“First Generation” Automated DNA Sequencing Technology

Barton E. Slatko1, Jan Kieleczawa2, Jingyue Ju3, Andrew F. Gardner1, Cynthia L. Hendrickson4, Frederick M. Ausubel5

1 New England Biolabs, Inc., Ipswich, Massachusetts, 2 Wyzer Biosciences, Cambridge, Massachusetts, 3 Center for Genome Technology & Biomolecular Engineering, Columbia University, New York, New York, 4 HudsonAlpha Institute for Biotechnology, Huntsville, Alabama, 5 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.2
DOI:  10.1002/0471142727.mb0702s96
Online Posting Date:  October, 2011
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Abstract

Beginning in the 1980s, automation of DNA sequencing has greatly increased throughput, reduced costs, and enabled large projects to be completed more easily. The development of automation technology paralleled the development of other aspects of DNA sequencing: better enzymes and chemistry, separation and imaging technology, sequencing protocols, robotics, and computational advancements (including base‐calling algorithms with quality scores, database developments, and sequence analysis programs). Despite the emergence of high‐throughput sequencing platforms, automated Sanger sequencing technology remains useful for many applications. This unit provides background and a description of the “First‐Generation” automated DNA sequencing technology. It also includes protocols for using the current Applied Biosystems (ABI) automated DNA sequencing machines. Curr. Protoc. Mol. Biol. 96:7.2.1‐7.2.28. © 2011 by John Wiley & Sons, Inc.

Keywords: dideoxy DNA sequencing; thermal cycle sequencing; BigDye terminator; high‐throughput DNA sequencing; genomics

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Step I: Template Preparations for Automated ABI 3100, 3500, or 3700 Series Capillary Machines
  • Basic Protocol 2: Step II: Standard Protocol for Preparing BigDye DNA Sequencing Reactions in Microcentrifuge Tubes or in 96‐Well Reaction Plates
  • Alternate Protocol 1: Use of ABI Prism dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit
  • Alternate Protocol 2: Diluted Premix and Reduced Sequencing Reaction Sizes
  • Basic Protocol 3: Step III: Thermal Cycle Sequencing
  • Alternate Protocol 3: 2‐Step Thermal Cycling Protocol
  • Alternate Protocol 4: Thermal Cycle Sequencing Protocol for Large Templates (BACs, YACs, Cosmids, and Fosmids)
  • Basic Protocol 4: Step IV: Purification of Sequencing Products from Unincorporated Terminator Dyes
  • Alternate Protocol 5: Ethanol/EDTA/Sodium Acetate Precipitation
  • Alternate Protocol 6: Purification of Thermal Cycle Sequencing Reactions Using Spin Columns
  • Alternate Protocol 7: Performing 96‐Well Spin Plate Purification
  • Alternate Protocol 8: SDS/Heat Protocol for Spin Columns and Plates
  • Alternate Protocol 9: Purification of Thermal Cycle Sequencing Reactions Using Magnetic Particles
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Step I: Template Preparations for Automated ABI 3100, 3500, or 3700 Series Capillary Machines

  Materials
  • ABI Ready Reaction Kit BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems).
  • DNA sequencing primer, 3‐ to 5‐µM stock
  • Microcentrifuge tubes or 96‐well V‐bottom microtiter plate
  • Plate sealers (see Strategic Planning)
  • Benchtop microcentrifuge for tubes or benchtop microtiter plate centrifuge (e.g., Beckman‐Coulter CS‐15)

Basic Protocol 2: Step II: Standard Protocol for Preparing BigDye DNA Sequencing Reactions in Microcentrifuge Tubes or in 96‐Well Reaction Plates

  • ABI PRISM dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems)

Alternate Protocol 1: Use of ABI Prism dGTP BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit

  • ABI Ready Reaction Kit BigDye Terminator v3.1 Cycle Sequencing Kit or ABI Ready Reaction Kit BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems)
  • 5× sequencing Buffer (ABI)

Alternate Protocol 2: Diluted Premix and Reduced Sequencing Reaction Sizes

  Materials
  • Tubes or microtiter plate containing sequencing reactions ( protocol 2 or Alternate Protocol protocol 31 or protocol 42)
  • Thermal cycler
  • Benchtop microcentrifuge (for tube reactions) or benchtop microtiter plate centrifuge (for microtiter plates)

Basic Protocol 3: Step III: Thermal Cycle Sequencing

  Materials
  • Tubes or microtiter reaction plates from the thermal cycler (see protocol 5 or Alternate Protocol protocol 63 or protocol 74)
  • 125 mM EDTA
  • 95% to 100% and 70% ethanol
  • Microcentrifuge (tube reactions) or benchtop centrifuge with microtiter plate carrier
  • Microtiter plate adhesive sealing tape
  • Speed vacuum concentrator (e.g., SpeedVac evaporator from Thermo Scientific) with holders for tubes or microtiter plates
NOTE: Absolute ethanol absorbs water from the atmosphere, gradually decreasing its concentration. This can lead to inaccurate final concentrations of ethanol, which can affect some sequencing results. 95% ethanol is usable, but it is crucial to make sure the final ethanol concentration for precipitation remains at 67% to 71%.

Alternate Protocol 3: 2‐Step Thermal Cycling Protocol

  • 3 M sodium acetate

Alternate Protocol 4: Thermal Cycle Sequencing Protocol for Large Templates (BACs, YACs, Cosmids, and Fosmids)

  • Ultrapure formamide for loading (optional)
  • Centri‐Sep spin columns (for microcentrifuge tubes; Princeton Separations)
NOTE: Perform the entire procedure without interruption to ensure optimal results. Do not allow the column to dry out.NOTE: Only process as many columns as can be handled conveniently at one time.

Basic Protocol 4: Step IV: Purification of Sequencing Products from Unincorporated Terminator Dyes

  • Performa Gel Filtration Kit (Edge Biosystems) or Centrisep‐96 (Princeton Separations)
  • Costar flat‐bottom plate (cat. no. 9017)
  • Centrifuge with cushioned microtiter plate carrier (typically supplied by centrifuge manufacturer)
  • Edge Biosystems 96‐well capillary plate (Costar, cat. no. 13506) or Costar polystyrene V‐bottom plate (cat. no. 3807)
  • Edge Biosystems Performa DTR 96 well short plates

Alternate Protocol 5: Ethanol/EDTA/Sodium Acetate Precipitation

  • 2.2% SDS in deionized water (weigh out 2.2 g SDS and bring volume to 100 ml).
  • Thermal cycler
CAUTION: SDS is highly airborne and an irritant. It is harmful if swallowed or inhaled, and may cause irritation to skin, eyes, and respiratory tract, as well as an allergic skin or respiratory reaction. SDS is a flammable solid. Weigh out in a hood or using a mask.

Alternate Protocol 6: Purification of Thermal Cycle Sequencing Reactions Using Spin Columns

  • 85% ethanol
  • Elution buffer: 0.1 M EDTA, pH 8.0 ( appendix 22) or reagent‐grade H 2O
  • Agencourt CleanSEQ SPRI magnetic bead kit (Beckman Coulter)
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Figures

Videos

Literature Cited

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