Mutation Detection Using Automated Fluorescence‐Based Sequencing

Kate T. Montgomery1, Oleg Iartchouck1, Li Li2, Anoja Perera3, Yosuf Yassin4, Alex Tamburino5, Stephanie Loomis1, Raju Kucherlapati1

1 Harvard Medical School – Partners Healthcare Center for Genetics and Genomics, Boston, Massachusetts, 2 Albany Medical College, Albany, New York, 3 Stowers Institute for Medical Research, Kansas City, Missouri, 4 Dana Farber Cancer Institute, Boston, Massachusetts, 5 GlaxoSmithKline MDR, Boston, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 7.9
DOI:  10.1002/0471142905.hg0709s57
Online Posting Date:  April, 2008
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Abstract

The development of high‐throughput DNA sequencing techniques has made direct DNA sequencing of PCR‐amplified genomic DNA a rapid and economical approach to the identification of polymorphisms that may play a role in disease. Point mutations as well as small insertions or deletions are readily identified by DNA sequencing. The mutations may be heterozygous (occurring in one allele while the other allele retains the normal sequence) or homozygous (occurring in both alleles). Sequencing alone cannot discriminate between true homozygosity and apparent homozygosity due to the loss of one allele due to a large deletion. In this unit, strategies are presented for using PCR amplification and automated fluorescence‐based sequencing to identify sequence variation. The size of the project and laboratory preference and experience will dictate how the data is managed and which software tools are used for analysis. A high‐throughput protocol is given that has been used to search for mutations in over 200 different genes at the Harvard Medical School – Partners Center for Genetics and Genomics (HPCGG, http://www.hpcgg.org/). Curr. Protoc. Hum. Genet. 57:7.9.1‐7.9.31. © 2008 by John Wiley & Sons, Inc.

Keywords: Genetic mutation; DNA sequencing; mutation identification; PCR amplification

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Primer Design
  • Basic Protocol 2: PCR Amplification of Genomic DNA
  • Alternate Protocol 1: PCR Amplification of Genomic DNA with GC‐Rich Regions
  • Basic Protocol 3: Purification of PCR Products by Enzymatic Cleanup
  • Alternate Protocol 2: Manual Purification of PCR Products Using AMPure System
  • Alternate Protocol 3: Automated Purification of PCR Products Using AMPure System
  • Basic Protocol 4: Fluorescence‐Based DNA Sequencing
  • Alternate Protocol 4: Fluorescence‐Based DNA Sequencing for GC‐Rich Regions
  • Basic Protocol 5: Manual Purification of Sequencing Reactions
  • Alternate Protocol 5: Automated Purification of Sequencing Reactions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Primer Design

  Materials
  • Computers and high‐speed access to the internet for Web‐based tools and information

Basic Protocol 2: PCR Amplification of Genomic DNA

  Materials
  • Molecular‐grade (autoclaved) water
  • 5 M Betaine solution (store at 4°C)
  • 5 U/µl AmpliTaq Gold DNA polymerase and 10× buffer (Applied Biosystems, store at −20°C)
  • 25 mM MgCl 2 (store at –20°C)
  • DMSO
  • 10 mM dNTP mixture (4 nucleotides, each at 10 mM)
  • Oligonucleotide primers (F and R) for PCR, with M13 tags at the 5′ end (see protocol 1):
  • 100 µM stocks in TE buffer, pH 7.4 (store at –20°C)
  • 20 µM working solutions in water (deionized, distilled, and autoclaved; store at −20°C)
  • 12.5 ng/µl genomic DNA for template ( appendix 3B), suspended in water (store at 4°C)
  • 15 ml‐conical tubes
  • Cooling block/ice bucket
  • Multichannel pipettors, 8‐ and 12‐channel
  • 384‐well skirted plates (Thermo‐Fast Diamond 384, cat. no. AB‐1111)
  • 0.2 ml‐strip tubes and caps
  • Seals for plates, heat or adhesive
  • Centrifuge with plate adaptors
  • Vortexer with plate adaptor
  • GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) or other instrument able to handle 384‐well plates (or similar thermal cycler for 96‐well plates)
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Alternate Protocol 1: PCR Amplification of Genomic DNA with GC‐Rich Regions

