SNP Genotyping Using the Sequenom MassARRAY iPLEX Platform

Stacey Gabriel1, Liuda Ziaugra1, Diana Tabbaa1

1 Broad Institute of MIT and Harvard, Cambridge, Massachusetts
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 2.12
DOI:  10.1002/0471142905.hg0212s60
Online Posting Date:  January, 2009
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Abstract

The method for SNP genotyping described in this unit is based on the commercially available Sequenom MassARRAY platform. The assay consists of an initial locus‐specific PCR reaction, followed by single base extension using mass‐modified dideoxynucleotide terminators of an oligonucleotide primer which anneals immediately upstream of the polymorphic site of interest. Using MALDI‐TOF mass spectrometry, the distinct mass of the extended primer identifies the SNP allele. Curr. Protoc. Hum. Genet. 60:2.12.1‐2.12.18. © 2009 by John Wiley & Sons, Inc.

Keywords: genotyping; SNP; MassARRAY; high‐throughput; PCR

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pre‐PCR: DNA and Oligo Pool Preparation
  • Basic Protocol 2: Amplification of Target Loci by PCR
  • Support Protocol 1: Post‐PCR: SAP Reaction Cleanup
  • Basic Protocol 3: Primer Extension
  • Support Protocol 2: Primer Extension Reaction Resin Cleanup
  • Basic Protocol 4: Spotting Primer Extension Products on SpectroCHIPs
  • Basic Protocol 5: Detection of Primer Extension Products by Mass Spectrometry
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pre‐PCR: DNA and Oligo Pool Preparation

  Materials
  • DNA source plate: 384‐well deep‐well PCR plate containing 2.5 ng/µl DNA of interest (see protocol 1; store at 4°C)
  • 100 mM dNTPs (Applied Biosystems; store at −20°C)
  • 25 mM MgCl 2 (QIAGEN; store at −20°C)
  • Ultrapure PCR‐grade H 2O (Invitrogen)
  • 5 U/µl HotStarTaq Plus DNA polymerase with 10× PCR buffer (QIAGEN; store at −20°C)
  • Forward and reverse primers: 1 µM each in multiplex pool (see protocol 1 and Background Information; store at 4°C)
  • 384‐well PCR reaction plate (Eppendorf twin.tec)
  • 96‐tip or 384‐tip pre‐PCR Tomtec or Hydra‐type (Matrix Technologies) robotic workstation (dedicated to DNA)
  • 96‐tip pre‐PCR Multimek robotic pipettor with stacker (Beckman‐Coulter) and disposable aerosol‐barrier “Beckman‐style” tips (LabSource)
  • 96‐well ABGene Thermo‐Fast skirted plates (AB‐0800)
  • 1.5‐ml microcentrifuge tubes or 15‐ or 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • MicroAmp adhesive plate sealers (Applied Biosystems)
  • Pre‐PCR tabletop centrifuge with microtiter plate carriers
  • ABI Viper dual or Thermo Scientific Hybaid 384‐well blocks, PC‐controlled thermal cyclers

Basic Protocol 2: Amplification of Target Loci by PCR

  Materials
  • 1× SAP buffer (Sequenom)
  • 1.7 U/µl shrimp alkaline phosphatase (SAP; Sequenom; store at 0°C)
  • PCR products in 384‐well PCR plates (see protocol 2)
  • 1.5‐ml microcentrifuge tubes or 15‐ or 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • 96‐well ABGene plates (AB‐0800)
  • 96‐tip post‐PCR Multimek robotic pipettor with stacker (Beckman‐Coulter)
  • MicroAmp adhesive plate sealers (Applied Biosystems)
  • Post‐PCR tabletop centrifuge with microtiter plate carriers
  • 37° and 85°C Precision incubators (VWR) or ABI or Hybaid thermal cycler with 384‐well blocks

