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

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)e85
JournalNucleic Acids Research
Volume42
Issue number10
DOIs
Publication statusPublished - 2014

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Transcription Factors
Binding Sites
Equipment and Supplies
DNA
DNA-Directed RNA Polymerases
Protein Interaction Mapping
Optical Tweezers
Atomic Force Microscopy
Protein Binding
Bacteriophages
Fluorescence
Escherichia coli

ASJC Scopus subject areas

  • Genetics

Cite this

Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices. / Sriram, K. K.; Yeh, Jia Wei; Lin, Yii Lih; Chang, Yi Ren; Chou, Chia Fu.

In: Nucleic Acids Research, Vol. 42, No. 10, 2014, p. e85.

Research output: Contribution to journalArticle

Sriram, K. K. ; Yeh, Jia Wei ; Lin, Yii Lih ; Chang, Yi Ren ; Chou, Chia Fu. / Direct optical mapping of transcription factor binding sites on field-stretched λ-DNA in nanofluidic devices. In: Nucleic Acids Research. 2014 ; Vol. 42, No. 10. pp. e85.
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