WO2012083198A2 - Methods and compositions for purifying dna - Google Patents

Abstract

Provided are methods and compositions for extracting or purifying a polynucleotide. Further provided are methods and compositions for extracting a lipopolysaccharide from a solution containing a polynucleotide. The methods may comprise contacting a cell or cell supsension with a composition comprising a non-ionic detergent (NID). The methods may comprise contacting a solution containing a polynucleotide with a composition comprising a Hofmeister series salt (HSS), and contacting the resulting solution with a composition comprising a non-ionic detergent (NID).

Description

METHODS AND COMPOSITIONS FOR PURIFYING DNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/459,737, filed December 17, 2010, and U.S. Provisional Patent Application No. 61/516,571 , filed April 5, 201 1. These documents are both incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grants awarded by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, and Center for Cancer Research. The Government has certain rights in this invention.

FIELD

[0003] This disclosure relates to methods and compositions for extracting and purifying DNA, especially for isolating DNA from lipopolysaccharides.

BACKGROUND

[0004] Purification of polynucleotides (e.g., DNA, plasmids, etc.) is a critical and routine procedure in molecular biology laboratories. Typically, purification methods, such as in plasmid DNA purification methods, involve the removal of contaminating macromolecules from cellular extracts. These contaminants may include chromosomal DNA, RNA, proteins, lipids, polysaccharides, and lipopolysaccharides (LPS). LPS (also called "endotoxins") are major components of the E. coli cell wall and often co-purify with DNA because they contain negatively-charged phosphate groups. Plasmid DNA contaminated with LPS may be toxic to cells in culture, and can activate inflammatory cascades in animals that may affect and alter experimental responses. Conventional methods for polynucleotide purification suffer from a number of shortcomings, including time- and labor-intensive protocols, inefficiency (e.g., material loss), cost (kits, columns, etc.), and/or do not adequately remove LPS from the polynucleotide solution. SUMMARY

[0005] In an aspect, the disclosure relates to a method for extracting a polynucleotide comprising contacting a cell or cell suspension with a composition comprising a non-ionic detergent (NID) under conditions that allow disruption of the cell membrane.

[0006] In a further aspect, the disclosure relates to a method for purifying a polynucleotide comprising (1 ) contacting a solution containing the polynucleotide with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition; (2) contacting the solution resulting from step (1 ) with a composition comprising a non-ionic detergent (NID); (3) adding a hydrophobic halogenated hydrocarbon to the solution resulting from step (2); and (4) centrifuging the solution resulting from step (3) to obtain a first aqueous phase comprising the polynucleotide. In some embodiments, the methods further comprise further comprising (5) repeating steps (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from step (4) to obtain a second aqueous phase comprising the polynucleotide. In some embodiments, the methods further comprise saturating the second aqueous phase comprising the polynucleotide resulting from step (5) with a hydrophobic halogenated hydrocarbon; (7) centrifuging the solution resulting from step (6) to form a supernatant and pellet; and (8) collecting the supernatant comprising the polynucleotide.

[0007] Another aspect of the disclosure provides a method for extracting a lipopolysaccharide from a solution comprising a polynucleotide, the method comprising (1 ) contacting the solution with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition; (2) contacting the solution resulting from step (1 ) with a composition comprising a non-ionic detergent (NID); (3) adding a hydrophobic halogenated hydrocarbon to the solution resulting from step (2); (4) centrifuging the solution resulting from step (3) to obtain a first aqueous phase comprising the polynucleotide; and (5) repeating steps (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from step (4) to obtain a second aqueous phase comprising the polynucleotide. In some embodiments, the methods further comprise saturating the second aqueous phase comprising the polynucleotide resulting from step (5) with a hydrophobic halogenated hydrocarbon; (7) centrifuging the solution resulting from step (6) to form a supernatant and pellet; and (8) collecting the supernatant comprising the polynucleotide. [0008] Another aspect of the disclosure provides a kit for purifying a polynucleotide from a composition, the kit comprising (a) a composition comprising a HSS and a buffer that maintains the pH at about 8-10 in the composition; and (b) a composition comprising a NID.

[0009] Another aspect of the disclosure provides a kit for extracting a polynucleotide from a cell or cell suspension, the kit comprising (a) a composition comprising a NID; and (b) optionally, a composition comprising an osmolyte.

[0010] The disclosure relates to other aspects and embodiments that will be apparent in light of the following detailed description and accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 is a gel comparing plasmid DNA isolated with methods using alkali compositions or NID compositions.

[0012] Figure 2 displays the effectiveness of various NIDs in removing protein contamination from plasmid DNA preparations.

[0013] Figure 3 is a schematic diagram of the steps in a large-scale method of purifying plasmid DNA using NIDs.

[0014] Figure 4 is a gel showing plasmid DNA purified with various different NIDs and Qiagen-tip 500 columns.

[0015] Figure 5 is a gel showing different plasmid DNA extracted with NIDs with different exposure times and extraction temperatures.

[0016] Figure 6 is a gel showing the digestion and ligation products of high-copy number and low-copy number plasmid DNA purified with either alkali or NID methods.

[0017] Figure 7 is a gel showing different digestion products plasmid DNA obtained with NID methods.

[0018] Figure 8 is a gel showing lambda phage DNA extracted with NID methods.

[0019] Figure 9 is a gel showing plasmid DNA molecular forms and images showing E. coli cell morphology after various DNA extraction procedures. DETAILED DESCRIPTION

[0020] In a broad sense, the disclosure relates to the unexpected and surprising finding that combinations of salt and non-ionic detergent compositions provide for simple, fast, and cost effective methods for extracting or purifying a polynucleotide. The disclosure also relates to the surprising identification of polynucleotide extraction methods and kits that comprise a non-ionic detergent composition and that provide for extracted polynucleotides that can be used directly in a number of molecular biological applications (e.g., sequencing, restriction mapping, etc.) These unexpected findings also provide for related methods for extracting a lipopolysaccharide from a composition comprising a biomolecule, such as a polynucleotide and/or a protein.

[0021] "Polynucleotide" as used herein means any nucleic acid molecules including, but not limited to, deoxyribonucleotides (DNA), ribonucleotides (RNA), single- and double-stranded DNA or RNA molecules, circular or linear DNA or RNA molecules, plasmid DNA, viral DNA/RNA, cDNA, mRNA, and genomic DNA.

[0022] In an aspect, the disclosure relates to a method for extracting a polynucleotide from a cell or a cell suspension. As used herein, extraction of a polynucleotide may refer to extraction of a polynucleotide from a cell or a cell suspension to form a cell lysate. In some embodiments the method comprises contacting the cell or cell suspension with a composition comprising a non-ionic detergent (NID).

