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Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification

Author:
van Bakel, Harm, van Werven, Folkert J., Radonjic, Marijana, Brok, Mariel O., van Leenen, Dik, Holstege, Frank C.P., Timmers, H.T. Marc
Source:
Nucleic acids research 2008 v.36 no.4 pp. e21
ISSN:
0305-1048
Subject:
transcription factors, DNA, polymerase chain reaction, yeasts, DNA microarrays, transcription (genetics), precipitin tests, binding sites, chromatin, RNA, DNA-directed RNA polymerase
Abstract:
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.
Agid:
2070983