Analysis of DNA Replication in Saccharomyces cerevisiae by Two‐Dimensional and Pulsed‐Field Gel Electrophoresis

Satoru Ide1, Takehiko Kobayashi2

1 National Institute of Genetics, Shizuoka, Japan, 2 The Graduate University for Advanced Studies, Sokendai, Mishima, Japan
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 22.14
DOI:  10.1002/0471143030.cb2214s49
Online Posting Date:  December, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Two methods to analyze DNA replication in budding yeast are described here. One is two‐dimensional (2D) gel electrophoresis, which has been used to analyze replication intermediates in yeast. The other method is pulsed‐field gel electrophoresis (PFGE) that monitors the replication status of a chromosome. These are convenient and easy, and they do not require specialized equipment. Curr. Protoc. Cell Biol. 49:22.14.1‐22.14.12. © 2010 by John Wiley & Sons, Inc.

Keywords: two‐dimensional gel electrophoresis; pulsed‐field gel electrophoresis; DNA replication; agarose plug; replicative intermediate

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Two‐Dimensional Agarose Gel Electrophoresis to Analyze DNA Structures
  • Support Protocol 1: DNA Preparation in Low‐Melting‐Point Agarose
  • Support Protocol 2: Digestion of DNA in Low‐Melting Agarose
  • Basic Protocol 2: Contour‐Clamped Homogeneous Electric Field Pulsed‐Field Gel Electrophoresis to Monitor Chromosome Replication Status
  • Support Protocol 3: DNA Preparation in Low‐Melting‐Point Agarose Gel for PFGE
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Two‐Dimensional Agarose Gel Electrophoresis to Analyze DNA Structures

  Materials
  • Agarose solution no. 1 for the first dimensional (1D) gel electrophoresis (see recipe)
  • Agarose solution no. 2 for the second dimensional (2D) gel electrophoresis (see recipe)
  • Restriction enzyme–digested DNA in agarose plug ( protocol 3)
  • 1× TBE ( appendix 2A)
  • Ethidium bromide
  • 0.2 N HCl
  • Denaturation solution: 1.5 M NaCl/0.5 N NaOH
  • Neutralization buffer: 1 M Tris⋅Cl (pH 7.4)/1.5 M NaCl
  • 20× SSC ( appendix 2A)
  • Phosphate‐SDS hybridization buffer (see recipe)
  • Radiolabeled DNA probe made with Rediprime II DNA Labeling System (GE)
  • 2% (w/v) SDS ( appendix 2A)
  • 12 × 14–cm gel casting tray
  • Well combs
  • Electrophoresis apparatus
  • Power supply
  • Hybond N+ nylon membrane (GE)
  • UV light
  • Stratalinker (Stratagene)
  • 200‐ml roller bottle
  • 65° and 68°C incubator
  • Plastic wrap
  • Imaging plate (Fuji film)

Support Protocol 1: DNA Preparation in Low‐Melting‐Point Agarose

  Materials
  • Cultures of yeast cells
  • 10% (w/v) sodium azide solution
  • Sorbitol solution (see recipe)
  • Zymolyase solution (see recipe)
  • Agarose solution no. 3 (see recipe)
  • ES solution: 0.5 M EDTA (pH 9.0)/1% (v/v) sodium N‐lauroyl sarcosinate
  • Proteinase K (Merck)
  • TE buffer, pH 9.0 ( appendix 2A)
  • 50‐ml tubes
  • 37° and 50°C incubators
  • Mold for making agarose plugs, 10‐mm (height) × 5‐mm (width) × 1.5‐mm (thickness; Bio‐Rad)

Support Protocol 2: Digestion of DNA in Low‐Melting Agarose

  Materials
  • Agarose plug ( protocol 2)
  • Restriction enzyme buffer (H buffer; Takara)
  • Restriction enzyme (e.g., BglII 30 U/µl to 60 U/µl; Takara)
NOTE: Restriction enzymes that digest the target region into small fragments (<10 kb) should be selected. Because the DNA fragment (<10kb) undergoing replication is shorter than 20 kb, it can be separated by the gel electrophoresis.

Basic Protocol 2: Contour‐Clamped Homogeneous Electric Field Pulsed‐Field Gel Electrophoresis to Monitor Chromosome Replication Status

  Materials
  • Yeast DNA in agarose plugs ( protocol 5)
  • Agarose solution for CHEF (200 ml): 1.0% (w/v) pulsed‐field certified agarose (Bio‐Rad)/0.5× TBE
  • 0.5× TBE ( appendix 2A)
  • Ethidium bromide
  • 43°C water bath
  • Gel casting tray
  • Well combs
  • Electrophoresis apparatus
  • CHEF mapper (Bio‐Rad)

Support Protocol 3: DNA Preparation in Low‐Melting‐Point Agarose Gel for PFGE

  • Agarose solution: 1.5% Sea plaque GTG agarose, 0.25 M EDTA (pH 8.0)
  • Synchronously grown yeast cells harvested from YPD medium (pH 4.0) containing 3.0 µM α‐factor.
  • 50 mM EDTA
  • LET buffer (see recipe)
  • ESP buffer (see recipe)
  • TE buffer, pH 9.0 ( appendix 2A)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Bell, L. and Byers, B. 1983. Separation of branched from linear DNA by two‐dimensional gel electrophoresis. Anal. Biochem. 130:527‐535.
   Brewer, B.J. and Fangman, W.L. 1987. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463‐471.
   Dijkwel, P.A. and Hamlin, J.L. 1997. Mapping replication origins by neutral/neutral two‐dimensional gel electrophoresis. Methods 13:235‐245.
   Ganley, A.R.D., Ide, S., Saka, K., and Kobayashi, T. 2009. The effect of replication initiation on gene amplification in the rDNA and its relationship to aging. Mol. Cell 35:1‐11
   Hennessy, K.M., Lee, A., Chen, E., and Botstein, D. 1991. A group of interacting yeast DNA replication genes. Genes Dev. 5:958‐969.
   Ide, S., Watanabe, K., Watanabe, H., Shirahige, K., Kobayashi, T., and Maki, H. 2007. Abnormality in initiation program of DNA replication is monitored by the highly repetitive rDNA array on chromosome XII in budding yeast. Mol. Cell. Biol. 27:568‐578.
   Nawotka, K.A. and Huberman, J.A., 1988 Two‐dimensional gel electrophoretic method for mapping DNA replicons. Mol. Cell. Biol. 8:1408‐1413.
   Schwartz, D.C. and Canter, C.R. 1984. Separation of yeast chromosome‐sized DNAs by pulsed field gradient gel electrophoresis. Cell 37:67‐75.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library