[0001] In certain embodiments, the methods comprise lysing or rupturing a cell in the presence of a non-ionic detergent (NID) under conditions that allow disruption of the cell membrane, for example, under enzymatic, chemical, or physical cell lysis conditions, such as are known in the art. Non-limiting examples of enzymatic cell lysis agents include lysozyme, lysostaphin, zymolase, cellulase, mutanolysin, glycanases, proteases, mannase, and the like. Non-limiting examples of chemical cell lysis agents include various organic solvents (e.g., acetone), ionic detergents (e.g., SDS), and the like. Non-limiting examples of physical cell lysis ("high-shear" mechanical methods) include sonication and French press techniques. The methods may optionally include adding a composition comprising an osmolyte selected from any common organic or inorganic osmolytes known in the art such as, for example, sugars, amino acids, polyols, methylamines, methylsolfonium compounds, salts, or a combination thereof. Sugars may include any sugar that is commonly used in an osmolytic solution such as, for example, glucose, trehalose, sucrose, fructose, and derivatives thereof. In some embodiments, the composition can comprise a Hofmeister series salt (HSS), as disclosed herein. The methods may further comprise incubating the solution for a duration and at a temperature effective to disrupt the cell membrane and extract polynucleotide from a cell in the presence of the NID. For example, the solution may be incubated at about 65°C for about 5 min, or at about 4°C for about 1 or about 2 hours. While the time and temperature for this incubation can be determined by one of skill in the art, typically, the incubation time decreases with increasing temperature, and increases with decreasing temperature. The methods may further comprise isolating the cell lysate comprising the polynucleotide, for example, by centrifugation.

[0002] Other embodiments relate to further purification steps of the extracted polynucleotide in the crude cell lysate, such as those methods described herein, or as otherwise known in the art, (e.g. alkali purification methods and kits).

[0003] In an aspect, the disclosure relates to a method for purifying a polynucleotide, wherein the method comprises a composition comprising a salt and a composition comprising a non-ionic detergent. In certain embodiments, the method comprises (1 ) contacting a solution containing a polynucleotide with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition; (2) contacting the solution resulting from (1 ) with a composition comprising a non-ionic detergent (NID); (3) adding a hydrophobic halogenated hydrocarbon to the solution resulting from (2); and (4) centrifuging the solution resulting from (3) to obtain a first aqueous phase comprising the polynucleotide. The hydrophobic halogenated hydrocarbon can be any such compound that can increase the density of the organic phase and may include, but is not limited to, chloroform, carbon tetrachloride, and bromoform. In certain embodiments, the method may further comprise heating at 55-75°C (e.g., about 70°C) the solution resulting from (3) before centrifuging in (4).

[0004] In certain embodiments, the method may further comprise (5) repeating steps (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from step (4) to obtain a second aqueous phase comprising the polynucleotide.

[0005] In certain embodiments, the method may further comprise (6) saturating the second aqueous phase comprising the polynucleotide resulting from (5) with a hydrophobic halogenated hydrocarbon (e.g., chloroform, carbon tetrachloride, and bromoform); (7) centrifuging the solution resulting from (6) to form a supernatant and pellet; and (8) collecting the supernatant comprising the polynucleotide. These steps may further remove any protein contamination from the polynucleotide solution. In certain embodiments, the method may further comprise heating the solution resulting from (6) before centrifuging in (7).

[0006] In certain embodiments, the method may further comprise (a) reconstituting the polynucleotide from (5) or (8) in a composition comprising a salt suitable for protein removal including, but not limited to, LiCI, NaCI, NH₄CI, and LiAc. Reconstituting includes any method known in the art to change the salt, buffer, or a combination thereof, of the polynucleotide solution. Exemplary methods include, but are not limited to, DNA precipitation such as ethanol precipitation or isopropanol precipitation, ultrafiltration, gel filtration, and dialysis. For example, the polynucleotide may be exchanged into a composition comprising LiCI. The method may further comprise (b) precipitating the polynucleotide from the solution resulting from (a) with isopropanol; and (c) centrifuging the isopropanol solution from (b) to pellet the polynucleotide and dissolving the polynucleotide pellet in a buffer or aqueous solution such as water.

[0007] In certain embodiments, the polynucleotide pellet of (c) may be dissolved in a buffer of about pH 8-10. In certain embodiments, the method may then further comprise adding an equal volume of a salt including, but not limited to, LiCI, NaCI, and NH₄CI. For example, an equal volume of 9 M LiCI, 5 M NaCI, or 3.5 M NH₄CI may be added.

[0008] In some embodiments, the solution comprising the polynucleotide may include any composition comprising a polynucleotide and other contaminants, where the solution includes, but is not limited to, a cell suspension, a completely or partially lysed cell suspension, a crude cell lysate, and a partially purified cell lysate. In some embodiments, the solution comprising the polynucleotide can relate to a polynucleotide contained within a cell in solution. Other contaminants may include, but are not limited to, other DNA (e.g., chromosomal DNA) or RNA molecules, protein, lipopolysaccharide, lipid, carbohydrate, polysaccharide, and other cellular debris.

[0009] Embodiments of the methods relate to buffers. Buffers may include any suitable buffer known in the art that is capable of maintaining the pH of the solution. Examples of suitable buffers may be found on the Sigma Alrich website (www.sigmaaldrich.com/life- science/core-bioreagents/biological-buffers/biological-buffer-products.html?TablePage=

15077099. Examples of buffers include tricine, BID-TRIS propane, BICINE, diethanolamine, TAPS, CH ES, TABS, and CAPSO. For example, the buffer in (1 ) may includeTris-HCI. For example, the buffer in (b) may include TE (Tris-EDTA).

[0010] Methods described herein may be effective to remove lipopolysacchande from a solution comprising a polynucleotide. The amount of lipopolysacchande in solution can be measured using any technique or expressed in any units that are known in the art. In some embodiments, endotoxin unit (EU) may be used to describe the amount of lipopolysacchande. The FDA defines the endotoxin unit (EU) as the endotoxin activity of Reference Endotoxin Standard. The EPA may change this standard often. Suitably, and as used in the Examples, the relation between EU and LPS amount may be 10-15 EU/ng LPS. For example, in embodiments, the solution resulting from (5) may comprise lipopolysacchande in an amount equal to or less than about 5 EU/mL. For example, the solution resulting from (8) may comprise lipopolysacchande in an amount equal to or less than about 5 EU/mL. For example, the solution resulting from (c) may comprise lipopolysacchande in an amount equal to or less than about 5 EU/mL. Methods described herein may result in a polynucleotide present in a composition comprising lipopolysacchande in an amount equal to or less than about 5 EU/mL, less than about 4 EU/mL, less than about 3 EU/mL, or less than about 2 EU/mL.