  • FailSafe PCR 2× PreMix D (Epicentre Biotechnologies, cat. no. SFP995D)
  • FailSafe PCR enzyme mix (Epicentre Biotechnologies, cat. no. FS99100)

Basic Protocol 3: Purification of PCR Products by Enzymatic Cleanup

  Materials
  • ExoSAP‐IT PCR Clean‐up Kit (USB) or elements of kit:
    • 10 U/µl exonuclease I (USB; alternate for kit)
    • 1 U/µl shrimp alkaline phosphatase (1 Unit/µl, USB; alternate for kit)
  • Dilution buffer: 50 mM Tris·Cl, pH 8.0
  • Plates of PCR products (see protocol 2 or protocol 3)
  • Molecular weight marker for gel electrophoresis, size range 100 to 1200 bp
  • Multichannel Pipettors, 8‐ and 12‐channel
  • Cooling block/ice bucket
  • Plate seals, adhesive or heat
  • Centrifuge with plate adaptors
  • Thermal cycler with heated lid
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Alternate Protocol 2: Manual Purification of PCR Products Using AMPure System

  Materials
  • Agencourt AMPure Magnetic Particle Solution
  • Plates of PCR products (see protocol 2 or protocol 3)
  • 70% ethanol (store up to 3 days in a tightly sealed container)
  • Elution buffer: autoclaved, reagent‐grade water (10 mM Tris acetate/0.1 mM EDTA, pH 8.0, may also be used)
  • 50‐ml reagent reservoirs
  • 96‐well round bottom microtiter plate, 300 µl capacity (e. g., Costar #07‐200‐105)
  • Multichannel pipettor
  • Vortexer with plate adaptor
  • Agencourt SPRIPlate 96R – Ring Magnet Plate
  • Plate seals, adhesive or heat

Alternate Protocol 3: Automated Purification of PCR Products Using AMPure System

  Materials
  • Agencourt AMPure Magnetic Particle Solution
  • 384‐well plates of PCR products (see protocol 2 or protocol 3)
  • 70% ethanol (store up to 3 days in a tightly sealed container)
  • Elution buffer: autoclaved, reagent‐grade water (10 mM Tris acetate/0.1 mM EDTA, pH 8.0, may also be used)
  • Biomek FX Laboratory Automation Workstation
  • 96‐well round‐bottom microtiter plate, 300 µl capacity (e. g., Costar no. 07‐200‐105)
  • Adhesive plate seals
  • Vortexer with plate adapters
  • 20‐µl nonbarrier tips
  • Agencourt SPRIPlate 384 Magnet Plates
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.7)

Basic Protocol 4: Fluorescence‐Based DNA Sequencing

  Materials
  • 5× Sequencing buffer (Applied Biosystems; store at –20°C)
  • DMSO
  • 20 pmol/µl M13 forward and reverse universal sequencing primers (store at −20°C):
    • M13 forward primer‐21 (18‐mer): 5′‐TGTAAAACGACGGCCAGT‐3′
    • M13 reverse primer‐24 (16‐mer): 5′‐AACAGCTATGACCATG‐3′
  • DNA Sequencing kit BigDye Terminator v3.1 (Applied Biosystems, Part no. 4337455, 4337456, 4337457, or 4337458)
  • BigDye Terminator v3.1 Sequencing Standard Kit(ABI Part no. 4336943)
  • pGEM‐3Zf(+) Vector, 20 µg (Fisher/Promega, no. P2271)
  • 15‐ml conical Tubes
  • Vortexer with plate adaptor
  • 0.2‐ml strip tubes and caps
  • Multichannel pipettors, 8‐ and 12‐channel
  • Heat or adhesive seals for plates
  • Centrifuge with plate adaptors
  • GeneAmp PCR System 9700 thermal cycler (Applied Biosystems) or other instrument able to handle 384‐well or 96‐well plates
  • ABI 3730xl Sequencer (or equivalent) and associated data collection computers with Windows operating system
  • 384‐well skirted plates (Thermo‐Fast Diamond 384 plates, AB‐1111) or other plates appropriate for sequence Analyzer

Alternate Protocol 4: Fluorescence‐Based DNA Sequencing for GC‐Rich Regions

  • DNA Sequencing kit dGTP BigDye Terminator v3.0 (Applied Biosystems)

