Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices

K. K. Sriram, Jia Wei Yeh, Yii Lih Lin, Yi Ren Chang, Chia Fu Chou

研究成果: 雜誌貢獻文章

9 引文 斯高帕斯(Scopus)

摘要

Mapping transcription factor (TF) binding sites along a DNA backbone is crucial in understanding the regulatory circuits that control cellular processes. Here, we deployed a method adopting bioconjugation, nanofluidic confinement and fluorescence single molecule imaging for direct mapping of TF (RNA polymerase) binding sites on field-stretched single DNA molecules. Using this method, we have mapped out five of the TF binding sites of E. coli RNA polymerase to bacteriophage λ-DNA, where two promoter sites and three pseudo-promoter sites are identified with the corresponding binding frequency of 45% and 30%, respectively. Our method is quick, robust and capable of resolving protein-binding locations with high accuracy (~ 300 bp), making our system a complementary platform to the methods currently practiced. It is advantageous in parallel analysis and less prone to false positive results over other single molecule mapping techniques such as optical tweezers, atomic force microscopy and molecular combing, and could potentially be extended to general mapping of protein-DNA interaction sites.

原文英語
頁(從 - 到)e85
期刊Nucleic Acids Research
42
發行號10
DOIs
出版狀態已發佈 - 2014

ASJC Scopus subject areas

  • Genetics

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