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Development of Real-time PCR Assays for the Detection of the pin II Terminator (tpinII) Used in GM Constructs and Its Donor Organism, Potato (Solanum tuberosum)

Debode Frederic, Zdeňková Kamila, Janssen Eric, Tizolova Anette, du Jardin Patrick, Berben Gilbert, Demnerova Kateřina

G-rich DNA sequences can form four-stranded G-quadruplex (G4) secondary structures and are linked to fundamental biological processes such as transcription, replication and telomere maintenance. G4s are also implicated in promoting genome instability, cancer and other diseases. Here, we describe a detailed G4 ChIP-seq method that robustly enables the determination of G4 structure formation genome-wide in chromatin. This protocol adapts traditional ChIP-seq for the detection of DNA secondary structures through the use of a G4-structure-specific single-chain antibody with refinements in chromatin immunoprecipitation followed by high-throughput sequencing. This technology does not require expression of the G4 antibody in situ, enabling broad applicability to theoretically all chromatin sources. Beginning with chromatin isolation and antibody preparation, the entire protocol can be completed in <1 week, including basic computational analysis.

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February, 2018


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