Basic Protocol 5: Manual Purification of Sequencing Reactions

  Materials
  • Agencourt CleanSEQ Magnetic Particle Solution
  • 85% ethanol (store up to 3 days in a tightly sealed container)
  • Ethanol buffer: molecular‐grade water (autoclaved, distilled)
  • 50‐ml reservoir
  • 96‐well round bottom microtiter plate, 300 µl capacity (e. g., Costar, no. 07‐200‐105)
  • Plate seals, adhesive or heat
  • Vortexer with plate adapter
  • Multichannel pipettors, 8‐ and 12‐channel
  • Thermo‐Fast Diamond 384 plate (Abgene Cat # AB‐1111)
  • Magnetic plate: Agencourt SPRIPlate 96R ring magnetic plate (for 96‐well format) or Agencourt SPRIPlate 384 magnetic plate (for 384‐well format)

Alternate Protocol 5: Automated Purification of Sequencing Reactions

  • 85% ethanol
  • 384‐well sequencing plates (see protocol 7 or protocol 8)
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Figures

Videos

Literature Cited

Literature Cited
   Bellissimo, D.B., Christopherson, P.A., Haberichter, S.L., Flood, V.H., Gill, J.C., Friedman, K.D., Abshire, T.C., DiPaola, J.A., Hoots, W.K., Leissinger, C., Lusher, J.M., Ragni, M.V., Shapiro, A.D., and Montgomery R.R. 2007. Novel VWF sequence variations identified in normal controls and index cases enrolled in the TS Zimmerman program for the molecular and clinical biology of VWD. Blood (ASH Annual Meeting Abstracts), Nov 2007; 110: 709.
   Kent, J. W. 2002. BLAT—The BLAST‐Like Alignment Tool. Genome Res. 12: 656‐664.
   Kuhn, R.M., Karolchik, D., Zweig, A. S., Trumbower, H., Thomas, D. J., Thakkapallayil, A., Sugnet, C. W., Stanke, M., Smith, K.E., Siepel, A., Rosenbloom, K.R., Rhead, B., Raney, B. J., Pohl, A., Pedersen, J. S., Hsu, F., Hinrichs, A. S., Harte, R. A., Diekhans, M., Clawson, H., Bejerano, G., Barber, G.P., Baertsch, R., Haussler, D., and Kent, W. J. 2007. The UCSC genome browser database: Update 2007. Nucleic Acids Res. 35D668‐D673.
   Nickerson, D. A., Tobe, V. O., and Taylor, S. L. 1997. PolyPhred: Automating the detection and genotyping of single nucleotide substitutions using fluorescence‐based resequencing. Nucleic Acids Res. 25: 2745‐2751.
   Roberts, A.E., Araki, T., Swanson, K.D., Montgomery, K. T., Schiripo, T. A., Joshi, V. A., Li, L., Yassin, Y., Tamburino, A.M., Neel, B.G., and Kucherlapati, R. S. 2007. Germline gain‐of‐function mutations in SOS1 cause Noonan syndrome. Nat. Genet. 39: 70‐74.
   Rozen, S. and Skaletsky, H. J. 2000. Primer 3 on the WWW for general users and for biologist programmers. In: Bioinformatics Methods and Protocols: Methods in Molecular Biology (S. Krawetz and S. Misener, eds.) pp. 365‐386. Humana Press, Totowa, N. J.
   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.
   Stephens, M., Sloan, J. S., Robertson, P.D., Scheet, P., and Nickerson, D. A. 2006. Automating sequence‐based detection and genotyping of SNPs from diploid samples. Nat. Genet. 38: 375‐381.
   Siepka, S.M., Yoo, S‐H., Park, J., Song, W., Kumar, V., Hu, Y., Lee, C., and Takahashi, J. S. 2007. Circadian mutant overtime reveals F‐box protein FBXL3 regulation of cryptochrome and period gene expression. Cell 129: 1011‐1023.
   Thomas, D. J., Trumbower, H., Kern, A.D., Rhead, B. L., Kuhn, R.M., Haussler, D., and Kent, W. J. 2007. Variation resources at UC Santa Cruz. Nucleic Acids Res. 35: D716‐D720.
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