Sandwich hybridization assay - SHA
The following text and the SHA animation (link below) illustrate the basics of sandwich hybridization assay, as performed by the ESP. It may be helpful to pause the animation at each step to read the associated text.
Animation of sandwich hybridization assay
(2.5 Mb quicktime movie opens in a separate window)
Steps in the SHA process:
- At the beginning, the capture probe—part of the printed probe array—is shown at lower left. It consists of a DNA probe (vertical green lines) chemically linked to biotin and mixed with the protein, streptdavidin (red dot), which binds the whole to the surface of the filter support (yellow line).
- The ESP collects a sample of water, concentrates the (particulate) organisms contained in the sample, and creates a nucleic acid extract. That extract is pumped into the puck, washing over the probe array. If the target rRNA is present (wavy white lines), it sticks to the capture probe while the rest of the extract sample is washed away. The sample extract is usually full of junk material, but the probes are specific, and very fine discrimination is possible.
- The next solution washed across the array substrate contains another probe (orange lines) —the digital signal probe—which attaches to a different region of the target molecule. This completes the "sandwich"—probe/target/probe.
- The signal probe generates a signal in the form of light, which is then recorded by a camera.
- The image is transmitted to shore via radio modem where it is processed to determine what sequences were detected and how intense the reaction was. The intensity of the light recorded is directly proportional to the amount of the target that is present. (Brighter spots indicate more target.)
Later, on shore, the assay can be verified by a matching 96-well bench run. In the bench-top assay, the presence of a target rRNA is indicated by a color change in the wells.
The advantage of this system is that sequences are captured directly from the homogenate (extract), which is not manipulated in any way. No purification is required, and the reagents are stable. This isn't the optimum method for desired results, but it was the best way to demonstrate a solution to doing this kind of chemistry on site. New, and more sophisticated, procedures are currently being developed.
Below is a static view of the complete process illustration.