Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification.
Nucleic Acids Res
; 36(4): e21, 2008 Mar.
Article
en En
| MEDLINE
| ID: mdl-18180247
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Factores de Transcripción
/
Proteínas Virales
/
ARN Polimerasas Dirigidas por ADN
/
Reacción en Cadena de la Polimerasa
/
Análisis de Secuencia por Matrices de Oligonucleótidos
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Inmunoprecipitación de Cromatina
Tipo de estudio:
Evaluation_studies
/
Prognostic_studies
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2008
Tipo del documento:
Article
País de afiliación:
Países Bajos