Polynucleotide capture materials, and methods of using same
AssigneeHandyLab, Inc.
InventorsSundaresh N. Brahmasandra, Elizabeth Craig
Life of this patent
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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 polyamidoamine (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.
Claims19
A method for isolating polynucleotides in a cell-containing sample from a polymerase chain reaction inhibitor selected from the group consisting of hemoglobin, peptides, faecal compounds, humic acids, mucousal compounds, DNA binding proteins, and saccharides, or any combination thereof, the method comprising:
- contacting the sample with a lysis solution containing a lysis reagent, said lysis reagent comprising a detergent, and a plurality of binding particles, wherein the binding particles comprise PAMAM(Generation 0) covalently bound to the surface of the binding particles, said lysis solution having a pH between 4 and 8;
- heating the sample contacted with the lysis reagent, thereby lysing cells contained in the sample in the lysis solution and concomitantly binding the polynucleotides to the PAMAM of the binding particles to create binding particles bound with polynucleotides and a remaining residual solution;
- compacting the binding particles bound with polynucleotides by bringing the binding particles bound with polynucleotides in contact with one another;
- removing a substantial portion of the residual solution;
- and
- contacting the compacted binding particles with a release solution having a pH of ≧9, thereby releasing the polynucleotides from the PAMAM of the binding particles.
A method for concentrating RNA from a sample containing polymerase chain reaction inhibitors, the method comprising:
- contacting between 500 μl and 1 ml of the sample with a plurality of RNA binding particles, wherein the binding particles retain the RNA in the sample as compared to the polymerase chain reaction inhibitors;
- magnetically compacting the plurality of binding particles having the one or more polynucleotides bound thereto into an effective volume between 50 nanoliters and 5 microliters;
- and
- releasing the one or more polynucleotides into <30 μl of a release solution comprising a buffered solution having a pH≧9.
A composition comprising:
- carboxyl modified microparticles;
- and
- PAMAM(Generation 0) covalently bound via one or more amine groups per molecule to one or more of the carboxylic acid groups on the microparticles.
Dependencies are inferred from the claim text.