Quadruple 9-mer-based protein binding microarray with DsRed fusion protein.

Full Abstract

BACKGROUND: The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner. RESULTS: We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor. CONCLUSION: Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA.

Author information

Author/s: Kim, Min-Jeong (MJ); Lee, Tae-Ho (TH); Pahk, Yoon-Mok (YM); Kim, Yul-Ho (YH); Park, Hyang-Mi (HM); Choi, Yang Do (YD); Nahm, Baek Hie (BH); Kim, Yeon-Ki (YK);

Affiliation: GreenGene Biotech Inc, Myongji University, Yongin 449-728, Korea. kim750a11(-atsign-)gmail.com

Journal and publication information

Publication Type: Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't

Journal: BMC molecular biology (BMC Mol Biol), published in England. (Language: eng)

Reference: 2009-; vol 10 (issue ) : pp 91

Dates: Created 2009/09/30; Completed 2009/11/03;

PMID: 19761621, status: MEDLINE (last retrieval date: 11/3/2009, IMS Date: )

Sourced from the National Library of Medicine. Abstract text and other information may be subject to copyright.

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