Detection Of Target DNA Sequences Research Articles

Emerging optofluidic technologies for point-of-care genetic analysis systems: a review

This review describes recently emerging optical and microfluidic technologies suitable for point-of-care genetic analysis systems. Such systems must rapidly detect hundreds of mutations from biological samples with low DNA concentration. We review optical technologies delivering multiplex sensitivity and compatible with lab-on-chip integration for both tagged and non-tagged optical detection, identifying significant source and detector technology emerging from telecommunications technology. We highlight the potential for improved hybridization efficiency through careful microfluidic design and outline some novel enhancement approaches using target molecule confinement. Optimization of fluidic parameters such as flow rate, channel height and time facilitates enhanced hybridization efficiency and consequently detection performance as compared with conventional assay formats (e.g. microwell plates). We highlight lab-on-chip implementations with integrated microfluidic control for "sample-to-answer" systems where molecular biology protocols to realize detection of target DNA sequences from whole blood are required. We also review relevant technology approaches to optofluidic integration, and highlight the issue of biomolecule compatibility. Key areas in the development of an integrated optofluidic system for DNA hybridization are optical/fluidic integration and the impact on biomolecules immobilized within the system. A wide range of technology platforms have been advanced for detection, quantification and other forms of characterization of a range of biomolecules (e.g. RNA, DNA, protein and whole cell). Owing to the very different requirements for sample preparation, manipulation and detection of the different types of biomolecules, this review is focused primarily on DNA-DNA interactions in the context of point-of-care analysis systems.

Ultrasensitive DNA Detection Using Oligonucleotide−Silver Nanoparticle Conjugates

Oligonucleotide-gold nanoparticle (OGN) conjugates are powerful tools for the detection of target DNA sequences due to the unique properties conferred upon the oligonucleotide by the nanoparticle. Practically all the research and applications of these conjugates have used gold nanoparticles to the exclusion of other noble metal nanoparticles. Here we report the synthesis of oligonucleotide-silver nanoparticle (OSN) conjugates and demonstrate their use in a sandwich assay format. The OSN conjugates have practically identical properties to their gold analogues and due to their vastly greater extinction coefficient both visual and absorption analyses can occur at much lower concentrations. This is the first report of OSN conjugates being successfully used for target DNA detection and offers improved sensitivity which is of interest to a range of scientists.

Photocleavable peptide-DNA conjugates: synthesis and applications to DNA analysis using MALDI-MS

The synthesis and characterization of photocleavable peptide-DNA conjugates is described along with their use as photocleavable mass marker (PCMM) hybridization probes for the detection of target DNA sequences by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Three photocleavable peptide-DNA conjugates were synthesized, purified, and characterized using HPLC and denaturing gel electrophoresis, as well as IR-MALDI and UV-MALDI. The hybridization properties of the conjugates were also studied by monitoring their thermal denaturation with absorption spectroscopy. No significant difference in the melting temperature ( T (m)) of the duplexes was observed between the unmodified duplex and the duplex in which one strand was modified with the photocleavable peptide moiety. These conjugates were evaluated as hybridization probes for the detection of immobilized synthetic target DNAs using MALDI-MS. In these experiments, the DNA portion of the conjugate acts as a hybridization probe, whereas the peptide is photoreleased during the ionization/desorption step of UV-MALDI and can serve as a marker (mass tag) to identify a unique target DNA sequence. The method should be applicable to a wide variety of assays requiring highly multiplexed DNA/RNA analysis, including gene expression monitoring, genetic profiling and the detection of pathogens.

Detection and quantification of plant viruses by PCR.

The polymerase chain reaction (PCR) is a technique that enables the specific amplification and hence detection of target DNA sequences from complex mixtures of nucleic acid. A combination of short, specific primers and thermostable DNA polymerases are used to amplify the target sequence, through repeated cycles of denaturation, reannealing, and DNA synthesis at high temperatures, allowing an exponential increase in the amount of the DNA of interest. By addition of a reverse-transcription (RT) step, PCR can also be applied to cDNA generated from RNA templates. Its extreme sensitivity and high specificity make it an unparalleled technique for the detection and characterization of rare messages, including viral infections that are difficult to detect and diagnose by serology or electron microscopy.

Electrochemically Active DNA Probes: Detection of Target DNA Sequences at Femtomole Level by High-Performance Liquid Chromatography with Electrochemical Detection

Electrochemically active DNA probes were prepared by linking a ferrocene unit with 5′-aminohexyl-terminated oligonucleotides. The DNA sequences of probes 5a, 5b, and 5c were 5′-T12-3′, 5′-T20-3′, and 5′-TGCAG TTCCG GTGGC TGATC-3′, respectively. Probe 5a could form a complex selectively with a single-strand poly(A) and a double-strand DNA fragment containing an A13 sequence and these complexes could be detected at femtomole levels by an electrochemical detector (ECD) on HPLC. The observed ECD response was proportional to the amount of the complex over the range 20-100 fmol. Probe 5c was capable of detecting femtomole levels of a restriction DNA fragment having oncogene v-myc. Moreover, probe 5b was able to detect picogram levels of mRNA taken from rat brain or yeast total cellular RNA. This proves that the electrochemically active DNA probes are useful in analyzing traces of DNA and RNA carrying the complementary sequence.