Plant Epigenetics Methods and Protocols

The discovery of DNA as the genetic material brought great hope to scientists all over the world. It was believed that many of the lingering questions in genetics and the mechanisms of heredity would fnally be answered. However, as often is the case in science, more qu- tions arose out of this discovery. What defnes a gene? What are the mechanisms of gene regulation? Further discovery and technological innovations brought about sequencing techniques that allowed the study of complete genomes from many organisms, including Arabidopsis and humans. Despite all the excitement surrounding these technologies, many features of the genome remained unclear. Peculiar characteristics in genome composition such as signifcant redundancy consisting of many repetitive elements and noncoding sequences, active transcriptional units with no protein product, and unusual sequences in promoter regions added to the mysteries of genetic make-up and gene regulation. Indeed, the more we discovered about the genome, the more diffcult it became to understand the complexity of cellular function and regulation. Out of the study of the intricacies of the genome and gene regulation, arose a new science that was independent of actual DNA changes, but critical in maintaining gene regulation and genetic stability. Epigenetics, literally translated as “above genetics,” is the science that describes the mechanisms of heritable changes in gene regulation that does not involve modifcations of DNA sequence. These changes may last through somatic cell division and, in some cases, throughout multiple generations.

Analysis of DNA Methylation in Plants by Bisulfite Sequencing.- Analysis of Bisulfite Sequencing Data from Plant DNA Using CyMATE.- Analysis of Locus-Specific Changes in Methylation Patterns Using a COBRA (Combined Bisulfite Restriction Analysis) Assay.- Detection of Changes in Global Genome Methylation Using the Cytosine-Extension Assay.- In Situ Analysis of DNA Methylation in Plants.- Analysis of Mutation/Rearrangement Frequencies and Methylation Patterns at a Given DNA Locus Using Restriction Fragment Length Polymorphism.- Isoschizomers and Amplified Fragment Length Polymorphism for the Detection of Specific Cytosine Methylation Changes.- Analysis of Small RNA Populations Using Hybridization to DNA Tiling Arrays.- Northern Blotting Techniques for Small RNAs.- qRT-PCR of Small RNAs.- Cloning New Small RNA Sequences.- Genome-Wide Mapping of Protein-DNA Interaction by Chromatin Immunoprecipitation and DNA Microarray Hybridization (ChIP-chip). Part A: ChIP-chip Molecular Methods.- Genome-Wide Mapping of Protein-DNA Interaction by Chromatin Immunoprecipitation and DNA Microarray Hybridization (ChIP-chip). Part B: ChIP-chip Data Analysis.- Metaanalysis of ChIP-chip Data.- Chromatin Immunoprecipitation Protocol for Histone Modifications and Protein–DNA Binding Analyses in Arabidopsis.- cDNA Libraries for Virus-Induced Gene Silencing.- Detection and Quantification of DNA Strand Breaks Using the ROPS (Random Oligonucleotide Primed Synthesis) Assay.- Reporter Gene-Based Recombination Lines for Studies of Genome Stability.- Plant Transgenesis.