Fluorescence Correlation Spectroscopy in Nucleic Acid Analysis

Abstract

Laser induced fluorescence correlation spectroscopy (FCS) is a new, developing branch in nucleic acid analysis. Interest in FCS has increased enormously over the past few years[3.1–3.3] as a result of the introduction of the confocal volume of detection [3.4–3.6]. Fluorescent molecules are observed in a very small volume element of ≈ 1 fl ( 10⁻¹⁵ 1) excited by a well-focused laser beam. The femtoliter “cavity”defined by the laser is either stationary or scans through the solution or matrix [3.7–3.13]. The emitted fluorescence bursts are detected from individual molecules. This enables a single fluorescent molecule in solution or on a matrix at room temperature to be measured with a negligible background. The principles of the identification nucleic acids target sites are linked closely to the concept of thermal fluctuations [3.14–3.17]. Due to thermodynamic fluctuations the number of molecules, for example, will fluctuate in the observed volume element. For small numbers Nof molecules, Poisson statistics are valid and the statistical parameters, mean value, variance and standard deviation, can be determined. The variance is directly related to the number of molecules. In other words, examination of the thermal fluctuations is sufficient to obtain the absolute number of molecules without any calibration. If the biochemical relaxation time τ_chem is much larger than the characteristic diffusion time τ_Diff, then the biochemical reaction is not equilibrated during diffusion through the three-dimensional volume element.