POLYNUCLEOTIDE CAPTURE MATERIALS, AND METHODS OF USING SAME
First Claim
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1. A method for isolating polynucleotides from a cell-containing sample in a process tube, the method comprising:
- contacting the sample in the process tube with a lysis solution having a pH between 4 and 8, thereby releasing polynucleotides from the cells in the cell-containing sample;
contacting the sample in the process tube with a plurality of magnetic binding particles coated in PAMAM, wherein the polynucleotides becomes reversibly bound to the PAMAM, thereby creating binding particles bound with polynucleotides and a solution containing residual cellular material in the process tube;
compacting the binding particles bound with the polynucleotides in the process tube by positioning a magnet in communication with the binding particles;
removing the solution containing residual cellular material from the process tube;
washing the binding particles with a solution having a pH <
9, thereby retaining the polynucleotides bound to the binding particles ; and
contacting the binding particles bound with polynucleotides with a solution having a pH≧
9 to release the polynucleotides from the PAMAM on the binding particles, thereby isolating the polynucleotides from the cell-containing sample.
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Abstract
Methods for processing polynucleotide-containing biological samples, and materials for capturing polynucleotide molecules such as RNA and/or DNA from such samples. The RNA and/or DNA is captured by polyamindoamine (PAMAM (Generation 0)) bound to a surface, such as the surface of magnetic particles. The methods and materials have high efficiency of binding RNA and of DNA, and of release, and thereby permit quantitative determinations.
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Citations
12 Claims
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1. A method for isolating polynucleotides from a cell-containing sample in a process tube, the method comprising:
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contacting the sample in the process tube with a lysis solution having a pH between 4 and 8, thereby releasing polynucleotides from the cells in the cell-containing sample; contacting the sample in the process tube with a plurality of magnetic binding particles coated in PAMAM, wherein the polynucleotides becomes reversibly bound to the PAMAM, thereby creating binding particles bound with polynucleotides and a solution containing residual cellular material in the process tube; compacting the binding particles bound with the polynucleotides in the process tube by positioning a magnet in communication with the binding particles; removing the solution containing residual cellular material from the process tube; washing the binding particles with a solution having a pH <
9, thereby retaining the polynucleotides bound to the binding particles ; andcontacting the binding particles bound with polynucleotides with a solution having a pH≧
9 to release the polynucleotides from the PAMAM on the binding particles, thereby isolating the polynucleotides from the cell-containing sample. - View Dependent Claims (2, 3, 4, 5, 6, 7)
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8. A method for separately isolating DNA and RNA from a cell-containing sample, the method comprising:
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contacting the sample with a lysis solution having a pH between 4 and 8, thereby releasing DNA and RNA from the cells in the cell-containing sample; contacting the sample with a plurality of binding particles, wherein the binding particles retain the DNA and RNA in the cell-containing sample; compacting the plurality of binding particles having the DNA and RNA bound thereto; releasing the RNA from the binding particles by contacting the compacted binding particles with a first release solution; removing the solution containing the released RNA, thereby isolating the RNA; and releasing the DNA from the binding particles by contacting the compacted binding particles with a second release solution, thereby isolating the DNA, wherein the pH of the second release solution is greater than the pH of the first release solution. - View Dependent Claims (9, 10, 11, 12)
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Specification