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Publication Date: 2015-Jun-25
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The Prior Art Database

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Isolation and purification of polynucleotides is an important aspect to a wide variety of molecular biology methodologies. Examples of such polynucleotides include double-stranded plasmid DNA, single-stranded phage DNA, chromosomal DNA, agarose gel-purified DNA fragments, RNA, aptamers, triplex forming agents, external guide sequences, catalytic oligomers, and ribozymes. Depending on their source, polynucleotides are typically found with significant quantities of contaminants from which they must be isolated or separated before being used in a desired molecular biological procedure.

Various methodologies have been developed for the purification of polynucleotides from contaminants. For example, methods have been developed that allow for the isolation of nucleic acids and fragments thereof using glass, silica, zeolite, etc., in the forms of filters, columns, or magnetically responsive particles to expedite separations (see, e.g., U.S. Pat. No. 5,075,430 by Little; U.S. Pat. No. 5,234,809 by Boom, et al.; U.S. Pat. No. 6,027,945 by Smith, et al.). Variously functionalized magnetic particles have also been reported (see, e.g., U.S. Pat. Nos. 5,705,628 and 5,898,071 by Hawkins; U.S. Pat. No. 7,378,035 by Margetts).

While existing technologies have varied utilities, improved methods and compositions for purifying nucleic acids that are rapid, simple to use, selective, and require little, if any, additional sample manipulation remains a challenging objective.  The experiments described below show improved performance in polynucleotide isolation using superparamagentic covalently derivitized with polyacrylic acid.

Comparison of PCR Product Purification: AMPure/Polybrene Magnetic Beads versus LodeStar/Polyquaternium-7 Magnetic Beads


Two sets of functionalized superparamagentic bead compositions were produced and compared in a method for purifying PCR products (double-stranded DNA). The PCR products ranged in size from 150 base pairs to 2.2 kilobases (kb). The first set of beads were AMPure beads (AP; Agencourt – Beckman-Coulter; AMPure beads are derivatized with non-polymeric carboxyl groups) modified with polybrene (as described in PCT application publication WO2007/136717) and the second set of beads were LodeStar 2.7 μm Carboxyl magnetic beads (LS; Agilent Technologies Inc.; LS beads are covalently derivitized with polyacrylic acid) modified with MIRAPOL® 550 (polyquaternium-7).

The AP and LS beads were functionalized with polybrene and MIRAPOL® 550, respectively, as follows.

AP or LS bead slurry (225 µL) were placed into respective microfuge tubes and the beads were collected on the side of the tubes by placing them on a magnetic stand.  After the solution phase (supernatant) was removed from each tube, the tubes were removed from the magnetic stand.  One (1) mL of 1% aqueous polybrene was added to the AP beads and 1 mL of 1% aqueous MIRA...