Microarrays of DNA or RNA polynucleotides or oligonucleotides are state-of-the-art biological tools used in the investigation and evaluation of genes for analytical, diagnostic, and therapeutic purposes. Microarrays typically comprise a plurality of oligomers, synthesized or deposited on a glass support or substrate in an array pattern. The support-bound oligomers are called “probes”, which function to bind or hybridize with a sample of DNA or RNA material under test, called a “target” in hybridization experiments. Some investigators also use the reverse definition, referring to the surface-bound oligonucleotides as targets and the solution sample of nucleic acids as probes. Further, some investigators bind the target sample under test to the microarray substrate and put the oligomer probes in solution for hybridization. Either of the “target” or “probes” may be the one that is to be evaluated by the other. Thus, either one could be an unknown mixture of polynucleotides to be evaluated by binding with the other. All of these iterations are within the scope of the present disclosure. In use, the array surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. The targets are typically labeled with an optically detectable label, such as a fluorescent tag, so that the hybridized targets and probes are detectable with scanning equipment. DNA array technology offers the potential of using a multitude (e.g., hundreds of thousands) of different oligonucleotides to analyze changing mRNA populations.
Hybridization of DNA microarrays typically employs a target solution having a suitable buffer. Such buffers often include anionic detergents such as sodium dodecyl sulfate, lithium lauryl sulfate, N-lauryl sarcoside, or non-ionic detergents such as Tween 20® or Triton X-102®. Known hybridization buffers provide at least some wetting and flow characteristics. Wetting and flow characteristics of the buffer, the target solution, or combinations thereof, can be significant when, for example, the DNA microarray slides that are used are hydrophobic. Appropriate flow characteristics can also be advantageous when, for example, it is desirable to use mixing or agitation to help distribute the target solution uniformly over the entire array surface over time. Such mixing can influence the hybridization kinetics and thermodynamics.
One way uniform mixing can be accomplished is by incorporating a bubble in the hybridization chamber along with the buffered target solution. Rotation of the hybridization chamber causes the bubble to move around the perimeter of the chamber, and aids in mixing the bulk solution. Due to the hydrophobic nature of some DNA microarray slides, their resistance to being wetted, their tendency to become dewetted once wet, and other factors, movement of this bubble can be problematic. For example, bubbles can form on the surface of the slide that are resistant to movement that can result in non-uniform exposure of the array to the target solution. In some instances “bubble scars” or defects can be observed where hybridization occurs very poorly or not at all due to non-uniform exposure of the array to the target solution. Other hybridization methods involving mixing do not use bubbles. However, appropriate flow and wetting characteristics can play a significant role in these methods as well.
The problem of sporadic or poor hybridization assay performance can be characterized by, for example, low-intensity or missing features on the microarray substrate, high backgrounds, and visually “blotchy” substrates. This problem can be observed using conventional hybridization conditions at hybridization temperatures of, for example, about 66° C. and a hybridization time of about 14 to 18 hours. The poor performance characteristics can be observed in as little as 6 hours of incubation time at high temperature in conventional buffer solutions.
Thus, there remains a need for materials, conditions and methods for hybridizing surfaces, such as DNA microarrays on siliceous substrates, that have improved hybridization assay performance and uniformity.