Pyrosequencing: Powerful and Quantitative Sequencing Technology

Martin Kreutz1, Norbert Hochstein1, Julia Kaiser1, Frank Narz1, Ralf Peist1

1 Qiagen GmbH, Hilden, Germany
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 7.15
DOI:  10.1002/0471142727.mb0715s104
Online Posting Date:  October, 2013
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Abstract

Pyrosequencing is a sequencing‐by‐synthesis method for DNA analysis that has emerged as a platform not only for de novo sequencing applications, but also for quantitative analysis of genomic methylation, single‐nucleotide polymorphisms, and allele quantification. In this unit, we describe a complete workflow from sample to result that is suitable for each of these applications. As cytosine conversion is a key element of successful methylation analysis using pyrosequencing, a support protocol for bisulfite treatment is also included. Curr. Protoc. Mol. Biol. 104:7.15.1‐7.15.23. © 2013 by John Wiley & Sons, Inc.

Keywords: pyrosequencing; DNA methylation; SNP; AQ; genome analysis; sequencing

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Pyrosequencing
  • Support Protocol 1: Cyostine Conversion for Methylation Analysis (only for CpG Analysis)
  • Support Protocol 2: Sample Lysis and Bisulfite Conversion of Unmethylated Cytosines in DNA Prepared from FFPE Tissue Samples
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Pyrosequencing

  Materials
  • Streptavidin Sepharose High Performance beads (GE Healthcare, cat. no. 17‐5113‐01)
  • PyroMark Q24 Advanced Reagents (Qiagen, cat. no. 970902) containing:
    • Binding Buffer
    • Annealing Buffer
    • Enzyme Mixture
    • Substrate Mixture
    • dATPαS
    • dGTP
    • dCTP
    • dTTP
  • High‐purity water (MilliQ 18.2 MΩ × cm or equivalent)
  • 5′‐biotinylated PCR product (5 to 20 µl of a 25 µl PCR per reaction; see unit 15.1 for PCR)
  • PyroMark Denaturation Buffer (Qiagen, cat. no. 979007)
  • PyroMark Wash Buffer (Qiagen, cat. no. 979008)
  • Sequencing primer of 15 to 20 bp length (with an annealing temperature of 45° to 55°C)
  • 70% ethanol
  • Sterile pipet tips with filters
  • PCR plate or tube strips
  • Adhesive foil or strip caps
  • Plate mixer for immobilization to beads
  • PyroMark Q24 Plate (Qiagen, cat. no. 979201)
  • PyroMark Q24 Advanced System (Qiagen, cat. no. 9002270), including:
  • PyroMark Q24 instrument
  • PyroMark Q24 Advanced Software
  • PyroMark Q24 Vacuum Workstation
  • Heating block capable of attaining 80°C
  • PyroMark Q24 Cartridge (Qiagen, cat. no. 979202)
  • PyroMark Q24 Advanced Software

Support Protocol 1: Cyostine Conversion for Methylation Analysis (only for CpG Analysis)

  Materials
  • DNA sample for methylation analysis
  • EpiTect Fast DNA Bisulfite Kit (Qiagen, cat. no. 59824) containing:
    • Bisulfite Solution
    • DNA Protect Buffer
    • RNase‐Free Water
    • MinElute DNA Spin Columns
    • Collection Tubes (2 ml)
    • Buffer BL (contains guanidine salts)
    • Buffer BW
    • Buffer BD
    • Buffer EB
    • Carrier RNA
  • 96% to 100% ethanol (molecular biology grade; do not use denatured alcohol, which contains other substances such as methanol or methylketone)
  • 0.2 ml PCR reaction tubes or 8‐well strips
  • Thermal cycler with heated lid

Support Protocol 2: Sample Lysis and Bisulfite Conversion of Unmethylated Cytosines in DNA Prepared from FFPE Tissue Samples

  Materials
  • FFPE slice (see, e.g., unit 14.1)
  • EpiTect Fast FFPE Bisulfite Kit (Qiagen, cat. no. 59844, parts 1 and 2) containing:
    • Deparaffinization Solution
    • Lysis Buffer FTB
    • Proteinase K
    • Bisulfite Solution
    • DNA Protect Buffer
    • RNase‐Free Water
    • MinElute DNA Spin Columns
    • Collection Tubes (2 ml)
    • Buffer BL (contains guanidine salts)
    • Buffer BW
    • Buffer BD
    • Buffer EB
    • Carrier RNA
  • Ethanol
  • 0.2‐ml PCR reaction tubes or 8‐well strips with caps
  • Thermal cycler with heated lid or heat blocks
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Figures

Videos

Literature Cited

Literature Cited
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