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Kinetic analysis of DNA hairpin folding by Fluorescence correlation spectroscopy (FCS) (CAT#: STEM-MB-1131-WXH)

Introduction

The hairpin-loop is a structural element that is present in DNA and RNA molecules in vivo as well as in vitro systems. It is known to play a key role in biological functions such as regulation of gene expression and facilitation of mutagenic events. DNA hairpins have also been used as highly sensitive reporters, so-called molecular beacons, for in vitro assays, and serve as model systems for secondary structure formation in DNA and RNA.




Principle

Fluorescence correlation spectroscopy (FCS) is a statistical analysis, via time correlation, of stationary fluctuations of the fluorescence intensity. Its theoretical underpinning originated from L. Onsager's regression hypothesis. The analysis provides kinetic parameters of the physical processes underlying the fluctuations. One of the interesting applications of this is an analysis of the concentration fluctuations of fluorescent particles (molecules) in solution. In this application, the fluorescence emitted from a very tiny space in solution containing a small number of fluorescent particles (molecules) is observed. The fluorescence intensity is fluctuating due to Brownian motion of the particles. In other words, the number of the particles in the sub-space defined by the optical system is randomly changing around the average number. The analysis gives the average number of fluorescent particles and average diffusion time, when the particle is passing through the space. Eventually, both the concentration and size of the particle (molecule) are determined. Both parameters are important in biochemical research, biophysics, and chemistry.

Applications

• Measurement of the diffusion coefficient of biomolecules
• Detection of translational diffusions
• Measurement of the biomolecular concentration in vitro or in vivo
• Quantification of the viscosity of a solution
• Monitoring the binding or unbinding of two kinds of biomolecules
• Probing the diffusion paths of different directions and mapping the intercellular obstacles

Procedure

1. Sample Preparation
2. Fluorescence correlation spectroscopy (FCS) testing
3. Data analysis

Materials

Fluorescence Correlation Spectrometer
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