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

  • Genetics and Genomics
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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.

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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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Acknowledgements

This study was supported by a Grant-in Aid for Scientific Research © (14540588) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

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Correspondence to H. Kodama.

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Communicated by H. Ebinuma

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Adachi, E., Shimamura, K., Wakamatsu, S. et al. Amplification of plant genomic DNA by Phi29 DNA polymerase for use in physical mapping of the hypermethylated genomic region. Plant Cell Rep 23, 144–147 (2004). https://doi.org/10.1007/s00299-004-0806-y

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  • DOI: https://doi.org/10.1007/s00299-004-0806-y

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