Experimental approach

Published ChlP-chip protocols are fairly consistent, despite the organism or factor of interest (6-8). A general scheme of the approach is depicted in Figure 13.1. Generally, cells are grown in the conditions that are relevant to the goal of the experiment and treated with formaldehyde to crosslink protein-DNA, as well as protein-protein, interactions. Although other crosslinking agents are available, formaldehyde is attractive because heating samples at 65°C reverses the crosslinking, allowing the DNA to be used in subsequent enzymatic reactions (4). Extracts are then prepared and the chromatin is either sonicated to shear the DNA to 500-700 base pairs for spotted arrays (9), or enzymatically treated to generate much smaller fragments for higher-resolution oligo arrays (10). Following clarification of the extracts, the protein-bound DNA is selected by immunoprecipitation with either antibodies specific for the protein of interest or antibodies specific for a tag if the protein has been tagged with a moiety such as myc or HA. The crosslinks are then reversed and the DNA is purified. The DNA is then subjected to a labeling reaction, slides are hybridized and scanned, the image is quantified and the data analyzed.

Prior to hybridization, the purified DNA is fluorescently labeled by generally one of two methods: either direct incorporation of a modified nucleotide or chemical coupling of a fluorescent molecule after incorporation of an aminoallyl-modified nucleotide (6, 8). The reference sample is usually labeled with one fluor, that is Cy3, while the experimental or enriched DNA sample is labeled with another fluor, Cy5 for example. The probes can then be combined and hybridized to a microarray. Both the hybridization buffer and reaction temperature must be optimize, as both will impact on stringency potentially altering the final outcome of the experiment. After several washing steps of varying stringency to remove unincorporated dye and non-hybridizing DNA, slides are scanned with lasers optimized for detection at specific wavelengths corresponding to the dyes. Most scanners are equipped with dual lasers, thus allowing for rapid simultaneous data acquisition of both background and experimental probes. The results of the hybridization will allow detection of enriched segments in the IP reaction, thereby identifying transcription factor binding sites.

0 0

Post a comment