Because of the large amount of genetic information which can be rapidly accessed, DNA microarrays have become indispensable tools for biological investigations. Despite their utility, however, a number of experimental difficulties have resulted in widely varying data quality between and even within the same laboratories. One of the more difficult challenges is that DNA probes of the same length can have widely varying hybridization or melting temperatures (Tm's). Surprisingly, although temperature gradient PCR devices exist for rapid optimization of reaction conditions, no such commercial devices exist for microarrays. The result is that suboptimal conditions are used for what are presently some of the most expensive experiments in molecular biology. ? ? This project focuses on the development of a hardware system for producing optimal """"""""temperature addresses"""""""" for every probe included in an arbitrary microarray design. This improvement will dramatically improve the accuracy and reproducibility of nucleic acid microarray data. By improving specificity of binding, the limit of detection for rare sequences in a sample should also be improved. Finally, the system will greatly increase flexibility regarding the ability to include sequences which would have been previously discarded from probe sets. As microarrays continue to find expanding applications in biological research, the developed system will represent an important advance in the field and should have considerable commercial attractiveness. In Phase II, we will integrate the temperature gradient hybridization hardware with a CCD-based imaging system for real-time image acquisition. Real-time imaging will allow kinetics to be followed as well as non-isothermal (time-varying gradients in this case) data to be acquired such as annealing kinetics, fluorescence melting curves, competitive binding assays, and """"""""on-chip"""""""" PCR, for example. By the end of Phase II, we will able to market our well-characterized equipment as well as the proof- of-principle data to manufacturers of microarray imaging and support equipment. ? ? DNA microarrays have become an indispensable tool for biological research, however a number of technical challenges have hampered their reliable use. This project focuses on developing the equipment for providing the optimal hybridization conditions for every probe on a microarray, as well as for imaging the surface reactions on the array in real-time. The engineering advances which will result will allow increased flexibility in microarray design for a variety of genotyping applications including gene expression analysis, chromosomal accessibility, sequencing by hybridization, and microbial identification. ? ? ?
