Detection of protein–DNA interaction with a DNA probe: distinction between single-strand and double-strand DNA–protein interaction

AUTOR(ES)

Ban, Changill

FONTE

Oxford University Press

RESUMO

A simple, direct method for the detection of DNA–protein interaction was developed with electrochemical methods. Single-stranded DNA (ss-DNA) probes were prepared through the chemical bonding of an oligonucleotide to a polymer film bearing carboxylic acid groups, and double-stranded DNA (ds-DNA) probes were prepared through hybridization of the complementary sequence DNA on the ss-DNA probe. Impedance spectroscopy and differential pulse voltammetry (DPV) distinguished the interaction between the DNA probes with mouse Purβ (mPurβ), an ss-DNA binding protein, and with Escherichia coli MutH, a ds-DNA binding protein. Impedance spectra obtained before and after the interaction of DNA probes with these proteins clearly showed the sequence-specific ss-DNA preference of mPurβ and the sequence-specific ds-DNA preference of MutH. The concentration dependence of proteins on the response of the DNA probes was also investigated, and the detection limits of MutH and mPurβ were 25 and 3 μg/ml, respectively. To confirm the impedance results, the variation of the current oxidation peak of adenine of the DNA probe was monitored with DPV. The formation constants of the complexes formed between the probe DNA and the proteins were estimated based on the DPV results.

Documentos Relacionados

• Single-strand interruptions in replicating chromosomes cause double-strand breaks
• A CT promoter element binding protein: definition of a double-strand and a novel single-strand DNA binding motif.
• A DNA fragment from the origin of single-strand to double-strand DNA replication of bacteriophage fd.
• A modified single-strand annealing model best explains the joining of DNA double-strand breaks mammalian cells and cell extracts.
• Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.