Reduction of dimensionality processes of DNA hybridization with immobilized oligonucleotide probes.

摘要

Binding or hybridization between either two complementary DNA molecules, DNA/RNA or DNA/protein is necessarv for gene regulations in cells, anti-sense technology and novel drug discovery. A number of companies manufacture a "DNA chip" with as much as 10,000 different sequences of single stranded (ss) DNAs on a small modified glass or silicon substrate. These DNA linked substrates are used as sensors for sequencing by hybridization, HIV sequence analysis etc. through the hybridization and detection of the complementary sequences in solution. However, the lack of a fundamental understanding about the behavior of a DNA molecule at the solid/liquid interface and how the physical parameters involved affect the rate of hybridization make quantitative application of these sensors difficult. The DNA-DNA hybridization sensor has been investigated experimentally and theoretically as a model system for understanding the biophysical properties of DNA molecules on a surface.; Building on a reduction of dimensionality model for ligand/receptor binding on a membrane surface, a mathematical model which describes DNA-DNA hybridization on a DNA bio-sensor surface was developed and used for predicting the number of DNA duplexes formed in experimental hybridization assay. The model included the effects of non-specific adsorption and surface diffusion of DNA molecules with different sizes on the time scale for signal detection from the sensor. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate.; Using total internal reflection (TIR) microscopy coupled with spot or pattern fluorescence recovery after photobleaching (FRAP) which was built for this study, the interfacial parameters including the desorption rate constants and surface diffusion coefficients of a 21 base long ss DNA molecule and bovine serum albumin (BSA) were measured on a amino-silanized glass which was used as a substrate for the bio-sensor. Further information on the structure of the adsorbed DNA molecules was obtained by performing independent adsorption measurements using large surface area porous glass beads.; A change in the chemical and electrostatic nature (e.g., acidity) of the surfaces or the solution may affect the magnitude of non-specific adsorption/desorption rates as well as the surface diffusion kinetics. Thus TIR/pattern or spot FRAP was carried on different types of silanized substrates at a range of ethanol contents which are well known to alter the behavior of DNA in solution. The data should provide guidance to the DNA chip and chromatography support designer so that he can use surface and solution chemistry to tailor the adsorption and diffusion behavior of DNA targets on different surfaces according to the requirements and specifications of different applications. In parallel with the experimental measurements of DNA with TIR/FRAP, a novel strategy for analyzing FRPLP data in the form of temporal fluorescence intensity profile was developed. This was based on a charge coupled device (CCD) camera and had the potential of distinguishing between key physical parameters in the complex adsorption/diffusion processes.