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.
Similar content being viewed by others
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
References
Assaad FF, Tucker KL, Signer ER (1993) Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol 22:1067–1085
Blanco L, Salas M (1984) Characterization and purification of a phage φ29-encoded DNA polymerase required for the initiation of replication. Proc Natl Acad Sci USA 81:5325–5329
Blanco L, Bernad A, Lazaro JM, Martin G, Garmendia C, Salas M (1989) Highly efficient DNA synthesis by the phage φ29 DNA polymerase. J Biol Chem 264:8935–8940
Dean FB, Nelson JR, Giesler TL, Lasken RS (2001) Rapid amplification of plasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Res 11:1095–1099
Esteban JA, Salas M, Blanco L (1993) Fidelity of φ29 DNA polymerase. J Biol Chem 268:2719–2726
Faruqi FA, Hosono S, Driscoll MD, Dean FB, Alsmadi O, Bandaru R, Kumar G, Grimwade B, Zong Q, Sun Z, Du Y, Kingsmore S, Knott T, Lasken RS (2001) High-throughput genotyping of single nucleotide polymorphisms with rolling circle amplification. BMC Genomics 2:4
Jorgensen RA, Cluster PD, English J, Que Q, Napoli CA (1996) Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs antisense constructs and single-copy vs complex T-DNA sequences. Plant Mol Biol 31:957–973
Kodama H, Hamada T, Horiguchi G, Nishimura M, Iba K (1994) Genetic enhancement of cold tolerance by expression of a gene for chloroplast ω-3 fatty acid desaturase in transgenic tobacco. Plant Physiol 105:601–605
Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225–232
Martienssen RA, Colot V (2001) DNA methylation and epigenetic inheritance in plants and filamentous fungi. Science 293:1070–1074
Murakami Y, Tsuyama M, Kobayashi Y, Kodama H, Iba K (2000) Trienoic fatty acids and plant tolerance of high temperature. Science 287:476–479
Nelson JR, Cai YC, Giesler TL, Farchaus JW, Sundaram ST, Ortiz-Rivera M, Hosta LP, Hewitt PL, Mamone JA, Palaniappan C, Fuller CW (2002) TempliPhi, phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing. Biotechniques 32:S44–S47
Paszkowski J, Whitham SA (2001) Gene silencing and DNA methylation processes. Curr Opin Plant Biol 4:123–129
Rabinowicz PD, Schutz K, Dedhia N, Yordan C, Parnell LD, Stein L, McCombie WR, Martienssen RA (1999) Differential methylation of genes and retrotransposons facilitates shotgun sequencing of the maize genome. Nat Genet 23:305–308
Schweitzer B, Kingsmore S (2001) Combining nucleic acid amplification and detection. Curr Opin Biotechnol 12:21–27
Walbot V, Warren C (1988) Regulation of Mu element copy number in maize lines with an active or inactive mutator transposable element system. Mol Genet Genomics 211:27–34
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.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by H. Ebinuma
Rights and permissions
About this article
Cite this article
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
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-004-0806-y