[001 1 ] Embodiments of the methods relate to compositions comprising a Hofmeister series salt (HSS). HSSs are known in the art and are typically ranked by anion or cation and the related ability of the anion or cation to stabilize a protein in solution. HSSs are typically identified as kosmotropes (decrease protein aqueous solubility) and chaotropes (increase aqueous protein solubility). In some embodiments, the method comprises a composition that includes a salt (HSS) having: (a) an anion selected from the group consisting of acetate, chloride, nitrate, bromide, chlorate, perchlorate, iodide, thiocyanate, and cyanate; or (b) a cation selected from the group consisting of potassium, sodium, lithium, magnesium, calcium, and guanidinium. HSS includes a salt comprising an anion and a cation such as F^", S0₄ ^2", HP0₄ ^2", acetate, CI^", N0₃ ^", Br, CI0₃ ^", I^", CI0₄ ^", SCN^", NH₄ ⁺, K⁺, Na⁺, Li⁺, Mg²⁺, Ca²⁺, and guanidinium. In certain embodiments, HSS does not include kosmotropic salts. In certain embodiments, HSS includes chaotropic salts. In certain embodiments, HSS includes neutral chaotropic salts. In certain embodiments, HSS includes LiCI, LiAc, K⁺, Na⁺, NH₄ ⁺, and guanidinium chloride (alternatively, guanidine hydrochloride). In certain embodiments, HSS includes LiCI, NaCI, LiAc, NH₄CI, and guanidinium chloride (alternatively, guanidine hydrochloride). The compositions may comprise HSS in an amount of at least about 0.05 M, at least about 0.06 M, at least about 0.07 M, at least about 0.08 M, at least about 0.09 M, 0.1 M, at least about 0.15 M, at least about 0.2 M, at least about 0.25 M, at least about 0.3 M, at least about 0.35 M, at least about 0.4 M, at least about 0.45 M, at least about 0.5 M, at least about 0.55 M, at least about 0.6 M, at least about 0.65 M, at least about 0.7 M, or at least about 0.75 M. The compositions may comprise HSS in an amount of less than about 2 M, less than about 1.9 M, less than about 1.8 M, less than about 1.75 M, less than about 1.7 M, less than about 1.65 M, less than about 1.6 M, less than about 1.55 M, less than about 1.5 M, less than about 1.45 M, less than about 1.4 M, less than about 1.35 M, less than about 1.3 M, less than about 1.25 M, less than about 1.2 M, less than about 1.15 M, less than about 1.1 M, less than about 1.0 M, less than about 0.95 M, less than about 0.9 M, less than about 0.85 M, or less than about 0.8 M. The compositions may comprise HSS in an amount of about 0.05 M to about 2 M, about 0.05 M to about 1.5 M, about 0.75 M to about 1.5 M, about 0.1 M to about 1.25 M, or about 0.1 M to about 1 M. In certain embodiments, the HSS composition is less dense than the NID composition, detailed below.

[0012] Embodiments of the methods relate to compositions comprising at least one non- ionic detergent (NID). In certain embodiments, NID includes surfactants having an HLB of less than 13. In certain embodiments, NID includes surfactants having an HLB of less than about 13, less than about 12.9, less than about 12.8, less than about 12.7, less than about 12.6, less than about 12.5, less than about 12.4, less than about 12.3, less than about 12.2, less than about 12.1 , less than about 12.0, less than about 1 1.9, less than about 11.8, less than about 11.7, less than about 1 1.6, less than about 1 1.5, less than about 1 1.4, less than about 1 1.3, less than about 1 1.2, less than about 1 1.1 , or less than about 1 1.0. In some embodiments, the HLB value is at least about 9.5-10.

[0013] The Hydrophilic-lipophilic balance (HLB) of a surfactant is a measure of the degree to which it is hydrophilic or lipophilic, determined by calculating values for the different regions of the molecule. HLB calculation is known in the art. For example, HLB may be calculated according to the Griffin method using the following equation:

HLB = 20 x (M_h / M) (1 ) wherein M_h is the molecular mass of the hydrophilic portion of the molecule, and M is the molecular mass of the whole molecule, giving a result on an arbitrary scale of 0 to 20. An HLB value of 0 would correspond to a completely hydrophobic molecule, and a value of 20 would correspond to a molecule made up completely of hydrophilic components. HLB may be calculated according to the Davies method using the following equation:

HLB = 7 + (m^*H_h) - (n^*H|) (2) wherein m is the number of hydrophilic groups in the molecule, H_h is the value of the hydrophilic groups in the molecule, n is the number of lipophilic groups in the molecule, and Hi is the value of the lipophilic groups in the molecule.

[0014] In certain embodiments, NID includes surfactants having a specific density greater than about 1.0. In certain embodiments, NID includes surfactants having a specific density of at least about 1.0, at least about 1.01 , at least about 1.02, at least about 1.03, at least about 1.04, at least about 1.05, at least about 1.06, at least about 1.07, at least about 1.08, at least about 1.09, at least about 1.1 , at least about 1.12, at least about 1.13, at least about 1.14, at least about 1.15, at least about 1.16, at least about 1.17, at least about 1.18, at least about 1.19, at least about 1.2, at least about 1.3, at least about 1.4, or at least about 1.5.

[0015] In certain embodiments, the NID may be selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert-octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene.

[0016] In certain embodiments, the acyl polyoxyethylene sorbitan ester may be a compound of Formula I:

wherein n-i + n₂ + n₃ + n₄ = x, wherein x is the number of oxyethylene units and R is alkyl, such that the HLB number is less than 13, as described above. In some embodiments, x is an integer from 1 to 20, and R is Ci-C₂o alkyl (including linear or branched alkyls).

[0017] In certain embodiments, the tert-octylphenyl polyoxyethylene ether may be a compound of Formula II:

wherein n is an integer such that the HLB number is less than 13, as described above. In some embodiments, n is an integer from 5 to 8.

[0018] In certain embodiments, the alkyl polyoxyethylene is a compound of Formula III:

R(OCH₂CH₂)_xOH (III) wherein x is the number of oxyethylene units and R is alkyl, such that the HLB number is less than 13, as described above. In some embodiments, x is an integer from 1 to 20, and R is CT C₂₀ alkyl (including linear or branched alkyls).

[0019] In certain embodiments, the alkyl phenylpolyoxyethylene is a compound of Formula IV:

wherein x is the number of oxyethylene units such that the HLB number is less than 13, described above. In some embodiments, x is an integer from 1 to 20.

[0020] Some examples of NIDs are shown in Table 1.

Table 1. Chemical structure and basic physicochemical properties of some NIDs.

Name Chemical Structure HLB CP (°C) Specific density

AcPOESEs: Acyl polyoxyethyelene sorbitan esters

Tween-20 C0₂R = laurate 16.7 76 1.1

Tween-80 C0₂R = oleate 15 65 1.07 ene ethers

[0021] In certain embodiments of the method, an NID may be added to a final concentration of about 0.1 % to about 10%, about 0.2% to about 9%, about 0.5% to about 8%, about 1 % to about 7%, about 2% to about 6%, or about 3% to about 5% by volume. In some embodiments, the NID may be added to a final concentration of at least about 0.1 %, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1 %, at least about 2%, or at least about 3% by volume. In some embodiments, the NID may be added to a final concentration of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, or less than about 5% by volume.

[0022] In another aspect, the disclosure relates to a method for extracting a lipopolysaccharide from a solution comprising a polynucleotide. In certain embodiments, the method may comprise (1 ) contacting the solution with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition; (2) contacting the solution resulting from (1 ) with a composition comprising a non-ionic detergent (NID); (3) adding a hydrophobic halogenated hydrocarbon that increases the density of the organic phase, including, but not limited to, chloroform, carbon tetrachloride, and bromoform to the solution resulting from (2); (4) centrifuging the solution resulting from (3) to obtain a first aqueous phase comprising the polynucleotide; and (5) repeating (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from (4) to obtain a second aqueous phase comprising the polynucleotide, as described above.

[0023] In some embodiments, the method may further comprise (6) saturating the second aqueous phase comprising the polynucleotide resulting from (5) with a hydrophobic halogenated hydrocarbon that increases the density of the organic phase, including, but not limited to, chloroform, carbon tetrachloride, and bromoform; (7) centrifuging the solution resulting from (6) to form a supernatant and pellet; and (8) collecting the supernatant comprising the polynucleotide, as described above.

