[Show abstract][Hide abstract] ABSTRACT: In molecular biology, formamide (FA) is a commonly used denaturing agent for DNA. Although its influence on DNA duplex stability in solution is well established, little is known about immobilized DNA on microarrays. We measured thermal denaturation curves for oligonucleotides immobilized by two standard protocols: thiol self-assembling and pyrrole electrospotting. A decrease of the DNA denaturation temperature with increasing FA fraction of the solvent was observed on sequences with mutations for both surface chemistries. The average dissociation temperature decrease was found to be -0.58+/-0.05 degrees C/% FA (v/v) independently of grafting chemistry and probe sequence.
[Show abstract][Hide abstract] ABSTRACT: Human Papilloma Virus (HPV) is a DNA virus belonging to the Papovavirus family. Genital HPV types have been subdivided into medium-low risk, and high-risk (HPV 16 and 18), frequently associated with cervical cancer. Three DNA-based piezoelectric biosensors were here developed for a quick detection and genotyping of HPV.
We developed a method for the detection and genotyping of HPV in human cervical scraping samples based on coupling DNA piezoelectric sensors with Polymerase Chain Reaction (PCR). The novelty of this work was the design and immobilisation of a degenerate probe (chosen in a conserved region of the viral genome) for the simultaneous detection of 16 virus strains and of two specific probes (chosen in a less-conserved region of the viral genome) for genotyping.
The three biosensors were optimised with synthetic oligonucleotides with good reproducibility (HPVdeg CV% (av) 9%, HPV16 CV%(av) 9%; HPV18 CV%(av) 11%) with a detection limit of 50 nM. Cervical scraping samples after PCR amplification (in 40-200 nM range), were tested without the need of label with high selectivity and reproducibility. The results were in agreement with a reference method used in routinary analysis.
Piezoelectric biosensors have proven to be suitable for detection and genotyping of HPV.
[Show abstract][Hide abstract] ABSTRACT: Piezoelectric sensing is here applied to point mutation detection in human DNA. The mutation investigated is in the TP53 gene, which results inactivated in most cancer types. TP53 gene maps on chromosome 17 (17p13.1). It contains 11 exons and codifies for the relative protein, involved in cell proliferation. The TP53 gene has a wide mutation spectrum that is related to different tumours. In particular, those occurring in the structurally important L2 and L3 zinc-binding domains, have been linked to patient prognosis and more strongly to radiotherapy and chemotherapy resistance in several major cancers. For this reason, the identification of these mutations represents an important clinical target and biosensors could represent good candidate for fast mutation screening. In this paper, a DNA-based piezoelectric biosensor for the detection of the TP53 gene mutation at codon 248 is reported. A biotinylated probe was immobilised on the sensor surface via dextran-streptavidin modified surfaces. The sensor was optimised using synthetic oligonucleotides. Finally, the sensor system was successfully applied to polymerase chain reaction (PCR)-amplified real samples of DNA extracted from two cell lines, one normal (wild-type) and one mutated, carrying the mutation at codon 248 of the TP53 gene. The results obtained demonstrate that the DNA-based piezoelectric biosensor is able to detect the point mutations in PCR-amplified samples showing the potentialities of this approach for routine analysis.