Chemistry: analytical and immunological testing – Including sample preparation – Liberation or purification of sample or separation of...
Reexamination Certificate
1998-12-02
2002-03-19
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Liberation or purification of sample or separation of...
C436S174000, C359S368000, C422S091000, C422S105000, C435S286200, C435S309100
Reexamination Certificate
active
06358749
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to an automated system for microdissection of a sample such as chromosomes or other biological material, and in particular, it relates to a robotic assisted microdissection system and method that significantly reduces the time and skill needed for cellular and sub-cellular dissections.
BACKGROUND OF THE INVENTION
Conventional surgical microdissection involves miniature tools performing cellular level dissection under a microscope. This can be an extremely tedious process. Miniature tools generally need to be fabricated in the laboratory by the researcher or technician using a micro-forge. The tools are then placed into the clamp of a micro-manipulator and moved about within the microscope's field of view. The micro-manipulator scales the operator's motion by some factor such as ten to one range. Even with the scaled movement of the micromanipulator, a great deal of practice and skill are needed to perform delicate operations such as chromosome dissection or in-vitro fertilization. Because microdissection is so physically demanding, it is a very low yield process (e.g., 4 per day), requiring frequent breaks from the work. In some situations large volumes of micro-dissected material are needed for significant (anywhere from 4 to 100) sampling to occur. For example, chromosome microdissection of a specific gene locus requires from 10 samples, up to 100 samples in order to gather enough material for subsequent processes. It can take up to a month for 100 microdissection samples to be taken. Manually positioning a micro-manipulator is a tedious process. A free standing micro-manipulator must be positioned by the operator sliding and bumping and tapping the base in an attempt to get the micro-tool point within the microscope's field of view. If a new micro tool is used after each operation, the sliding, bumping and tapping process must be repeated each time.
Prior to the use of PCR in micro cloning, the construction of microdissection libraries had several drawbacks; e.g. the size of each such library was generally small; e.g., such a library contained at most only a few hundred clones of the microdissected material. However, for a dissected region of 10-20 mb, many more micro clones may be required to adequately generate libraries of the dissected region. Furthermore, since unstained and unbanded chromosome preparations were preferably used in microdissection, the identification of individual chromosomes and the chromosome bands was difficult and less accurate. Although suitable translocation stocks in mouse or human/rodent cell hybrids could be used, the prospect of a wider application to many other genomes was severely curtailed. Finally, the need to dissect several hundred chromosome segments for micro cloning would discourage investigators to use microdissection as a general method.
Focused UV laser microbeams have been used in conjunction with microdissection (e.g. Ponelies et al., 1989). In this procedure, all unwanted nuclei and chromosome regions on the slide are destroyed by a laser beam, leaving only the needed region intact which is then picked up and transferred to an Eppendorf tube for PCR.
Various other micromanipulating devices are known that utilize various means for manipulating material. For example, Parvin et al., U.S. Pat. No. 5,671,086 uses electrophoresis wherein electrodes are coupled to the stage of a microscope in an array whereby the electrode array allows for distinct manipulations of the electric field for accurate manipulations of an object. Kleindiek, U.S. Pat. No. 5,568,004 utilizes an electromechanical positioning device that is moved for course and fine adjustment by the operator when viewing a subject through a microscope, preferably using a piezo-tube connected to variable voltages to cause movements of a tip means. Higuchi et al., U.S. Pat. No. 5,225,750 disclose a micro-injection apparatus utilizing a micromovement device which relies upon a piezo electric/electro strictive element, which, in response to an electrical signal, causes a micromovement of a microsyringe.
Currently, the major obstacle to more widespread adoption of surgical microdissection technology is the extreme difficulty of the actual chromosome microdissection itself. This procedure takes a very experienced and skilled cytogeneticist. It requires continuous practice and a very significant amount of training. Surgical microdissection is also slow compared to possible automated procedures. It requires extreme care with regard to contamination. Finally, the preparation of the micro-tools requires for the procedure is also currently imprecise and requires an experienced individual. Only a few laboratories have the necessary resources and demand for the technique to have established it themselves.
SUMMARY OF THE INVENTION
Molecular analysis and high resolution physical mapping of specific genomic regions in any organism require large numbers of probes from the region of interest. A direct approach is to use microdissection techniques to physically remove the critical region followed by a micro cloning procedure to construct a region-specific library. These region-specific libraries are useful in a wide variety of studies, particularly for high resolution genome analysis and to facilitate positional cloning of important disease genes mapped to a specific region. Some examples of applications include: (i) isolation of large insert clones, like Pl, for contig assembly to cover a given genomic region; (ii) isolation of highly poly-morphic markers for linkage analysis; (iii) isolation of large numbers of single-copy micro clones as STSs for high density coverage of the region, (iv) isolation of region-specific CDNA clones as candidate genes for positional cloning, and (v) serving as chromosome painting probes to analyze complex chromosomal abnormalities.
The present invention is directed to an automated system to carry out microdissection for use by scientists who are not well equipped in genome analysis but are anxious to clone disease genes as their primary interest. This system is useful in laboratories for production of both microdissection painting probes for specific regions of the human or any other genome, including the genomes of plants, mice, and other model organisms currently of interest, e.g., aradopsis and Rugu rubripes (the Japanese pufferfish). The reliability and ease of operation of the automated system makes it widely available to laboratories that occasionally need microdissection technology but are not in a position to establish the technology as it currently stands due to the extremely demanding technical aspects of the conventional procedure.
Before the completion of the sequencing project for the entire human genome, there will be continuing needs for region-specific libraries and clones in specific regions for contig construction, refined physical mapping, linkage analysis, candidate gene isolation, etc. , all of which are essential to a successful cloning of a critical disease gene.
The automated system of the present invention greatly simplifies other microdissection procedures, for example microdissection of tumor specimens to separate the tumor cells from the normal surrounding tissue, and in vitro fertilization. Therefore, the automated system described herein has wide biomedical application. The present invention provides a device and method that makes it possible to construct microdissection libraries efficiently with high quality.
One aspect of the present invention relates to a method for removing select portions of a chromosome comprising calibrating a microdissection workstation by determining the position of a micro-tool using at least two CCD cameras and recording a zero position for the micro-tool. The micro-tool is then moved to a desired microscope objective (e.g., the crosshairs of the microscope) and the position of the micro-tool in such location is recorded. The micro-manipulator then releases the first micro-tool and selects a second micro-tool. Using the CCD cameras, th
Ludlow Jan
Ozo Diversified Automation, Inc.
Sheridan & Ross P.C.
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