Chemistry: analytical and immunological testing – Automated chemical analysis – With sample on test slide
Reexamination Certificate
1999-09-03
2002-05-28
Bhat, Nina (Department: 1761)
Chemistry: analytical and immunological testing
Automated chemical analysis
With sample on test slide
C436S048000, C422S065000, C422S067000, C435S289100
Reexamination Certificate
active
06395554
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to an apparatus and method for use in a slide loading/unloading system and in particular, to a microarray loading/unloading system.
BACKGROUND
Deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and proteins are complex molecules that are integral to every living organism. DNA contains information required to define a structure and process of a cell. RNA transfers that information by becoming a template for protein synthesis and enabling protein synthesis process. Proteins initiate and control all functions within a cell. Because of the fundamental nature of these molecules to biology, researchers have developed methods of experimenting and characterizing their structure. One method that is commonly used is called “hybridization”.
Hybridization takes advantage of the complimentary nature of RNA and DNA to characterize their sequence. Typically, a reference strand of DNA (the “target”) is bound to a substrate. One or more types of DNA under test (the “probes”) are labeled with either radioactive or fluorescence tags. The probes are then mixed with the target. Probe molecules with a sequence similar that of to the target will bind or hybridize to the target molecules. Dissimilar probe molecules will not bind and be washed away in a subsequent rinsing process. By measuring the quantity of bound probe molecules, a researcher can determine the likeness between the probe and target. This method is used to measure a variety of biological characteristics including gene expression, genotype, and gene sequence.
Hybridization experiments are normally conducted in large quantities in order to be generally useful. There are approximately 100,000 genes in the human genome, several thousand of which are considered in a typical study. Technologies to allow for massive parallel hybridization experiments have been developed.
One such technology is the microarray. A microarray is a substrate, typically a one-by-three inch glass slide, that is “spotted” with an array of reference target genes, typically in the form of DNA. Several thousand to several tens of thousands of genes (or partial genes) in the order of 100-114 microns in diameter are generally spotted onto a typical microarray. This allows a researcher to compare the probe DNA to many targets simultaneously. The result is the ability to characterize the gene profile of a tissue or cell type under a specified condition.
Spotting is accomplished by using an instrument called an “arrayer”. A typical arrayer is a robot that can spot 40 to 100 microarrays in an automated fashion. Arrayers are usually kept in an environment in which humidity is controlled, since it affects the rate of evaporation of the solution to be spotted. This is particularly important where there is significant evaporation before the solution is transferred to the substrate. Cleanliness will directly affect the quality of any microarray because the information gleaned from the microarray is the image captured at the surface of the substrate. Any artifact, such as dust or fingerprints, will degrade the fidelity of the microarray. Thus, microarrays are manually loaded into and removed from the arrayer which may affect the quality of the spotted microarray.
The next step of the microarray process is to introduce the probe DNA. The DNA is mixed in a buffer solution to enable its transfer. A small amount of probe solution is placed on the surface of the microarray. A thin piece of glass is then used to sandwich the probe with the microarray causing the probe to spread across the region that contains the target. The probes are typically labeled with fluorescence tags. The fluors convert incident light, referred to as “excitation light”, into fluoresced light, referred to as “emitted light”. However, the fluors are generally susceptible to damage caused by ambient light and thus, such light should be avoided. Excitation caused by ambient light degrades the efficiency of fluors prior to scanning with an imager. This damage is called photobleaching.
Next, the target and probe are hybridized. This is accomplished by putting the microarray into a humid, thermally controlled environment where it is “baked”. During this stage, the target and probe molecules with similar structures bind. After hybridization, the thin piece of glass is removed and the microarray is rinsed to wash away the non-binding probe DNA. At this point, the microarray is susceptible to damage from dirt, heat and light. Image degradation caused by contamination from handling continues to be a problem. Heat can cause the probe and target to become disassociated. Further, the fluorescent labels on the probe DNA are susceptible to photobleaching.
Finally, the microarray is imaged. The substrate is loaded into an microarray imager which excites the fluors and senses the emitted light. The resulting image is analyzed to determine the density of probe DNA that hybridized to each target DNA spot. This information is used to characterize the state and structure of the probe DNA. Any dirt or marks will contribute to fluorescence which is not related to DNA density. As a result, these artifacts will reduce the accuracy of the calculations.
After the process is complete, the microarrays are often saved for later inspection. They need to be properly stored if they are to be useful in later imaging.
If the procedure above is performed manually, the procedure will be costly and time consuming. A researcher or technician is required to handle each microarray at every step in the process. The imaging process is particularly labor intensive. Each slide is inserted by hand for single slide imaging while the user waits to load the next slide. The problem becomes acute for high volume users. Research facilities that process several hundred microarrays a day will need to hire several technicians just to keep up with the imaging.
There is a further problem with tracking microarrays. In a typical lab there are many avenues of research being pursued simultaneously by separate research groups. Since the microarrays are identical to the naked eye, they are difficult to sort manually. Even if they are labeled for tracking, the large number of microarrays being used will result in some confusion and errors during the process.
As described above, microarrays are prone to damage each time that they are handled. Everything ranging from dust to fingerprints cause image artifacts that pollute the final result. Statistically, when a large number of microarrays are processed, the amount of degradation will be proportional to the amount of handling of each slide.
Microarrays are susceptible to damage if they are not stored properly. Everything from dust to light can affect the data that is gleaned from the microarray.
There are automated microarray scanning systems available currently. These systems, however require the substrate to be placed in a metal “sub-frame” or “clip” prior to loading it into the storage mechanism. Both the substrate and frame are then located into the scanning field and subsequently scanned. This approach is forgiving of dimensional variability in substrates. Further, it provides for metal-on-metal wear surfaces, which provide for a more tolerant system design. However, as throughput demands increase, the sub-frame approach becomes limiting.
Typically, high-throughput microarray applications process batches of microarrays numbering from 10 to 100, and this is increasing. Sub-frames are less attractive to the user because of the added manual labor placing each microarray into a sub-frame. Additionally, the user needs to gather sufficient sub-frames to process a batch of microarrays. Sub-frames also increase the amount of space required by each microarray in a storage mechanism and may make a system built to handle 100 or more microarrays unmanageable to use.
Downstream microarray quantitation processes require that microarray be accurately placed in a scanning field repeatedly. Sub-frames have been shown to be inadequate in this respect.
SUMMARY
In accordance with the inve
Doane Sean R.
Gyorke, Jr. George
Milkowski Robert T.
Regan Donald T.
St. Cyr Paul L.
Bhat Nina
Cesari and McKenna LLP
Packard Instrument Company
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