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Novel technology (Optical-based)

Introduction to Novel Technology

The increasing demands placed upon trace nucleic acid detection technologies have driven the development and application of novel technologies that have the potential to reliably detect single target nucleic acid molecules within challenging matrices.

It has proven difficult in the past to identify and separate biomolecules at very low concentrations. However, recent developments have improved the sensitivity of detection such that single-molecule detection has been achieved. The ability to detect such low concentrations of DNA will have applications in many fields including medical diagnosis and forensic analysis.

Current trace detection technologies are predominantly nucleic amplification based, employing core methodologies such as the polymerase chain reaction (PCR), which have the potential of amplifying a single target molecule. However, an amplification-based approach suffers from specific drawbacks such as cross amplification and matrix effects.

Emerging trace nucleic acid detection technologies hold the promise of enhanced sensitivities without prior target amplification. Single molecule detection represents the ultimate sensitivity limit in biochemical analysis. However, the developmental nature of these technologies means that few are currently in routine use or commercially available.

Optical-based Technologies:

In order to visualise nucleic acids, fluorescent dyes can be attached to them. Technical advances have made it possible to detect a single dye molecule under biologically relevant conditions. The detection of single fluorophores has become possible with the development of ultra-sensitive detectors and detection schemes. These detection schemes have been applied to a variety of areas such as capillary electrophoresis and hybridisation studies.

Increasing the sensitivity of fluorescence detection to the limit of an individual fluorophore allows for an assay on a single-cell or a single-DNA fragment basis without any individual amplification.

1. Surface Enhanced Raman Spectroscopy (SERS)

Raman is a novel technology for the identification and analysis of molecular species (similar to FT-IR spectroscopy). Raman spectroscopy is based on the detection of scattered light-the Raman effect, which forms a characteristic Raman spectrum � in effect a molecular fingerprint. Under normal conditions, Raman emissions are weak and the inherent fluorescence of the analyte can be sufficient to mask the Raman spectrum. However, it was discovered that the Raman effect is enhanced when the analyte interacts with a roughened metal surface such as silver or gold. The resultant technique was termed surface enhanced Raman spectroscopy (SERS), and demonstrates an enhancement of 1X10e6 over conventional Raman emissions.

Key features of the SERS technique:
1) detect target molecules down to the single molecule level
2) potentially quantitative
2) simultaneous detection of multiple probe labels, which allows the technique to be adapted to a multiplex format.

A manufacturer of such technology include Avalon Instruments(http://www.avaloninst.com/)

2. Fluorescence Correlation Spectroscopy (FCS)

Fluorescence Correlation Spectroscopy (FCS) is a spectroscopic technique for the study of molecular interactions in solution. FCS utilises laser excitation to monitor the random motion of fluorescently labelled molecules in extremely small, confocally defined volume elements.

FCS can successfully be applied to extremely dilute samples (1x10-8 to 1x10-15 M), allowing the fluctuations of individual molecules to be observed. These molecular fluctuations provide information on the rate of diffusion and diffusion time of the target molecule, thereby enabling the detection of inter-molecular interactions such as probe-target hybridisation.

FCS has the potential to rapidly characterise the hybridisation of nucleic acids in solution without the need for prior target amplification or free probe separation. At nanomolar concentrations it is possible to quantify the stoichiometry, kinetics and thermodynamics of molecular interactions.
(Click for Zeiss (http://www.zeiss.com/) Confocor 2-Combi Fluorescence Correlation Microscope - 12KB)

For more information on these and other novel technologies visit the UK Bio-Measurement Network or the Review of Technologies for low level measurement page and down load the following report from the Document Library:

'Survey on emerging technologies potential for highly sensitive detection.'

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Nucleic acid measurements

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