Single-stranded DNA loops as fiducial markers for exploring DNA-protein interactions in single molecule imaging.
作者： Oliver Chammas , Daniel J. Billingsley , William A. Bonass , Neil H. Thomson
DOI: 10.1016/J.YMETH.2013.03.002
关键词:
摘要: A polymerase chain reaction (PCR) based method of adding a single-stranded DNA (ssDNA) hairpin loop to one end linear double-stranded (ds) templates was developed. The structure serves as fiducial marker in single molecule imaging by atomic force microscopy (AFM) and can be applied study DNA–protein interactions. nucleic acid end-labels allow discrimination the polarity template AFM while limiting non-specific interactions which might occur from non-nucleic labels. Homo-polynucleotide ssDNA loops made up 20 base-pairs (bp) for each four bases (A, T, G, C) were investigated determine effects sequence on labelling. products produced with high efficiency yield readily distinguished dsDNA height diameter AFM. application transcription firing Escherichia Coli RNA (RNAP) λPR promoter direction loop-labelled end. captured elongating complexes RNAP, arresting preventing dissociation. dual role retainer previously active RNAP will mechanisms gene expression studied sensitivity This enable insight into molecular convergent or tandem configurations.
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