Plant Cell Reports

, Volume 23, Issue 3, pp 144–147 | Cite as

Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region

Genetics and Genomics

Abstract

Plant genomes contain a heavily methylated region in which cytosines are methylated in both the symmetrical and asymmetrical sequences. The physical mapping of such a hypermethylated region is difficult because many restriction enzymes are sensitive to methylated cytosine residues in their recognition sites. The Phi29 DNA polymerase provides an efficient and representative amplification of the genomic DNA that is methylation-free. Using this amplified genomic DNA, we were able to show that a heavily methylated genomic DNA region becomes amenable to physical mapping with any restriction enzymes. This protocol will be especially useful for analysis of the heavily methylated region of plant genomes.

Keywords

DNA methylation Phi29 DNA polymerase Physical mapping Rolling circle amplification Transgene-induced gene silencing 

Abbreviations

18:2

Linoleic acid

18:3

α-Linolenic acid

CaMV

Cauliflower mosaic virus

PTGS

Post-transcriptional gene silencing

RCA

Rolling circle amplification

TGS

Transcriptional gene silencing

WT

Wild type

ω-3

Refers to the position of the double bond from the methyl end of a fatty acid

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Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • E. Adachi
    • 1
  • K. Shimamura
    • 1
  • S. Wakamatsu
    • 2
  • H. Kodama
    • 1
  1. 1.Department of Bioproduction Science, Faculty of HorticultureChiba UniversityChibaJapan
  2. 2.Department of Biology, Faculty of ScienceKyushu UniversityFukuokaJapan

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