Support Protocol 1: Post‐PCR: SAP Reaction Cleanup

  Materials
  • Ultrapure PCR‐grade H 2O (Invitrogen)
  • iPLEX enzyme (Sequenom)
  • 10× iPLEX buffer (Sequenom)
  • iPLEX Extension Mix (Sequenom)
  • Extend primers: from 5 to 10 µM each in multiplex pool
  • PCR products in 384‐well PCR plates, cleaned up by SAP reaction (see protocol 3)
  • 1.5‐ml microcentrifuge tubes or 15‐ or 50‐ml conical polypropylene centrifuge tubes (e.g., Falcon)
  • 96‐well ABGene plates
  • 96‐tip post‐PCR Multimek SpectroPREP (Sequenom) with stacker
  • MicroAmp adhesive plate sealers (Applied Biosystems)
  • Post‐PCR tabletop centrifuge with microtiter plate carriers
  • ABI or Hybaid Thermal cycler with 384‐well blocks

Basic Protocol 3: Primer Extension

  Materials
  • Ultrapure PCR‐grade H 2O (Invitrogen)
  • SpectroCLEAN resin (Sequenom; store at room temperature)
  • Post–primer extension products in 384‐well plates (see protocol 4), kept at 4°C
  • Clean plastic bottle with cap
  • Turbulator nonmagnetic mixing device (ACME Automation), firmly positioned on the Multimek deck (e.g., Position 1)
  • 96‐tip post‐PCR Multimek SpectroPREP (Sequenom) with stacker
  • Disposable “Beckman‐style” tips for Multimek
  • Post‐PCR tabletop centrifuge with microtiter plate carriers
  • TiterTop plate sealers (LabSource)
  • Plate rotator (VWR)

Support Protocol 2: Primer Extension Reaction Resin Cleanup

  Materials
  • 100% and 50% ethanol
  • Plates from primer extension, cleaned up with resin and centrifuged (see protocol 5)
  • 3‐point calibrant (Sequenom)
  • SpectroPOINT nanoliter sample dispensing instrument with appropriate software (Sequenom)
  • SpectroCHIPs (Sequenom)
  • Adhesive plate sealers (TiterTop from LabSource)

Basic Protocol 4: Spotting Primer Extension Products on SpectroCHIPs

  Materials
  • Compact mass spectrometer (Sequenom)
  • Spotted SpectroCHIPs (see protocol 6)
  • Scout plate (Sequenom)
  • Tweezers
  • Router.exe, FlexControl, ServerControl, SpectroCaller 3.4 (Sequenom), SpectroAcquire 3.4, and Typer ChipLinker software
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Figures

Videos

Literature Cited

   Gabriel, S.B., Schaffner, S.F., Nguyen, H., Moore, J.M., Roy, J., Blumenstiel, B., Higgins, J., DeFelice, M., Lochner, A., Faggart, M., Liu‐Cordero, S.N., Rotimi, C., Adeyemo, A., Cooper, R., Ward, R., Lander, E.S., Daly, M.J., and Altshuler, D. 2002. The structure of haplotype blocks in the human genome. Science 296:2225‐2229.
   Tang, K., Fu, D.J., Julien, D., Braun, A., Cantor, C.R., and Koster, H. 1999. Chip‐based genotyping by mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 96:10016‐10020.
Key References
   International Multiple Sclerosis Genetics Consortium, Hafler, D.A., Compston, A., Sawcer, S., Lander, E.S., Daly, M.J., De Jager, P.L., de Bakker, P.I., Gabriel, S.B., Mirel, D.B., Ivinson, A.J., Pericak‐Vance, M.A., Gregory, S.G., Rioux, J.D., McCauley, J.L., Haines, J.L., Barcellos, L.F., Cree, B., Oksenberg, J.R., and Hauser, S.L. 2007. Risk alleles for multiple sclerosis identified by a genomewide study. N. Engl. J. Med. 357:851‐862. Epub 2007 Jul 29.
  Useful reference using the iPLEX platform.
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