[0024] In some embodiments, the method may further comprise (a) reconstituting the polynucleotide from (5) or (8) in a composition comprising LiCI; (b) isopropanol precipitating the polynucleotide from the solution resulting from (a); and (c) centrifuging the isopropanol solution from (b) to pellet the polynucleotide and dissolving the polynucleotide pellet in a buffer, as described above.

[0025] Methods disclosed herein may be used for any scale of extraction and/or purification. For example, methods disclosed herein may be used for large-scale, mini, midi, or maxi preparations.

[0026] In another aspect, the disclosure relates to a kit for extracting a polynucleotide from a cell. In certain embodiments, the kit may comprise a composition comprising a HSS and a buffer that maintains the pH at about 8-10 in the composition; and a composition comprising a NID. In certain embodiments, the kit may further comprise instructions for use.

[0027] In another aspect, the disclosure relates to a kit for purifying a polynucleotide from a composition. In certain embodiments, the kit may comprise a composition comprising a HSS and a buffer that maintains the pH at about 8-10 in the composition; and a composition comprising a NID. In certain embodiments, the kit may further comprise instructions for use.

[0028] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise noted. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to illustrate aspects and embodiments of the disclosure and does not limit the scope of the claims. [0029] It also is understood that any numerical range recited herein includes all values from the lower value to the upper value. For example, if a concentration range is stated as 1 % to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1 % to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this application.

[0030] The Examples that follow merely serve to illustrate and clarify some of the aspects and embodiments that have been described above. Accordingly, the Examples should not be interpreted as limiting the scope of the disclosure or the appended claims.

EXAMPLES

Example 1 : Materials and methods

[0031] NIDs, salts, and endotoxin removal solution (ERS) were purchased from Sigma- Aldrich (St. Louis, MO) or Mallinckrodt Baker (Phillipsburg, NJ). RNase A was purchased from 5 PRIME (Gaithersburg, MD). Antibiotics were purchased from USB (Cleveland, Ohio), Sigma- Aldrich, and EMD/Calbiochem (Gibbstown, NJ). Bacterial media, including LB and Super Broth (SB), were obtained from Quality Biological (Gaithersburg, MD). DH5a and XL1-Blue were supplied by Invitrogen (Carlsbad, CA) and Stratagene (La Jolla, CA), respectively. The plasmid pLTM 330 (6.5 Kb) was kindly provided by L. Tessarolo (NCI, Frederick, MD); pLTM 330 is a pBluescript-based plasmid and high-copy number. The following high copy number plasmids were used: 1 ) pUC19 (2.7Kb), purchased from Invitrogen (Carlsbad, CA); 2) pCYPAC3 (18.8 Kb), a pUC-based plasmid, kindly provided by S. O'Brien (NCI, Frederick, MD); 3) pLTM330 (6.5 Kb), a pBluescript-based plasmid, kindly provided by L. Tessarollo (NCI, Frederick, MD); and 4) B254 (6.06 Kb), a pBluescript-based plasmid, kindly provided by E. Leibold (University of Utah, Salt Lake City, UT). pEL04 (5.07 Kb, ts pSC101 oriR), a low copy number plasmid (Qiagen® Plasmid Purification Handbook 3^rd Edition, Nov 2005, pg. 12), was kindly provided by NCI-

E. coli cells UB-61 lysogenic for a heat-inducible bacteriophage lambda (cl857 ind 1 Sam7) were kindly provided by S. Casjens (University of Utah). All enzymes were purchased from New England Biolabs (Ipswich, MA), except Pstl (Promega), and Sstl, and Xhol (Invitrogen). Enzymatic reactions were performed at 37°C for 1 hour if not otherwise specified. DNA quantification

[0032] DNA was quantified using a two wavelength spectrophotometric method on nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA).

Protein quantification

[0033] The BCA Protein Assay Kit (Pierce, IL) was used for total protein quantification with the following modifications: the ratio of sample to working reagent was from 1 :4 to 1 :5. Samples were incubated at 75°C for 15 min. Readings were taken in the protein concentration range of 1-40 μg/mL. The following BSA standards were used in duplicate: 40 μg/mL, 20 μg/mL, 10 μg/mL, 5 μg/mL, 3 μg/mL, 2 μg/mL, 1 μg/mL, and 0 μg/mL. The average ± standard deviation for the linear correlation coefficient was 0.9 ± 0.003.

LPS quantification

[0034] The ToxinSensor Assay Kit (Genscript, NJ) was used to determine LPS levels. The assay utilized chromogenic substrate and LAL (Limulus Amebocyte Lysate from Limulus polyphemus) reagent clotting upon exposure to endotoxin. The absorbance of the released chromophore measured the endotoxin concentration. The test's results are expressed in endotoxin units (EU). According to the manufacturer's information, 10-15 EU corresponds to 1 ng of LPS.

Alkali DNA isolation and purification

[0035] For the alkali isolation/purification in Figure 1 , the bacterial pellet was resuspended in 333 μί buffer P1 (Qiagen). Subsequently, 333 μί buffer P2 was added to the suspension and gently mixed. Finally, 333 μί buffer P3 was added and mixed. Cellular debris was removed by centrifugation, and DNA was precipitated with 0.7 V of isopropanol. The DNA pellet was rinsed with 70% of ethanol.

[0036] The alkali isolation and purification for Figure 2 and Table 4 were performed using the QIAfilter Maxi kit (Qiagen, Germantown, MD) according to manufacturer's instructions. Alkali lysates were purified by Qiagen-tip 500 columns. NID large-scale plasmid isolation procedure

[0037] Bacteria were harvested from LB/SB cultures by centrifugation. If the pellet was large, it was loosened by mixing with a small spatula and vortexing until a bacterial slurry was attained. The bacterial pellet was resuspended in the large-scale extraction buffer. Lysozyme (250 g/mL), RNase A 25 μg/mL (optional), and 0.5% IGEPAL CA-630 (or Triton X-100) from 10% stock were added to the bacterial suspension. The suspension was mixed by gently inverting the tube to avoid unnecessary foaming. The suspension was incubated in a water bath at 65°C for 10 min for bacterial suspension volumes up to about 30 mL, or up to 30 min for larger volumes. The cellular debris was spun down at 30,000 rpm for 30 min or until a compact pellet of bacterial debris was formed. 20-30 μg/mL RNase A was added to cleared extracts and incubated at 37°C for 15 min. The cleared extracts were precipitated with 0.6 volumes isopropanol at 3000-4000 rpm for 15 min at room temperature, and then the pellets were rinsed with 70% ethanol and dissolved in TE buffer. The composition of the large-scale extraction buffer was 5% sucrose, 50 mM EDTA, 50 mM Tris pH 8, and 1 M KCI or 1.25 M NH₄CI.

NID large-scale plasmid purification procedure

[0038] The bacterial strain XL1-Blue was used to identify the best purification conditions (1.5 mL for every 100 mL bacterial culture, using 0.2 M GuHCI, 100 mM Tris pH 9 with either Triton X-1 14 or IGEPAL CA-520). Under these conditions, we confirmed similar results using DH5a. The procedure was performed according to the steps detailed in Figure 3. The parts of the procedure in dashed boxes were optional. Note that positive displacement devices were better for dispensing viscous NIDs.

