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Compositions, methods, and devices for isolating biological materials

A composition and microbial technology, applied in biochemical equipment and methods, analytical materials, microbial measurement/inspection, etc., can solve problems such as unbound double-stranded DNA

Inactive Publication Date: 2010-03-17
3M INNOVATIVE PROPERTIES CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, dsDNA has been found not to bind to the column matrix

Method used

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  • Compositions, methods, and devices for isolating biological materials
  • Compositions, methods, and devices for isolating biological materials
  • Compositions, methods, and devices for isolating biological materials

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0330] Example 1: Preparation of metal ion-mediated magnetic particles

[0331]Metal ion-mediated magnetic microparticles (used as immobilized metal support materials) were obtained from magnetic particles with a diameter of about 1 μm with surface carboxylic acid groups (DYNABEADS MYONE carboxylic acid, available from Invitrogen, Carlsbad, CA) or from Thermo Scientific (called Prepared for SERA-MAG magnetic particles of Seradyn, Indianapolis, IN). Carboxylated magnetic particles are placed in a test tube, magnets are used to attract them to the tube wall for washing, liquid is removed by aspiration, the liquid space is replaced with wash solution, the tube is removed from the magnetic field, and the tube is agitated to resuspend the particles.

[0332] Before the metal ion treatment, the magnetic microparticles were washed twice with 0.1 M MES buffer (containing 0.1% TRITON X-100) at pH 5.5, and then resuspended in the same buffer. After washing, 0.2 mL per mg of magnetic ...

example 2

[0333] Example 2: Comparison of Metal Ions for DNA Capture and Release

[0334] In this experiment, 40 μg of Ga(III)-microparticle-1 and 40 μg of Fe(III)-microparticle-1 from Example 1 were used in a split experiment to combine 10 5 cfu equivalent of MRSA DNA (about 1.8 ng). The supernatant was named SN0. Then, the microparticles were washed twice with MES buffer, and the supernatants of each time were collected (named SN1 and SN2, respectively). To elute bound DNA, resuspend the microparticles in 20 mM sodium phosphate buffer (PO 4 , pH 8.5), and heated to 95°C for 5 minutes. The supernatant (designated SN3) was collected for mecA-FAM RT-PCR analysis.

[0335] Real-time PCR amplification of the mecA gene was performed on 5 microliters of each sample (SN3) using the following optimal concentrations of primers, probes and enzymes and thermocycling. The sequences of all primers and probes listed below are given in 5'→3' orientation and are known and described in Francois,...

example 3

[0340] Example 3: Quantification of DNA binding and elution efficiency by PicoGreen assay

[0341] PicoGreen is a common method for quantifying dsDNA in solution (Nakagawa et al., Biotech & Bioeng. 2006, 94(5), 862-868). Lambda DNA was chosen as a model to demonstrate capture and release efficiencies. Lambda DNA from the PicoGreen assay kit (Invitrogen, Carlsbad, CA) was diluted two-fold from 8 μg / mL to 0.25 μg / mL in 1×TE buffer (10 mM Tris-HCl, pH 8.0). 100 µL of each DNA solution was added to 100 µL of 0.1 M MES buffer (pH 5.5) containing 400 µg Ga(III)-microparticle-2, followed by thorough mixing for 10 minutes. Microparticles were then washed twice with MES buffer. 100 μl of 20 mM sodium phosphate buffer (pH 8.5) was added and the suspension was heated at 65° C. for 5 minutes to release the DNA from the microparticles.

[0342] In another experiment, DNA was first denatured at 95°C for 5 min and then immediately placed on ice to generate single-stranded DNA. Single-s...

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Abstract

Compositions, methods, devices, and kits, which include an immobilized-metal support material comprising a substrate having a plurality of -C(O)O<-> or -P(O)(-OH)2-x(-O<->)x groups bound to the substrate and a plurality of metal ions, M<y+>, bound to the -C(O)O<-> or -P(O)(-OH)2-x(-O<->)x groups; wherein M is selected from the group consisting of zirconium, gallium, iron, aluminum, scandium, titanium, vanadium, yttrium, and a lanthanide; y is an integer from 3 to 6; and x is 1 or 2, and to which microorganisms and polynucleotides bind, and which can be used for separating and optionally assaying microorganisms and / or a polynucleotide from a sample material are disclosed.

Description

[0001] This application claims priority to US Provisional Application No. 60 / 913,812, filed April 25, 2007, which is hereby incorporated by reference in its entirety. Background technique [0002] When performing methods of detecting or analyzing biological material, it is useful or even necessary to separate biological material (eg, cells, viruses, and polynucleotides) from a sample. In some methods, microorganisms are isolated from a sample, and enumerative or non-enumerative methods are used to determine the total number of microorganisms or to identify at least some of the microorganisms. Enumerated methods used are (for example) standard plate counts, Escherichia coli, yeast and mold counts, bioluminescent assays and impedance or conductivity measurements; non-enumerated methods used are (for example) selective differential Adhesion method, DNA hybridization method, agglutination method and enzyme immunoassay method. Identification of polynucleotides or portions of polyn...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/24C12Q1/68
CPCC12N15/1006G01N33/569C12Q1/24C12Q1/6806
Inventor 夏文生保罗·N·霍尔特拉尼亚宁·V·帕塔萨拉蒂曼基里·T·克什尔萨格尔
Owner 3M INNOVATIVE PROPERTIES CO