Improved genome-wide localization by ChIP-chip using double-round T7 RNA polymerase-based amplification
| Autor: | Harm van Bakel, H. T. Marc Timmers, Frank C. P. Holstege, Dik van Leenen, Mariel O. Brok, Marijana Radonjic, Folkert J. van Werven |
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| Rok vydání: | 2008 |
| Předmět: |
Chromatin Immunoprecipitation
Binding Sites Saccharomyces cerevisiae Proteins Inverse polymerase chain reaction Multiple displacement amplification Recombinase Polymerase Amplification DNA-Directed RNA Polymerases Genomics Biology Polymerase Chain Reaction Molecular biology ChIP-sequencing Viral Proteins genomic DNA Real-time polymerase chain reaction Genetics medicine RNA Methods Online T7 RNA polymerase Promoter Regions Genetic Chromatin immunoprecipitation Oligonucleotide Array Sequence Analysis Transcription Factors medicine.drug |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkm1144 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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