Standard alkali DNA isolation

[0039] Alkali isolation was performed according to the original description by Birnboim and Doly (Birnboim HC, Doly J. Nucleic Acids Res. 1979, 7, 1513-1523).

NID miniprep plasmid isolation procedure

[0040] The following procedure was used for the NID miniprep plasmid isolation: 1. 1.5-2 mL of bacterial cultures were pelleted at 6000-7000 rpm for 1 min. 2. After drawing 150 μΙ_ extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet resuspended.

3. The bacterial suspension was incubated at 65°C for 5 min.

4. Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The pellet was removed with a toothpick.

5. 100-120 μΙ_ isopropanol was added, followed by mixing and centrifugation of the solution at 7000 rpm for 10 min at room temperature.

6. DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. After discarding the supernatant, the DNA was centrifuged after adding 70% ethanol. Ethanol was removed, and the DNA pellet was dissolved in 20-50 μΙ_ TE buffer.

[0041] The composition of the extraction buffer was 5 % sucrose, 20-50 mM EDTA, 50 mM Tris pH 8, 0.75 M NH₄CI, 0.5 % IGEPAL CA-630 (or Triton X-100), lysozyme 100 g/mL, and RNase A 25 μg/mL. Addition of 20-50 mM CaCI₂ to the extraction buffer reduces extraction of chromosomal DNA and large plasmids, but greatly facilitates formation of cellular debris during sedimentation. A 100x enzyme stock containing 10 mg/mL lysozyme and 2.5 mg/mL of RNase A prepared in 50% glycerol and 50 mM Tris pH 8 was stored at -20°C and used repeatedly.

Densitometry

[0042] Image densitometry/gel quantification analysis was performed using Image J (Abramoff MD, Magalhaes PJ, Ram SJ. Biophotonics International 2004, 11, 36-42). All gel images were calibrated in OD units using Kodak No. 3 Calibrated Step Tablet.

DNA sequencing

[0043] DNA sequencing was performed using the ABI BigDye Terminator Cycle Sequencing Kit v1.1 (NID DNA) or v3.1 (NID crude lysates) according to the manufacturer's instructions on a Gene Amp 9700 PCR machine. The primers used were M13f (GTA AAA CGA CGG CCA GT) (SEQ ID NO: 1 ) for sequencing B256 plasmid, and r pLTM330_3617 (GCT GGT TCT TTC CGC CTC A) (SEQ ID NO: 2). The sequence fragments were detected on an ABI 3130XL Genetic Analyzer. Samples were then analyzed and base-called by Applied Biosystems DNA Sequencing Analysis Software V5.2 (Applied Biosystems, Foster City, CA). Mathematical model

[0044] To estimate time-efficiency of the NID relative to the alkali procedure, the total NID and alkali procedure completion times (T_tot) were considered as the sum of the operational time (time dependent on the number of samples, T_op) and the preparation/working equipment times (time independent on the number of samples, time idle, T_id).

T_op + T_id. T_op is a product of k (a constant, average time for isolating 1 sample) by n (number of samples).

/ n + Ti_d where T_id is a constant (c).

1 + c// n, showing that as the number of samples increases T_tot/T_op approaches 1. In other words, as n increases, T_tot and T_op can be used interchangeably.

[0045] k and the standard error were estimated by linear regression analysis (T_op as a function of n) using the data presented in Table 5, forcing the fitting line to intersect the point of origin. Microsoft Excel LINEST function was used, with const is FALSE and stats is TRUE.

Bacteriophage lambda particle isolation

[0046] Lysogenic E. coli UB-61 cells were grown at 30°C until OD₆₀₀ = 1. The culture was divided into 12-15 ml. aliquots and heat-shocked for 10 min at 42-43°C, then re-combined and incubated at 37°C for 7-8 hours. One or two 5 ml. aliquots were lysed with chloroform (full lysis sample). The other 5 ml. aliquots were spun down and the cells were resuspended in the following lambda NID extraction buffer: 5% sucrose, 50 mM Tris pH 8, 2 M NH₄CI, 50 mM CaCI₂, lysozyme 120 μg/mL, and 0.5% Tween 80. No EDTA was included in this buffer. Bacteria were incubated at 40°C for 45 min with occasional mixing to prevent bacterial sedimentation, and centrifuged at 12-15,000 rpm for 5-10 min. Easy cell sedimentation and absence of viscous pellets suggested no lysis of bacteria. The supernatant was precipitated with 1 :1 volume 1.25 M NH₄CI and 20% PEG 8000 after 1 hour incubation in ice.

[0047] The full lysis samples were precipitated with the following solution: 1 M NaCI and 10% PEG 8000 added as powder.

[0048] The phage containing pellets were processed according to Sambrook et al. with minor modifications (Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 1989. New York Cold Spring Harbour Lab. Press). Shortly, they were gently resuspended in 0.2-0.5 mL modified SM buffer (25 mM Tris pH 7.6, 75 mM NaCI, 10 mM MgCI₂) containing >5 Kunitz units/mL DNasel and incubated at 37°C for 1 hour. Subsequently, 15 mM EDTA was added. DNA was extracted with phenol/chloroform and before isopropanol precipitation the aqueous phase was mixed with 20 μg linear polyacrylamide carrier to ensure quantitative DNA recovery (Gaillard C, Strauss F. Ethanol precipitation of DNA with linear polyacrylamide as carrier. Nucleic Acids Res. 1990, 18, 378). After centrifugation supernatant was carefully aspirated and the pellets were dissolved in 40 μΙ_ TE containing 30 μg/mL RNase A.

Plasmid DNA miniprep according to Godson and Sinsheimer using Brij-58 (Godson GN, Sinsheimer RL. Biochim. Biophys. Acta. 1967, 149, 476-488)

[0049] Bacterial cells from 2 ml. cultures were resuspended in 60 μΙ_ Godson's sucrose solution (25% w/v sucrose in 10 mM Tris pH 8.1 ). Cells were kept on ice throughout the procedure. We mixed in advance 7.5 μΙ_ 16 mM EDTA and 7.5 μΙ_ 0.85 mg/mL lysozyme in 250 mM Tris 8.1. This solution was mixed with the bacterial suspension and incubated on ice for 1 min. The bacterial suspension was then transferred to a lytic mixture containing 15 μΙ_ 5 % Brij- 58 in 10 mM Tris pH 7.4 and 60 μΙ_ deionized water and incubated on ice for 10 min. We excluded 10 mM MgS0₄ from the original lytic mixture and increased the last incubation time from 2 to 10 min to increase the amount of plasmid DNA extracted. The mixture was then centrifuged at 5000 RPM (3000xg) for 5 min, and the cleared lysate was precipitated with 150 μΙ_ isopropanol. DNA pellets were resuspended in 35 μΙ_ TE buffer containing 30 μg/μL RNase A. The insoluble material was spun down, and the cleared DNA solution was loaded on a gel.

