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

Plant Cell Rep. 2004 Sep;23(3):144-7. doi: 10.1007/s00299-004-0806-y. Epub 2004 May 28.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Chromosome Mapping / methods*
  • Cytosine / metabolism
  • DNA / genetics
  • DNA / metabolism
  • DNA Methylation
  • DNA, Plant / drug effects
  • DNA, Plant / genetics*
  • DNA-Directed DNA Polymerase / metabolism
  • DNA-Directed DNA Polymerase / pharmacology*
  • Gene Silencing
  • Genome, Plant
  • Nucleic Acid Amplification Techniques / methods*
  • Plants, Genetically Modified / enzymology
  • Plants, Genetically Modified / genetics
  • Tobacco / enzymology
  • Tobacco / genetics*
  • Transgenes / genetics

Substances

  • DNA, Plant
  • Cytosine
  • DNA
  • DNA-Directed DNA Polymerase