DNA microarray location analysis of DNA-binding proteins using chromatin immunoprecipitation (ChIP–chip) is making important contributions to the investigation of DNA-bound proteins such as the regulatory factors that control gene expression. ChIP-chip studies have already been successfully used for high-resolution mapping of nucleosome positions (1
), for identification of transcription factor binding sites (2
) and for elucidating how histone modifications relate to transcription (3
). Increased resolution of microarray technology and ongoing optimization of experimental protocols will boost the efficiency of this technique, further increasing its already widespread use.
The basic principle of ChIP is straightforward and typically involves in vivo
coupling of proteins to DNA using a crosslinking agent such as formaldehyde (4
). DNA is then fragmented, by sonication or nuclease digestion, followed by immunoprecipitation with specific antibodies against the protein of interest, thereby enriching for crosslinked genomic fragments. After reversal of the crosslinks, the immunoprecipitated DNA is purified, labeled and hybridized onto a DNA microarray (6
). Probes corresponding to regions in the genome bound by the protein of interest will show enrichment for the immunoprecipitated sample compared to input. Alternative methods for microarray detection of protein–DNA interactions exist, such as tethering DNA-binding proteins to DNA adenine methyltransferase, resulting in a localized methylation around genomic binding sites (7
). Similar to ChIP, in DamID the location of genomic binding sites can then be inferred following isolation of the methylated DNA fragments and hybridization to whole-genome DNA microarrays.
A crucial step in these techniques is obtaining sufficient material (~1–5 μg) for DNA microarray hybridization. Whereas immunopreciptiation of abundant proteins such as histones often readily provides the required amounts of bound DNA, the yields for transcription factors with a limited number of genomic binding sites is in the sub-nanogram (ng) range (8
). One way to address this problem is to increase the amount of starting material, but for transcription factors with only a few genomic binding sites this is hardly feasible. Therefore, the amount of ChIP material for hybridization is often increased by amplification. PCR-based methods like ligation-mediated PCR (LM-PCR) or whole-genome amplification display an unwanted bias toward certain DNA sequences. This is exaggerated by the exponential nature of the amplification. Previously, it was shown that a linear amplification method based on T7 RNA polymerase transcription prevents this bias (9
). Using this protocol, 2.5 ng of endonuclease-digested genomic DNA was increased to 10 μg amplified RNA (aRNA). Several groups have now successfully used T7 amplification for ChIP-chip analysis of nucleosomes and histone modifications (10
). However, for transcription factors, single round T7 amplification still yields insufficient amounts of aRNA for microarray hybridizations.
Here, we present a two-step T7 amplification protocol that can accurately and reproducibly amplify as little as 0.4 ng of chromatin immunoprecipitated DNA for microarray analysis. Importantly, in our experiments, the double-round T7 amplification method also shows improved signal-to-noise ratios when compared to conventional LM-PCR, resulting in increased sensitivity and specificity to detect genomic regions bound by DNA-sequence-specific transcription factors.