Plasmid DNA miniprep according to Clewell and Helinski using Brij-58 and DOX[19]

[0050] Bacterial cells from 2 ml. cultures were resuspended in 47 μΙ_ Clewell's sucrose solution (25% sucrose in 0.05 M Tris, pH 8.0). Cells were kept on ice throughout the procedure. Subsequently, 10 μΙ_ 5 mg/mL lysozyme in 250 mM Tris pH8 was added, and the solution was kept on ice for 5 min. 19 μΙ_ 0.25 M EDTA was added and incubation continued for another 5 min. 76 μΙ_ detergent mixture (1 % Brij-58, 0.4% DOX, 62.5 mM EDTA, 50 mM Tris, pH 8.0) was then added to the 76 μΙ_ bacterial suspension and kept on ice for 5-10 min. The mixture was then centrifuged at 48,000g at 2°C for 25 min. The cleared lysate was precipitated with 150 μΙ_ isopropanol, and the DNA pellets were resuspended in 35 μΙ_ TE buffer containing 30 μg/μL RNase A. The insoluble material was spun down and the cleared DNA solution was loaded on a gel. Plasmid DNA miniprep according to Summerton et al. using Brij-58 and DOX in highly hypertonic salt-sucrose solutions (Summerton J, Atkins T, Bestwick R. Anal. Biochem. 1983, 133, 79-84)

[0051] Bacterial cells from 2 mL cultures were resuspended in 60 μΙ_ DEPC/Summerton's sucrose solution (1 μΙ_ DEPC/500 μΙ_ Summerton's sucrose solution: 100 mM Tris pH 8.1 , 30% sucrose, 100 mM EDTA). The cells were incubated on ice for 5 min. Subsequently, 15 μΙ_ lysozyme (6 mg/mL in water) was added, and incubation continued for 20 min in ice. An equal volume (75 μΙ_) ice cold salt-detergent solution (0.4% DOX, 1 % Brij-58, 2 M NaCI) was added, gently mixed, and incubated at 25°C for 20 min without mixing. The mixture was then centrifuged at 40,000g for 30 min at 0°C. The cleared lysate was precipitated with 150 μΙ_ isopropanol, and the DNA pellets were resuspended in 35 μΙ_ TE buffer containing 30 μg/μL RNase A. The insoluble material was spun down and the cleared DNA solution was loaded on a gel.

Imaging of bacterial suspensions

[0052] 20-40 μΙ_ bacterial cell lysates from the various isolation methods were placed on slides and mounted with cover slips. Images were recorded using either an Olympus IX81/Metamorph v6.2r6 microscope (100x objective in oil) or a Leica DMR A/Metamorph v6.3r1 (63x objective in oil).

Example 2: Effective scaling-up of the NIP isolation procedure

[0053] To assess how effectively the NID plasmid DNA isolation procedure could be scaled- up and how small a volume of lysis buffer can be used without loss of DNA quality and quantity, we analyzed the effects of increasing bacterial cell concentration on the amount and molecular forms of plasmid DNA extracted by alkali or NIDs. The pLTM 330 plasmid was used in these and all subsequent experiments. We used 333 μΙ_ of each alkali method solution and 1 mL of the NID extraction buffer to achieve the same 1 mL final lysate volume because the NID method uses a single extraction buffer whereas the alkali method uses three solutions. Referring to Figure 1 , cells were harvested from 10 mL (lanes 1 , 2), 20 mL (lanes 3, 4), 30 mL (lanes 5, 6) and 40 mL (lanes 7, 8) bacterial cultures. The samples from the Alkali prep DNA are shown in lanes 1 , 3, 5, and 7. The samples from the NID prep DNA are shown in lanes 2, 4, 6, and 8. A 5 μί aliquot of either undiluted (10 mL culture), 2-fold diluted (20 mL culture), 3-fold diluted (30 mL culture), or 4-fold diluted (40 mL culture) plasmid DNA was loaded in the lane. For both methods, 1 :10 lysis buffer to culture volume ratios produced the largest amount of DNA per culture volume (Figure 1 , lanes 1 ,2), whereas increasing bacterial culture volumes from 10 mL to 40 mL decreased the yield of DNA, including fast migrating and slow migrating DNA forms, per culture volume unit (Figure 1 , lanes 3-8). However, the yield of fast migrating forms was far higher with the NID method at any culture volume. There was almost no fast migrating DNA when a 40 mL culture volume was used in the alkali method (Figure 1 , lane 7), but a 1 :20 ratio still produced sufficient amount of DNA in the NID method (Figure 1 , lane 4). These results showed that compared to alkali, the NID method was more effective in large-scale plasmid isolation procedures over a wide range of bacterial cell concentrations.

Example 3: Hofmeister series salts decrease plasmid DNA protein contamination

[0054] We tested the effect that HSS has on the purification of plasmid DNA from protein contamination in large-scale preps. Cleared lysates from 25 mL LB cultures were generated according to the NID large-scale procedure. The lysate was treated with RNase A 20 μg/mL for 15 min at 37°C, followed by incubation for 15 min at 55°C. Lysates were precipitated with isopropanol (1st precipitation). Pellets were reconstituted in 0.5 mL of the different salt solutions and precipitated further (2nd and 3rd precipitation). Pellets were rinsed with 70% ethanol before dissolving them in 100 μί TE. All insoluble residues were removed before measuring protein concentration. We determined protein concentration in the DNA sample after treatment with RNase and the first isopropanol DNA precipitation (IDP) (Figure 2, row 1 ). Next, we compared the ability of some chaotropic (Figure 2, rows 2-3), neutral (Figure 2, rows 4-7), or kosmotropic salts (Figure 2, row 8) to decrease protein co-precipitation from plasmid DNA/salt/isopropanol solutions. For all salts, protein contamination decreased after the third IDP (Figure 2, rows 2- 8). Neutral salts (LiCI) removed protein more effectively (Figure 2, rows 4-7). In particular, 4.5 M LiCI at pH 9 eliminated protein most efficiently (Figure 2, row 5), while chaotropic and kosmotropic salts removed protein, however with less efficiency (Figure 2, rows 2, 3 and 8). We also tested the combination of a relatively low salt concentration second IDP (0.5 M NaCI) with a high salt concentration third IDP (4.5 M LiCI), and found that this approach was effective in removing protein (Figure 2, row 5 vs. 9). These results suggested that several HSS work generally, and that several neutral salts, such as LiCI, were highly effective in purifying plasmid DNA from protein contamination. Example 4: Hofmeister series salts regulate the phase separation properties and CP of

