Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification.
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.
van Bakel
Harm
H
Department of Physiological Chemistry, University Medical Center Utrecht, 3584 CG Utrecht, The Netherlands.
van Werven
Folkert J
FJ
Radonjic
Marijana
M
Brok
Mariel O
MO
van Leenen
Dik
D
Holstege
Frank C P
FC
Timmers
H T Marc
HT
D001665:Binding Sites; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D012321:DNA-Directed RNA Polymerases; D023281:Genomics / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D012313:RNA / Q000032:analysis / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014764:Viral Proteins
C500382:GSM1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases