Methods for nucleic acid capture

a nucleic acid and capture method technology, applied in the field of molecular biology, to achieve the effect of improving quality and yield, fast, reliable and efficien

Inactive Publication Date: 2015-08-13
ZYMO RES CORP
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  • Abstract
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  • Application Information

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Benefits of technology

[0008]In some embodiments, the present invention provides a fast, reliable, and efficient method for the isolation of substantially purified genomic or plasmid DNA. For example, in some aspects, methods detailed herein are based on an alkaline lysis procedure. In certain aspects, a process is provided that increases the quality and yield of DNA, or other nucleic acid isolated, in a small elution volume.
[0036]In still a further embodiment there is provided a method of isolating plasmid DNA by alkaline lysis, comprising (a) resuspending cells comprising a plasmid (e.g., bacterial cells) in a first aqueous solution; (b) lysing a sample with a second solution; (c) neutralizing the sample and precipitating genomic DNA and proteins with a third solution; (d) capturing of the plasmid DNA to a mineral matrix with a phase separation reagent comprising a cationic surfactant of Formula I; (e) treating the captured plasmid DNA with a salt solution, thereby enhancing the retention of captured nucleic acid; (f) washing the captured plasmid DNA with an organic wash solution; and (g) eluting the plasmid DNA, thereby isolating the plasmid DNA. The use of enzymatic, chemical lysis techniques, or physical lysis such as heat could also be used for isolation of plasmid DNA. In still a further embodiment there is provided a method of isolating plasmid DNA, comprising (a) obtaining a plasmid DNA sample (e.g., a cell lysate produced by enzymatic, chemical or physical lysis); (b) capturing of the plasmid DNA to a mineral matrix with a phase separation reagent comprising a cationic surfactant of Formula I; (c) treating the captured plasmid DNA with a salt solution, thereby enhancing the retention of captured nucleic acid; (d) washing the captured plasmid DNA with an organic wash solution; and (e) eluting the plasmid DNA, thereby isolating the plasmid DNA.
[0039]In some embodiments, methods provided herein employ silica based chromatography that allows for elution into small volumes of water, TE, or elution buffer. However, other forms of chromatography are also contemplated. The resulting nucleic acid whether it be genomic DNA, plasmid DNA, cell-free DNA, or RNA, is suitable for any molecular biological application, including, PCR, restriction digestions, transfection, sequencing, transcriptions, ligations, cloning, among others sensitive applications. The stability of DNA including genomic DNA and plasmid DNA isolated by this novel method is enough that they may be stored for prolonged times at room temperature.

Problems solved by technology

Cloning procedures, for example, are often complex and involve numerous steps; therefore, methods that reliably isolate pure DNA, and other nucleic acids, in significant quantities are desired.

Method used

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Examples

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example 1

Titration of NaCl with 1-decyl-3-methylimidazolium chloride (DMIC)

[0132]A solution of plasmid DNA (pDNA) and degraded RNA was used to test the ability of 1-decyl-3-methylimidazolium chloride (DMIC) to selectively bind DNA in the presence of varying concentrations of salt. The plasmid DNA used was pGEM, which is a 3.2 kb plasmid. The degraded DNA used was RNA digested with RNAse A, which runs as a smear below the lkb ladder on an agarose gel.

[0133]A TE solution (50 mM Tris-HCl pH 8.0 / 10 mM EDTA) containing pDNA and RNAse A degraded RNA was mixed with P4. The P4 solution (1% DMIC with titration of 0-2M NaCl) was added to the aqueous solution containing the pDNA and degraded RNA and mixed thoroughly by inversion (1 ml of P4 was added for every 3 ml of sample volume). The sample was subsequently loaded onto a glass fiber matrix in a spin column, and the captured nucleic acids were washed with 700 μl 0.5 M NaCl / 80 mM Tris pH 8.5 / 0.5 mM EDTA and subsequently washed with 700 μl 95% ethanol...

example 2

Titration of NaCl with 1,3-didecyl-2-methylimidazolium chloride (DDMIC)

[0134]A solution of plasmid DNA (pDNA) and degraded RNA was used to test the ability of 1,3-didecyl-2-methylimidazolium chloride (DDMIC) to selectively bind DNA in the presence of varying concentrations of salt. The plasmid DNA used was pGEM, which is a 3.2 kb plasmid. The degraded DNA used was RNA digested with RNAse A, which runs as a smear below the 1 kb ladder on an agarose gel.

[0135]A TE solution (50 mM Tris-HCl pH 8.0 / 10 mM EDTA) containing pDNA and RNAse A degraded RNA was mixed with P4. The P4 solution (1% DDMIC with titration of 0-2M NaCl) was added to the aqueous solution containing the pDNA and degraded RNA and mixed thoroughly by inversion. (1 ml of P4 was added for every 3 ml of sample volume.) Next, P4 buffer (1% didecylimidazole with titration of 0-2 M NaCl) was added to the cleared solution and mixed thoroughly by inversion. After loading the solution onto a glass fiber matrix in a spin column, th...

example 3

Titration of NaCl with cetylpyridinium bromide (CPB)

[0137]A solution of Cetylpyridinium bromide (CPB) was evaluated with increasing concentrations of sodium chloride to determine the optimal concentrations of the salt and phase separation reagent for selective capture of plasmid DNA while removing degraded RNA. For this, a culture containing JM109 transformed with pGEM was grown overnight for 16 hours. The cells contained in 35 ml of culture were pelleted and suspended in 5 ml P1 buffer. The cells were then lysed using 5 ml P2 buffer for 3 minutes and the solution was neutralized and genomic DNA / proteins were precipitated using 5ml P3 buffer that contained 1.5 M potassium acetate at about a pH 4.9. The precipitated cellular debris were cleared by using a glass fiber filter. 5 ml P4 buffer (0.25%-1% CPB with titration of 0-1.75 M NaCl) was added prior to loading the solution onto a glass fiber matrix in a spin column. The matrix was washed with 700 μl 0.5 M NaCl / 80 mM Tris pH 8.5 / 0.5...

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Abstract

Solutions, reagents, and methods for nucleic acid purification. In certain aspects, cationic surfactant and, optionally, an anionic surfactant solutions are provided which can be used for phase separation and capture of nucleic acids, such as plasmid or genomic DNA, to a solid phase carrier, such as a mineral matrix.

Description

[0001]This application claims the benefit of U.S. Provisional Patent Application No. 61 / 937,824, filed Feb. 10, 2014; 62 / 013,668, filed Jun. 18, 2014; and 62 / 079,358, filed Nov. 13, 2014, each of which is incorporated herein by reference, in its entirety.BACKGROUND OF THE INVENTION[0002]1. Field of the Invention[0003]The present invention relates generally to the field of molecular biology. More particularly, it concerns nucleic acid purification, particularly the isolation of DNA.[0004]2. Description of Related Art[0005]The present invention relates to the purification of nucleic acids from source materials, especially genomic and plasmid DNA. In the case of genomic DNA, modern molecule biological techniques require substantially purified DNA samples and, in some cases it is highly desirable to purify genomic material having a limited amount of retained RNA and / or plasmid DNA. Likewise, in the purification of plasmid DNA from bacterial lysates, plasmid purity is critical for downst...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/10
CPCC12N15/1006
Inventor KEMP, RYANCLAYPOOL, JONATHAN A.VAN EDEN, MARC E.JIA, XI-YU
Owner ZYMO RES CORP
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