Nucleic Acids Research Advance Access originally published online on January 7, 2008
Nucleic Acids Research 2008 36(4):e21; doi:10.1093/nar/gkm1144
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Nucleic Acids Research, 2008, Vol. 36, No. 4 e21
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification
Department of Physiological Chemistry, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands
*To whom correspondence should be addressed. Tel: +31 30 2538186; Fax: +31 30 2538479; Email: f.c.p.holstege{at}umcutrecht.nl Correspondence may also be addressed to Marc Timmers. Tel: +31 30 2538981; Fax: +31 30 2539035; Email: h.t.m.timmers{at}umcutrecht.nl
Received August 27, 2007. Revised November 15, 2007. Accepted December 10, 2007.
Chromatin immunoprecipitation combined with DNA microarrays (ChIP-chip) is a powerful technique to detect in vivo protein–DNA interactions. Due to low yields, ChIP assays of transcription factors generally require amplification of immunoprecipitated genomic DNA. Here, we present an adapted linear amplification method that involves two rounds of T7 RNA polymerase amplification (double-T7). Using this we could successfully amplify as little as 0.4 ng of ChIP DNA to sufficient amounts for microarray analysis. In addition, we compared the double-T7 method to the ligation-mediated polymerase chain reaction (LM-PCR) method in a ChIP-chip of the yeast transcription factor Gsm1p. The double-T7 protocol showed lower noise levels and stronger binding signals compared to LM-PCR. Both LM-PCR and double-T7 identified strongly bound genomic regions, but the double-T7 method increased sensitivity and specificity to allow detection of weaker binding sites.
Present address: Harm van Bakel, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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