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Conjugated polymer particles and methods for their preparation

A polymer particle and conjugation technology, applied in the field of conjugated polymer particles and its preparation, can solve the problems of high signal-to-noise ratio, low precision, low signal, etc.

Active Publication Date: 2018-02-09
LIFE TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such inefficiencies result in lower densities of conjugated polynucleotides or random regions lacking desired polynucleotides
Such inefficiencies can lead to weak separations, low precision in detection methods, and low signal or high signal-to-noise ratios in sequencing technologies

Method used

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  • Conjugated polymer particles and methods for their preparation
  • Conjugated polymer particles and methods for their preparation
  • Conjugated polymer particles and methods for their preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] like Figure 4 As shown in , oligonucleotides were directly conjugated to methanesulfonyl-activated Dynal particles. Although functional groups are shown as being on the surface of the particle, functional groups may be present throughout the particle. Methanesulfonyl chloride-activated microgels were prepared via the Ugelstad process, a seeded emulsion polymerization. The particles thus formed were washed in N-methyl-2-pyrrolidone (NMP) in formulation for conjugation with ion-exchanged single-stranded DNA.

[0075] The sodium salt of the 5'-NH2-C6-30-mer oligonucleotide was dissolved in 0.1 M tetrabutylammonium acetate and injected onto a reverse phase HPLC column. Elution was performed with a 0.1 M tetrabutylammonium acetate mobile phase. Fractions containing nucleic acid were pooled, lyophilized to a dry powder, and resuspended in pure N-methyl-2-pyrrolidone (NMP).

[0076] Put five million particles (5.0x10 9 ) was dispersed in 350 uL of anhydrous NMP and vorte...

Embodiment 2

[0080] like Figure 5 Particles were conjugated through a series of substitutions as shown in . Although functional groups are shown as being on the surface of the particle, functional groups may be present throughout the particle.

[0081] To a methanesulfonyl chloride-activated hydrogel solution in NMP (2 billion, 1 ml) in a 2 ml centrifuge tube, a saturated solution of tetramethylammonium azide in NMP (800 uL) was added, and the reaction mixture was Stir overnight at 60°C. The reaction mixture was centrifuged at 21300 rcf for 10 minutes and the supernatant was removed. The resulting pellet was resuspended in NMP and centrifuged to remove the supernatant. Repeat this process twice.

[0082] The resulting pellet was resuspended in deionized (DI) water (1 ml). The reaction mixture was centrifuged at 21300 rcf for 10 minutes and the supernatant was removed. This process was repeated 2 more times.

[0083] 1 ml of TCEP solution (DI water, 1 M) was added to the pellet, and...

Embodiment 3

[0087] Example 3 Activation of amino-hydrogels and conjugation of hydrogels to amine-terminal DNA probes.

[0088] Add solid disuccinyl to a solution (600 μL) of 100 million amino-hydrogels (diameter = 0.55 μm, 23 million amines / μm) in anhydrous, amine-free N-methylpyrrolidone (NMP). Amino suberate (22.1 mg, 60 μmol) followed by tributylamine (14 μL, 60 μmol). After stirring at 60°C for 1 hour, the hydrogel was isolated by centrifugation (30 minutes at 21300 rcf). The hydrogel pellet was diluted with amine-free anhydrous NMP (1 ml) and isolated by centrifugation; this washing process was repeated 2 times and the final pellet was resuspended in NMP (600 μL). The hydrogel suspension was treated with acetic anhydride (30 μL, 317 μmol) and tributylamine (30 μL, 126 μmol) and stirred at room temperature for 2 hours. The resulting hydrogel was separated by centrifugation (30 min at 21300 rcf) and the pellet was diluted with amine-free anhydrous NMP (1 ml) and separated by centrifu...

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Abstract

The invention discloses a method of conjugating a substrate comprising exchanging a counterion bound to a biomolecule with a lipophilic counterion to form a biomolecular complex, dispersing the biomolecular complex in a non-aqueous solvent, and The biomolecular complex is coupled to the substrate in the presence of an aqueous solvent. The substrate or solid support is a polymer particle, the biomolecule to be immobilized on the particle is an oligonucleotide, the lipophilic counterion is tetrabutylammonium ion, and the non-aqueous solvent is N-methylpyrrolidone (NMP). Oligonucleotide-coated particles are used in nucleic acid isolation or nucleic acid sequencing reactions.

Description

[0001] This patent application claims the benefit of US Provisional Application No. 61 / 597,064, filed February 9, 2012, which is hereby incorporated by reference in its entirety. technical field [0002] The present disclosure relates generally to methods of conjugating substrates and substrates formed by such methods. Background technique [0003] Conjugation of polynucleotides to substrates has become the focus of various industries. Substrates including conjugated polynucleotides are useful in separation techniques, genetic marker detection and sequencing. [0004] For example, substrates conjugated to polynucleotide probes can be used to capture genetic markers for detection. Exemplary genetic markers can be associated with disease variants within genes, disease-causing bacteria or viruses, or alleles that can be used for identification in humans. Substrates comprising conjugated probes complementary to genetic markers can capture such genetic markers, and a variety of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6834C12Q2523/101C12Q2563/149C12Q2565/119C12Q1/6813C08F8/40C12Q1/6876
Inventor S.曼钦A.布兰查德L.安德鲁兹S.坎D.格雷姆亚钦斯基A.卢伊C.斯托拉克泽克T.索科尔斯基
Owner LIFE TECH CORP
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