NIDs

[0055] To test how HSS regulate N ID-dependent phase separation, we measured cloud point (CP) and used a fat-soluble tracking dye (Sudan IV) and the re-clouding test. Results are shown in Table 2. 0.5 ml. of different NIDs stained with Sudan IV were tested with or without co-solutes. Cellular extracts were prepared from 40 ml. LB cultures according to the NID large- scale isolation procedure. The Tween 20 solutions had pH 7 and low HLB NID solutions had pH 9 (adjusted with 100 mM Tris). CP was determined visually by monitoring the transition temperature between one and two-phase state of the solution. Both the dye and the re-clouding tests were performed by heating NID solutions 15°C above CP followed by centrifugation. For CP results, "a" indicates phase separation was not stable upon cooling; "b" indicated no one- phase state was observed between 4-100°C; and "c" indicates no two-phase state was observed between 4-100°C. "NS" indicates non-soluble particles did not allow test. We concluded that phase separation was incomplete when either Sudan IV was visible in the aqueous phase or the solution became cloudy upon re-heating. Addition of neutral or chaotropic HSS to high HLB NIDs, such as Tween 20, produced either unstable phase separation or no phase separation (results not shown), which precluded using these combinations. Addition of kosmotropic HSS (NaAc and NH₄Ac) to Tween 20 decreased CP and produced a stable but incomplete phase separation, as shown by the tracking dye and the re- clouding tests (NaAc, Table 2A, row 3), or the re-clouding test (NH₄Ac, Table 2A, row 5). Triton X-1 14, a lower HLB NID, had a lower CP and stable and complete phase separation in kosmotropic (results not shown), neutral (LiCI) or chaotropic (GuaHCI) HSS (Table 2A, rows 9, 1 1 ). Another lower HLB NID, IGEPAL CA-520, had limited solubility in water, but strong chaotropes such as GuaSCN made it soluble in this solution with a CP of 34°C (Table 2A, row 14). At higher GuaSCN concentrations, CP increased from 34°C to >100°C, virtually abolishing phase separation of IGEPAL CA-520 (Table 2A, row 15). Neutral or chaotropic HSS increased the volume of the organic phase. Trace chloroform improved phase separation and increased the volume of the aqueous phase when low HLB NIDs were mixed with neutral or chaotropic HSS (Table 2B). These results showed that HSS could modulate the solubility and phase separation of NIDs. Table 2. Hofmeister series salts regulate the phase separation properties of NIDs.

Example 5: Lower HLB NIDs in neutral/chaotropic HSS solutions minimize LPS contamination

[0056] LPS (EU/mL, numerator) and protein concentration ^g/nnL, denominator) were determined in DNA generated by NID isolation and purified by NIDs and HSS. Five replicas of each sample were prepared from 30 mL LB cultures according to the NID large-scale isolation procedure followed by NID purification. Prior to the second IDP, salt concentrations in DNA solutions were adjusted as follows: NH₄Ac solutions to 2 M, NaAc solutions to 0.75 M, and 0.2 GuaHCI solutions to 0.5 M NaCI (0.75 M LiCI solutions remained unchanged). The third IDP was performed in 4.5 M LiCI, 100 mM Tris pH 9 solutions for all samples. Results are shown in Table 3. The sign + or - in the first column indicates whether the sample was treated with chloroform. The addition of neutral or chaotropic HSS to low HLB NIDs effectively minimized LPS contamination in plasmid DNA. LPS contamination was higher with high HLB NIDs (Tween 20) and kosmotropic HSS (Table 3A). Moreover, kosmotropic HSS, such as NH₄Ac or NaAc, appeared to have relatively increased LPS contamination with any NID, although to a lower degree with low HLB NIDs (Table 3B, C). Lower HLB NIDs, such as Triton X-100, Triton X-1 14, and IGEPAL CA-520, minimized LPS contamination when mixed with either neutral (LiCI) or chaotropic (GuaHCI) HSS (Table 3B, C). DNA contamination with LPS did not increase when trace chloroform was used to recover the aqueous phase after purification by lower HLB NIDs in neutral or chaotropic HSS solutions (Table 3C). These results showed that low HLB NIDs and neutral/chaotropic HSS were highly effective in purifying plasmid DNA from LPS.

Table 3. The addition of neutral or chaotropic HSS to low HLB NIDs minimizes LPS contamination in plasmid DNA.

NID Tween 201

4M NH₄Ac 2 M NaAc

LPS (EU/mL) 39,370 ± 1 1 ,310 24,380 ± 7,920 protein (pg/mL) 3.6 ± 0.6 3.7 ± 1.1

NID Triton X-100/+

0.25 M NaAc 0.75 M NaAc 1.5 M NaAc

LPS (EU/mL) 2.6 ± 0.6 2.9 ± 1.3 447 ± 125

protein (pg/mL) N/A N/A 18.1 ± 6.7 16.7 ± 4.8

Example 6: NID-purified large-scale plasmid DNA contains low levels of protein and LPS

[0057] Next, we tested different combinations of plasmid DNA isolation/purification procedures in a larger scale format (100 mL culture). 100 mL LB culture bacteria were lysed either by alkali (according to the QIAfilter maxi kit) or the NID large-scale isolation procedure. Alkali lysates were purified by Qiagen-tip 500 columns or NID purification. NID lysates were purified by ERS, NID purification procedure, or Qiagen-tip 500 columns. The NID lysates were adjusted to pH 7 before column loading. NID purification was implemented in a 1.5 mL final volume (0.2 M GuaHCI, 100 mM Tris pH 9 with either Triton X-114 or IGEPAL CA-520). Before the second IDP, 0.5 M NaCI was added to NID purified samples. The third IDP was performed in 4.5 M LiCI, 100 mM Tris pH 9 solutions. All DNA was dissolved in 250 μΙ_ TE and aliquots used to measure LPS levels, total protein, and DNA concentrations. In Table 4, "^*" indicates either XL1-Blue or DH5a were used for these experiments and gave similar results; Ave LPS_a are LPS units according to Cotton M. et al. (Cotten et al. 1994), LAL 10 EU/1 ng LPS; Ave LPS_b are LPS units according to the Qiagen's handbook, LAL 1.8 EU/1 ng LPS.

[0058] LPS levels prior to the purification procedure were similar in the alkali and NID procedure: 698, 600±13,400 EU/mL and 732,100±22,900 EU/mL, respectively. However, protein concentration prior to the purification procedure was higher in the NID compared to the alkali procedure (1045±45 μg/mL vs. 518±60 μg/mL). Samples isolated and purified by NIDs had a low protein level, but samples isolated by alkali and purified by either IGEPAL CA-520 or column contained the lowest protein contamination (Table 4, rows 2, 3 vs. 4, 5, 8). Column purification was ineffective in removing protein from N ID-extracted samples (Table 4, rows 6, 8). Adjustment of the NID cleared lysates to pH 7 (pH for the column washing buffer) did not reduce DNA contamination by either protein or LPS (Table 4, row 7). To facilitate comparison of the data, we converted the LPS units (EU/mL DNA solution) into the units used by other investigators (Ευ/θμς DNA) (Cotten M, Baker A, Saltik M, Wagner E, Buschle M. Gene Ther. 1994, 1, 239-46), or by Qiagen (EW\ vg DNA) (EndoFree Plasmid Purification Handbook, Qiagen). The LAL activity of our assay kit was 10-15 EU/1 ng LPS, the kit used by Cotton et al. had similar LAL activity (8-10 EU/1 ng LPS), while the LAL activity of the Qiagen kit was about 6.9 times lower (1.8 EU/1 ng LPS). To convert our data to the units reported by Cotton et al., the average LPS (EU/mL) was divided by the DNA concentration ^g/mL) and multiplied by 6. To convert our data to the units used in the Qiagen kit, the average LPS (EU/6 μg DNA) was divided initially by 6.9 and then by 6. The endotoxin removal solution (ERS) was not efficient in LPS removal from NID DNA (Table 4, row 1 ). The lowest amount of LPS contamination was detected in samples purified by NIDs after either alkali or NID isolation (Table 4, rows 2-5). Taken together, these data showed that large-scale NID isolation and purification were highly efficient for protein and LPS removal, and demonstrated that specific NIDs such as IGEPAL CA- 520 were more effective than columns for the purification of plasmid DNA from LPS. Table 4. NID-purified large-scale plasmid DNA contains low levels of protein and LPS.

