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Method for using aqueous two-phase system for the isolation, purification and/or concentration of short nucleic acid fragments

A nucleic acid fragment and aqueous two-phase technology, which is applied in biochemical equipment and methods, analytical materials, organic chemistry, etc., can solve unsatisfactory problems

Active Publication Date: 2020-06-30
相达生物科技国际有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The need to prepare biological samples specifically for the isolation, purification, and concentration of nucleic acid fragments of 250bp or less is important in both research and industry, but is largely unsatisfactory with existing methods
Although various methods have been described to achieve species separation in aqueous systems using different chemical formulations, no successful attempts have been made to exploit this possibility for separation, purification and concentration in a time-effective, cost-effective, simple and rapid manner Nucleic acid fragments of 250bp or less and provide excellent yields, which the present invention has demonstrated to have achieved

Method used

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  • Method for using aqueous two-phase system for the isolation, purification and/or concentration of short nucleic acid fragments
  • Method for using aqueous two-phase system for the isolation, purification and/or concentration of short nucleic acid fragments
  • Method for using aqueous two-phase system for the isolation, purification and/or concentration of short nucleic acid fragments

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0116] Embodiment 1-use two-phase system to selectively separate and concentrate short-chain nucleic acid fragments (

[0117] A DNA ladder (GeneRuler 1kb plus DNA Ladder, Thermo Fisher Scientific) was added to 1 mL of 11% (w / w) polyethylene glycol (PEG) 6000 and 20% (w / w) K 2 HPO 4 In an aqueous two-phase system composed of PBS solution, the final DNA concentration was 1 μg / mL. After thorough vortexing, the mixture was centrifuged at 10,000 rcf for 10 s for phase separation. The volume ratio of the top phase to the bottom phase is about 1:3. Extract the top and bottom phases and transfer to new tubes separately. The extracted phases were subjected to ethanol precipitation and the precipitates were separated by gel electrophoresis in order to visualize the DNA size distribution in each phase as image 3 shown. Most nucleic acids larger than 250 bp are assigned to the bottom phase (right lane), while nucleic acids smaller than 250 bp are assigned to the top phase (left l...

Embodiment 2

[0121] Example 2 - Selective separation and concentration of short-chain nucleic acid fragments (

[0122] A DNA ladder (GeneRuler 1 kb plus DNA Ladder, Thermo Fisher Scientific) was added to 500 ul plasma samples. Add the standard-added plasma sample to 500 μl of 15% (w / w) polyethylene glycol (PEG) 1000 and 15% (w / w) K 2 HPO 4 In a two-phase aqueous system composed of MilliQ aqueous solution, the final DNA concentration was 1 μg / mL. After thorough vortexing, the mixture was centrifuged at 10,000 rcf for 10 s for phase separation. The volume ratio of the top phase to the bottom phase is approximately 1:1.

[0123] Extract the bottom phase and add another compound consisting of 11% (w / w) polyethylene glycol (PEG) 6000 and 20% (w / w) K 2 HPO 4 Composition of ATPS solution. After thorough vortexing, the mixture was centrifuged at 10,000 rcf for 10 s for phase separation. The volume ratio of the top phase to the bottom phase is about 1:3. Extract the top and bottom phases...

Embodiment 3

[0125] Example 3 - Comparison of the two-phase aqueous system with the QIAamp Blood DNA mini kit (Qiagen)

[0126] Digested DNA plasmids of different sizes (250, 200, 150, 100, 75, 50, 25 bp) were added to plasma samples to give a final DNA concentration of 100 ng / mL. Add 1 mL of the resulting standard-spiked plasma sample to 1 mL of 15% (w / w) polyethylene glycol (PEG) 1000 and 15% (w / w) K 2 HPO 4 A two-phase aqueous system consisting of MilliQ aqueous solution. After thorough vortexing, the mixture was centrifuged at 10,000 rcf for 10 s for phase separation. The volume ratio of the top phase to the bottom phase is approximately 1:1.

[0127] Extract the bottom phase and add another compound consisting of 8% (w / w) polyethylene glycol (PEG) 6000 and 22% (w / w) K 2 HPO 4 Composition of ATPS solution. After thorough vortexing, the mixture was centrifuged at 10,000 rcf for 10 s for phase separation. The volume ratio of the top phase to the bottom phase is about 1:5. Extra...

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Abstract

The present invention relates to methods of using aqueous two-phase system (ATPS) for the isolation, concentration and / or purification of short nucleic acid fragment having about or less than 250 basepairs (bp). In one embodiment, the present invention provides a composition and kit for the purification of short nucleic acid fragments having about or less than 250 base pairs from nucleic acid-containing biological materials. In another embodiment, the present invention provides uses of certain salts and / or polymers in a two-phase system for the purification of short nucleic acid fragments having about or less than 250 base pairs from nucleic acid containingbiological materials.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Serial No. 62 / 560,180, filed September 18, 2017, the entire contents and disclosure of which is incorporated into this application by reference. technical field [0003] The present invention relates to a method for separating, concentrating and / or purifying short-chain nucleic acid fragments in an aqueous two-phase system (ATPS). In particular, the present invention provides a method, a kit, and ATPS components for isolating, concentrating and / or purifying short-chain nucleic acid fragments from biological material. This application cites various publications, the entire contents of which are hereby incorporated by reference. Background technique [0004] Since the discovery of the polymerase chain reaction, or PCR for short, in 1983, whole genomes have been sequenced, enabling numerous discoveries in basic research in the life sciences and key medical developments. In order...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6809G01N33/48G01N33/53G01N33/543
CPCC12N15/1003C07H21/04C12N15/1006
Inventor 招彦焘
Owner 相达生物科技国际有限公司