Electrochemical DNA hybridisation assays allow the measurement of probe-target binding in a label free fashion for biosensor and healthcare detection, particularly when using electrochemical impedance spectroscopy (EIS). Typically in EIS assay development a short artificial oligonucleotide is used to test sensor performance and consideration is not given to binding real world samples which can contain DNA fragments of varying length. This paper investigates the effect of solution side DNA overhangs on the EIS signal. Firstly, by using a surface tethered 15 nucleotides (nt) PNA probe and a series of artificial DNA targets with increasingly long overhangs it was possible to measure differences in the EIS signal brought about by target length. It was found that an overhang of 1515 nt nt gave the most effective enhancement of signal. This enhancement was attributed to the overhang reducing the access of the redox couple to the electrode surface. Overhangs of 45 and 8515 nt nt caused a smaller enhancement of signal which can be attributed to the reduced hybridisation efficiency of longer strands. Secondly, EIS responses from fragmented Methicillin resistant Staphylococcus aureus (MRSA) bacterial genomic DNA (gDNA) were measured and it was found that in the case of the MRSA gDNA, progressive fragmentation of long DNA sequences (>1000 bp) to less than 100 bp coincided with enhancement of the EIS signal. From the results obtained with oligonucleotides it is inferred that the creation of shorter solution side overhangs in real world MRSA samples boosts the electrochemical signal observed upon DNA binding. (C) 2014 Elsevier B.V. All rights reserved.