System and method for manipulating magnetically responsive...

Classifying – separating – and assorting solids – Magnetic – Paramagnetic

Reexamination Certificate

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C210S222000, C210S695000, C073S061420, C422S105000, C436S526000

Reexamination Certificate

active

06672458

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a system and method for manipulating magnetic particles in a fluid sample to efficiently and effectively collect DNA or RNA that has been bound to the particles. More particularly, the present invention relates to a system and method employing movable magnets for holding and releasing magnetic particles in a fluid sample so that DNA or RNA bound to the magnetic particles can be separated from the fluid sample.
2. Description of the Related Art
A variety of molecular biology methodologies, such as nucleic acid sequencing, direct detection of particular nucleic acids sequences by nucleic acid hybridization, and nucleic acid sequence amplification techniques, require that the nucleic acids (DNA or RNA) be separated from the remaining cellular components. This process generally includes the steps of collecting the cells in a sample tube and lysing the cells with heat and reagent which causes the cells to burst and release the nucleic acids (DNA or RNA) into the solution in the tube. The tube is then placed in a centrifuge, and the sample is spun down so that the various components of the cells are separated into density layers within the tube. The layer of the nucleic acids can be removed from the sample by a pipette or any suitable instrument. The samples can then be washed and treated with appropriate reagents, such as fluorescein probes, so that the nucleic acids can be detected in an apparatus such as the BDProbeTec™ ET system, manufactured by Becton Dickinson and Company and described in U.S. Pat. No. 6,043,880 to Andrews et al., the entire contents of which is incorporated herein by reference. Although the existing techniques for separating nucleic acids from cell samples may be generally suitable, such methods are typically time consuming and complex. Furthermore, although the centrifuging process is generally effective in separating the nucleic acids from the other cell components, certain impurities having the same or similar density as the nucleic acids can also be collected in the nucleic acid layer, and must be removed from the cell sample with the nucleic acids.
A technique has recently been developed which is capable of more effectively separating nucleic acids from the remaining components of cells. This technique involves the use of paramagnetic particles, and is described in U.S. Pat. No. 5,973,138 to Mathew P. Collis, the entire contents of which is incorporated herein by reference.
In this technique, paramagnetic particles are placed in a buffer solution along with cell samples. After the cell samples are lysed to release the nucleic acids, a acidic solution is mixed with the particles and the nucleic acids are reversibly bound to the paramagnetic particles. The paramagnetic particles can then be separated from the remainder of the solution by known techniques such as centrifugation, filtering or magnetic force. The paramagnetic particles to which the nucleic acids are bound can then be removed from the solution and placed in an appropriate buffer solution, which causes the nucleic acids to become unbound from the magnetic particles. The paramagnetic particles can then be separated from the nucleic acids by any of the techniques described above.
Examples of systems and method for manipulating magnetic particles are described in U.S. Pat. Nos. 3,988,240, 4,895,650, 4,936,687, 5,681,478, 5,804,067 and 5,567,326, in European Patent Application No. EP905520A1, and in published PCT Application WO 96/09550, the entire contents of each of said documents being incorporated herein by reference.
Although the mangnetic or paramagnetic particle manipulating techniques can be effective in separating and harvesting nucleic acids from cell samples, a need exists for an improved technique for manipulating the magnetic or paramagnetic particles to provide an even more effective method of separation.
SUMMARY OF THE INVENTION
An object of the present invention is to provide an improved system and method for manipulating magnetically responsive particles, such as iron oxide particles, magnetic, ferromagnetic or paramagnetic particles, or any other particles that are responsive to a magnetic field, to which nucleic acid molecules are bound in a solution to effectively separate the nucleic acid molecules from the remaining components of the solution.
A further object of the present invention is to provide a system and method that is capable of altering the temperature of a cell solution to perform a lysing technique which enables nucleic acid molecules to become bound to magnetically responsive particles in the solution, as well as being capable of manipulating the magnetically responsive particles to appropriately separate the nucleic acid molecules from the remaining components of the solution.
A further object of the present invention is to provide a system and method for use in a nucleic acid assay preparation system, that is capable of heating and cooling sample solutions as appropriate to perform a lysing technique, and which is further capable of manipulating magnetically responsive particles to which nucleic acid molecules of the lysed cell samples become bound, so that the assay preparation system can properly wash the nucleic acid molecules and place the nucleic acid molecules in a sample assay.
These and other objects are substantially achieved by providing a system and method for manipulating nucleic acid molecule-bound magnetically responsive particles in a sample solution to separate the molecules from the remaining components in the solution. The system and method includes a tube receiver for receiving at least one sample tube containing a cell solution, magnetically responsive particles such as iron oxide particles, and an acidic solution. The tube receiver is adapted for use with a system for preparing nucleic acid assays. The tube receiver includes a heating and cooling unit, such as a thermoelectric element, which is capable of heating the cell solution to lyse the cell and enable the nucleic acid molecules to become bound to the magnetically responsive particles. The thermoelectric elements can also be used to quickly cool the solution as necessary. The tube receiver further includes movable magnets which can be moved proximate to the outer wall of the tubes to attract the molecule-bound magnetically responsive particles to the sides of the tubes, while the assay preparation system removes the remainder of the cell solution and washes the particles. The movable magnets can then be moved away from the tubes so that the molecule-bound magnetically responsive particles are released from the walls of the tubes, so that the assay preparation system can eject an elution reagent, such as a suitable buffer solution, which causes the nucleic acid molecules to become unbound from the magnetically responsive particles. The tube receiver further includes electromagnets which are activated to provide an alternating magnetic field to the tubes to degauss the magnetically responsive particles to allow the magnetically responsive particles to mix with the elution reagent. The movable magnets can then be moved proximate to the sample tubes to adhere the magnetically responsive particles to the walls of the sample tubes while the assay preparation system aspirates the nucleic acid molecules from the sample tubes. The assay preparation system can then place the nucleic acid molecules in the appropriate microtiter trays for reading by an assay reading system.


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