Detection of highly repeated sequences in non-amplified genomic DNA by bulk acoustic wave (BAW) affinity biosensor

A DNA-based sensor for the label-free, real-time detection of highly repeated sequences in non-amplified DNA is reported. The transduction principle is a piezoelectric quartz crystal and the immobilisation chemistry is based on the direct coupling of thiol-modified probes on the gold electrode surface. The sequence chosen as probe is internal to a region of the satellite 13 DNA from Bos taurus. The sensor was developed using bovine DNA commercially available, alone or mixed at different percentages with DNA of a different origin (porcine DNA: Sits scrofa). Real samples (DNA extracted from bovine animal muscle) have also been tested. The sample pre-treatment consisted only in a fragmentation with restriction enzymes, followed by an optimised denaturation step. No amplification by polymerase chain reaction was carried out. The developed biosensor resulted very specific reproducible (CV% of 11%) both with synthetic oligonucleotides and with genomic non-amplified DNA. In both cases, the tested negative controls did not give significant frequency shifts. Moreover, the signals obtained with samples containing bovine DNA, alone or mixed with DNA of a different origin, are comparable. The sensor could represent a method alternative to traditional biomolecular techniques, for the identification of species-specific DNA sequences, directly in enzymatically digested DNA, bypassing the amplification step. (C) 2004 Elsevier B.V. All rights reserved.