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Method for Using Aqueous Two-Phase System for the Isolation, Purification and/or Concentration of Short Nucleic Acid Fragments

Pending Publication Date: 2020-07-02
PHASE SCI INT LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention allows for easy and high-purity and concentration of target molecules without the need for additional purification or concentration steps. This is compatible with various downstream applications such as amplification, sequencing, labeling, and detection.

Problems solved by technology

Thus the extraction method for the isolation of cfDNA from biological samples can significantly affect the yield of cfDNA which may further affect characteristics of different tests and degrees of clinical validation.
The challenge here is to isolate, purify, and concentrate the very rare and sparse nucleic acid fragments against a complex background of biological matrix.
Often times the yield of relevant fragments is so low that subsequent PCR results may not have sufficient diagnostic sensitivity and specificity.
Both of the processes disclosed the use of ATPS for the plasmid DNA purification but none of them were designed for isolating nucleic acid with a length of 250 bp or below.
The need for preparing biological samples specifically for isolating, purifying, and concentrating nucleic acid fragments of 250 bp or smaller is very significant in both the arena of research and industry, but this need is largely unmet by existing methods.

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
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Effect test

example 1

Selective Isolation and Concentration of Short Nucleic Acid Fragments (<250 bp) from PBS Solution Using Aqueous Two-Phase System

[0115]The DNA ladder (GeneRuler 1 kb plus DNA Ladder, Thermo Fisher Scientific) was spiked into 1mL of aqueous two-phase system composed of 11% (w / w) polyethylene glycol (PEG) 6000 and 20% (w / w) K2HPO4 in PBS solution to make a final DNA concentration of 1 μg / mL. After vortexing thoroughly, the mixtures were centrifuged for 10 s at 10000 rcf for phase separation. The volume ratio of top phase to bottom phase was around 1:3. The top and bottom phases were extracted and transferred to new tubes respectively. The extracted phases were subjected to ethanol precipitation and the precipitates were separated by gel electrophoresis so as to visualize the DNA size partition in each phase, as shown in FIG. 3. Most of the nucleic acids larger than 250 bp partitioned into the bottom phase (right lane) while nucleic acids smaller than 250 bp partitioned into the top pha...

example 2

Selective Isolation and Concentration of Short Nucleic Acid Fragments (<250 bp) from Plasma Sample Using Aqueous Two-Phase System

[0117]The DNA ladder (GeneRuler Low Range DNA Ladder, Thermo Fisher Scientific) was spiked into 500 ul of plasma sample. The spiked plasma sample was added in to 500 ul of aqueous two-phase system composed of 15% (w / w) polyethylene glycol (PEG) 1000 and 15% (w / w) K2HPO4 in MilliQ water to make a final DNA concentration of 1 μg / mL. After vortexing thoroughly, the mixtures were centrifuged for 10 s at 10000 rcf for phase separation. The volume ratio of top phase to bottom phase was around 1:1.

[0118]The bottom phase was extracted and added into another ATPS solution composed of 11% (w / w) polyethylene glycol (PEG) 6000 and 20% (w / w) K2HPO4. After vortexing thoroughly, the mixtures were centrifuged for 10 s at 10000 rcf for phase separation. The volume ratio of top phase to bottom phase was around 1:3. The top and bottom phases were extracted and transferred to...

example 3

Comparison of the Aqueous Two-Phase System with the QIAamp Blood DNA Mini Kit ((Hagen)

[0120]Digested DNA plasmids of different sizes (250, 200, 150, 100, 75, 50, 25 bp) was spiked into plasma sample to make a final DNA concentration of 100 ng / mL. 1mL of the resulting spiked plasma sample was added into 1 mL of aqueous two-phase system composed of 15% (w / w) polyethylene glycol (PEG) 1000 and 15% (w / w) K2HPO4 in MilliQ water. After vortexing thoroughly, the mixtures were centrifuged for 10 s at 10000 rcf for phase separation. The volume ratio of top phase to bottom phase was around 1:1.

[0121]The bottom phase was extracted and added into another ATPS solution composed of 8% (w / w) polyethylene glycol (PEG) 6000 and 22% (w / w) K2HPO4. After vortexing thoroughly, the mixtures were centrifuged for 10 s at 10000 rcf for phase separation. The volume ratio of top phase to bottom phase was around 1:5. The top phase was extracted and transferred to new tube.

[0122]Another extraction was conducted...

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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 base pairs (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 containing biological materials.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims benefit of U.S. Ser. No. 62 / 560,180 filed Sep. 18, 2017.[0002]The entire contents and disclosures of the preceding applications are incorporated by reference into this application.FIELD OF THE INVENTION[0003]This invention relates to a method for the isolation, concentration and / or purification of short nucleic acid fragments in an aqueous two-phase system (ATPS). In particular, the present invention provides a method, a kit and ATPS components for the isolation, concentration and / or purification of short nucleic acid fragments from biological materials. The present application cites various publications, the entire contents of which are incorporated herein by reference into this application.BACKGROUND OF THE INVENTION[0004]Since the discovery of polymerase chain reaction or simply PCR in 1983, whole genomes have been sequenced and numerous discoveries in the fundamental understanding of life sciences and key medic...

Claims

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Application Information

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IPC IPC(8): C12N15/10C07H21/04
CPCC07H21/04C12N15/1006C12N15/1003C12Q2527/125
Inventor CHIU, YIN TO
Owner PHASE SCI INT LTD