DNA Sequencing: An Outsourcing Guide

Jeffrey W. Touchman1

1 Arizona State University, Tempe, Arizona
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 12.1
DOI:  10.1002/9780470089941.et1201s02
Online Posting Date:  December, 2009
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

DNA sequencing, the process of determining the precise order of nucleotides in a DNA strand, is a fundamental and ubiquitous method in molecular biology. Examples of its many uses include characterization of unknown DNA, mutation detection, allele identification, and clone construct confirmation. Advances in sequencing technology, motivated by the successful International Human Genome Project, have resulted in unprecedented access to high‐quality and cost‐effective automated analysis. It is increasingly common for researchers to delegate routine DNA sequencing needs to dedicated laboratories or companies specializing in this technology. In this unit, we discuss key protocols for maximizing the success of DNA sequencing when working with such service providers and describe detailed troubleshooting advice for analyzing anomalous results. Curr. Protoc. Essential Lab. Tech. 2:12.1.1‐12.1.19. © 2009 by John Wiley & Sons, Inc.

Keywords: DNA sequencing; capillary electrophoresis; PCR cleanup; plasmid preparation; chromatogram

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Overview and Principles
  • Strategic Planning
  • Safety Considerations
  • Protocols
  • Basic Protocol 1: Isolating Plasmid DNA by Alkaline Lysis Miniprep for Use as Sequencing Template
  • Basic Protocol 2: Treating PCR Products with Exonuclease I and Shrimp Alkaline Phosphatase to Purify Sequencing Template
  • Alternate Protocol 1: PEG Purification of PCR Products for DNA Sequencing
  • Reagents and Solutions
  • Understanding Results
  • Troubleshooting
  • Variations
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Isolating Plasmid DNA by Alkaline Lysis Miniprep for Use as Sequencing Template

  Materials
  • Bacterial culture containing plasmid of interest
  • 2× YT medium (see recipe) with antibiotic appropriate for the plasmid used
  • GET buffer (see recipe)
  • Lysis solution (see recipe)
  • Potassium acetate solution (see recipe)
  • Absolute (100%) and 70% (v/v) ethanol
  • 100 µg/ml DNase‐free RNase A (Invitrogen) in standard TE buffer, pH 8.0 (see unit 3.3 for TE buffer)
  • Phenol/chloroform (1:1 v/v), pH 7.5 saturated with 10 mM Tris⋅Cl, pH 7.5 (unit 3.3), room temperature
  • 3 M sodium acetate, pH 5.2 (unit 3.3)
  • Sequencing TE buffer (0.1 mM Tris⋅Cl, pH 7.5/0.1 mM EDTA, pH 8.0)
  • 14‐ml round‐bottom polypropylene culture tubes
  • 37°C shaking incubator
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifuge, capable of at least 12,000 × g
  • Vortex mixer
  • Additional reagents and equipment for culture of bacteria (unit 4.2) and spectrophotometrically quantitating DNA (unit 2.2)

Basic Protocol 2: Treating PCR Products with Exonuclease I and Shrimp Alkaline Phosphatase to Purify Sequencing Template

  Materials
  • Template DNA of interest (e.g., protocol 1)
  • Molecular‐weight markers (e.g., molecular‐weight marker III; Roche)
  • 10 U/µl exonuclease I (Exo I; GE Healthcare)
  • 1 U/µl shrimp alkaline phosphatase (SAP; GE Healthcare)
  • Distilled, nuclease‐free water (Invitrogen)
  • 0.2‐ml or 0.5‐ml thin‐walled PCR reaction tubes (appropriate for the thermal cycler block)
  • Thermal cycler (see unit 10.2)
  • Additional reagents and equipment for performing PCR (unit 10.2) and agarose gel electrophoresis (unit 7.2)

Alternate Protocol 1: PEG Purification of PCR Products for DNA Sequencing

  Materials
  • Template DNA of interest (e.g., protocol 1)
  • 5 M NaCl
  • Standard TE buffer (unit 3.3)
  • 40% (w/v) PEG 8000/10 mM MgCl 2
  • Absolute (100%) ethanol
  • Sequencing TE buffer (0.1 mM Tris, pH 7.5/0.1 mM EDTA, pH 8.0)
  • 1.5‐ml microcentrifuge tubes
  • Vortexer
  • Microcentrifuge capable of at least 12,000 × g
  • SpeedVac (ThermoSavant)
  • Additional reagents and equipment for performing PCR (see unit 10.2) and spectrophotometrically quantitating DNA (unit 2.2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bergett, S. and Rosteck, J.P.R. 1994. Use of dimethylsulfoxide to improve fluorescent Taq cycle sequencing. In Automated DNA Sequencing and Analysis Techniques. (M. Adams, ed.) Academic Press, New York.
   Birnboim, H.C. and Doly, J. 1979. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7:1513‐1523.
   Hillier, L. and Green, P. 1991. OSP: A computer program for choosing PCR and DNA sequencing primers. PCR Methods Appl. 1:124‐128.
   Riethman, H., Birren, B., and Gnirke, A. 1997. Preparing, manipulating, and mapping of HMW DNA. In Analyzing DNA: A Laboratory Manual. Genome Analysis. (B. Birren, E. Green, S. Klapholz, R. Myers, and J. Roskams, eds.) pp. 83‐248. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Rozen, S. and Skaletsky, H. 2000. Primer3 on the WWW for general users and for biologist programmers. Methods Mol. Biol. 132:365‐386.
   Sanger, F., Nicklen, S., and Coulson, A.R. 1977. DNA sequencing with chain‐terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74:5463‐5467.
   Slatko, B., Albright, L., Tabor, S., and Ju, J. 1999. DNA sequencing by the dideoxy method. Curr. Protoc. Mol. Biol. 47:7.4A.1‐7.4A.39.
   Voytas, D. 2000. Agarose gel electrophoresis. Curr. Protoc. Mol. Biol. 51:2.5A.1‐2.5A.9.
   Watson, J.D. and Crick, F.H. 1953. Molecular structure of nucleic acids: A structure for deoxyribose nucleic acid. Nature 171:737‐738.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library