 5-7 Run Parameters for Specific Sequencing Chemistries. 5-8 6 Optimizing Software Settings. 6-1 Introduction . 6-1 Choosing a Run Module . 6-2 Choosing a Dye Set/Primer (Mobility) File. 6-3 Choosing the Correct Basecaller . 6-6 Creating an Instrument (Matrix) File. 6-7 Setting the Data Analysis Range . 6-15 7 Data Evaluation and Troubleshooting . 7-1 Overview . 7-1 Data Evaluation . 7-2 Practical Examples of Data Evaluation . 7-10 Troubleshooting Sequencing Reactions. 7-16 Troubleshooting DNA Sequence Composition Problems . 7-30 Troubleshooting Sequencing Data. 7-39 Troubleshooting Gel Electrophoresis on the ABI 373 and ABI PRISM 377 . 7-44 Troubleshooting Capillary Electrophoresis on the ABI PRISM 310. 7-55 Troubleshooting Software Settings . 7-62 A Gel Preparation . A-1 Introduction . A-1 Protocol and Run Conditions for 19:1 Polyacrylamide Gels . A-2 Protocol and Run Conditions for 29:1 Polyacrylamide Gels. A-6 Protocol and Run Conditions for Long Ranger and PAGE-PLUS Gels . A-10 Preparing APS, TBE Buffer, and Deionized Formamide . A-15 ii B IUB Codes . .B-1 C References . .C-1 D Technical Support . .D-1 How to Get Help. D-1 E Part Numbers. .E-1 ABI PRISM DNA Sequencing Kits and Reagents. .E-1 ABI PRISM 310 Genetic Analyzer . .E-5 ABI PRISM 377 DNA Sequencer . .E-8 ABI 373 DNA Sequencer . .E-9 Documentation and Software . .E-10 Index iii Introduction 1 1 New DNA Sequencing Chemistry Guide Purpose Since the original DNA Sequencing Chemistry Guide was published in early 1995, PE Applied Biosystems has released two new instrument platforms, five new sequencing chemistries, and a new sequencing enzyme. To accommodate this new information, we have written the Automated DNA Sequencing Chemistry Guide. This updated guide provides the following: ♦ An introduction to automated DNA sequencing ♦ Descriptions of PE Applied Biosystems sequencing instruments, chemistries, and software ♦ Detailed protocols for preparing DNA templates, performing cycle sequencing, and preparing the extension products for electrophoresis.