Example 7: NID isolation and purification generates higher yields of high quality DNA compared to alkali isolation and column purification

[0059] To evaluate the effects of various plasmid DNA isolation/purification combinations on the degree of bacterial chromosomal contamination and plasmid DNA molecular structure, we studied the DNA reported in Table 4. As some NIDs absorb at 280 nm (Grant DA, Hjerten S. Biochem. J. 1977, 164, 465-8), we included A₂₆o/A₂8o ratios to ensure that no significant NID contamination occurred and that DNA spectrophotometric quality was similar using different methods.

[0060] Plasmid DNA was obtained by 5 different methods, with 2 replicas shown for each method. In Figure 4A, lanes 1-6 show DNA isolation by alkali and purification by column. In Figure 4B, lanes 1-4 show DNA isolation by alkali and purification by NID (IGEPAL CA-520). In Figure 4C, lanes 1-4 show DNA isolation by NID (IGEPAL CA-630) and purification by NID (IGEPAL CA-520). In Figure 4D, lanes 1-2 show DNA isolation by NID (IGEPAL CA-630) and purification by NID (Triton X-1 14), and lanes 3-4 show DNA isolation by alkali and purification by NID (Triton X-114). Bacterial genomic DNA was identified as the slow migrating band lost following digestion with Exonuclease λ (Figure 4A, lane 3). Denatured single stranded DNA was identified as the fast migrating form lost upon treatment with Mung bean nuclease (Figure 4A, lane 4; Figure 4B, lane 3). CCC plasmid DNA was identified as the intense form lost by treatment with nicking endonuclease Nt.BbvCI (Figure 4A, lane 5). Linear DNA (Figure 4A, lane 4; Figure 4B, lane 3; Figure 4C, lane 3) was identified as the form migrating similar to DNA linearized using the restriction endonuclease Agel (Figure 4A, lane 6; Figure 4B, lane 4; Figure 4C, lane 4).

[0061] Isolation by the alkali method and purification by column produced various plasmid DNA molecular forms (Table 4, Row 8 and Figure 4A, lanes 1 , 2). Plasmid DNA isolated and purified by NIDs was enriched in total DNA, including CCC DNA. We used various endonucleases to identify these forms. DNA digestion by exonuclease λ showed that the upper band represented bacterial genomic DNA (Figure 4A, lane 3). The lower "irreversibly denatured" band represented single stranded DNA, as identified by elimination of this band after treatment with Mung bean nuclease (Figure 4A, lane 4) (Kedzierski W, Laskowski M, Sr. J. Biol. Chem. 1973, 248, 1277-80). The most intense band represented covalently closed circular (CCC) DNA because it disappeared after treatment with nicking endonuclease (Figure 4A, lane 5). This treatment also showed that the upper portion of the middle band in lane 4 represented plasmid DNA in a relaxed form. Finally, treatment with restriction endonuclease showed that the lower portion of the middle band in lane 4 most likely represented linearized plasmid DNA (Figure 4A, lane 6). Similar DNA forms were present when DNA was isolated by alkali and purified by NIDs, specifically IGEPAL CA-520 (Table 4, Row 5 and Figure 4B, lanes 1-4). DNA isolated by NIDs (IGEPAL CA-630) and purified by NIDs (IGEPAL CA-520) had less genomic DNA and did not contain "irreversibly denatured" DNA (Table 4, Row 3 and Figure 4C, lanes 1- 4). In addition, it was enriched in total DNA, including CCC DNA and DNA in the relaxed form (Figure 4C, lanes 1-4). Enrichment with total DNA was also evident in the NID isolated DNA compared to alkali DNA when Triton X-114 was used for purification (Table 4, Row 2 and Figure 4D, lanes 1 , 2 vs. lanes 3, 4). High salt concentrations used in NID isolation/purification procedures did not inhibit linearization of plasmid DNA by the salt-sensitive endonuclease Age I (Figure 4ABC, lanes 4). Overall, these experiments showed that plasmid DNA isolation and purification using specific NIDs generated higher yields of high quality DNA compared to alkali isolation and Qiagen column purification. Example 8: Effective plasmid DNA extraction using NIDs, osmolytes, and elevated temperatures

[0062] To evaluate how different NIDs perform in plasmid DNA extraction, we used various NIDs mixed with lysozyme-treated DH5a cells harboring pUC19. Extractions of either pUC19 (Figure 5, lanes 1-8 and 10-15) or pCYPAC3 plasmids (Figure 5, lane 9) were carried out. Transformed DH5a cells were grown in 1.5 mL LB cultures and resuspended in 150 μί 50 mM Tris pH 8, 10 mM EDTA, with or without co-solutes. 500 μg/mL lysozyme was also added, and the cells were incubated as specified below. Salt concentrations were adjusted to 0.5 M NaCI in all extracts, except the ones loaded in lanes 10 and 12-15. Extracts were cleared by centrifugation, and precipitated with 150 μί isopropanol. DNA pellets were dissolved in 40 μί TE buffer, 40 μg/mL RNase A. 10 μί aliquots of the solutions containing either the pCYPAC3 (lane 9) or pUC19 plasmids (all other lanes) were loaded on the gel. Exposure times and temperatures of extraction are shown above the lanes. For lanes 1-9, extraction with the indicated NID was done in the absence of co-solutes. For lanes 10-15, the included co-solute is indicated. In Figure 5, Lane 1 : 0.5% IGEPAL CA-630; Lane 2: 0.5% TX-100; Lane 3: 0.5% IGEPAL CA-720; Lane 4: 0.5% Tween-80; Lane 5: 0.5% Tween-20; Lane 6: 2% IGEPAL CA- 720; Lane 7: 4% IGEPAL CA-720; Lane 8: 0.5% Tween 80; Lane 9: 0.5% Tween 80; Lane 10: 0.5% Tween 20/0.5 M KCI; Lane 1 1 : 0.5% Tween 20/22.5% sucrose; Lane 12: 0.5% IGEPAL CA-630/0.5 M NH₄CI; Lane 13: 0.5% TX-100/0.5 M NH₄CI; Lane 14: 0.5% TX-100/0.5 M NaCI; and Lane 15: 0.5% TX-100/0.5 M NaAc.

[0063] Plasmid DNA was extracted effectively when E. coli were exposed to NIDs with HLBs <15 (IGEPAL CA-720, Triton X-100, and IGEPAL CA-630) for 2 hours at 4°C (Figure 5, lanes 1-3), but other NIDs, such as Tween 80 and Tween 20, were ineffective at these conditions (Figure 5, lanes 4-5). A 0.5% NID concentration was more effective for DNA extraction compared to increasing NID concentrations up to 4% (shown for IGEPAL CA-720, Figure 5, lanes 3, 6, 7). The efficiency of DNA extraction increased markedly at 65°C and was similar for all NIDs (shown here only for Tween 80, Figure 5, lane 8). Under these conditions, plasmid DNA up to 19 kb (pCYPAC3) was also extracted well (Figure 5, lane 9). To decrease extraction time, we tested the effects of osmolytes. The addition of various osmolytes (sucrose, NaCI, KCI, N H₄CI, and NaAc) to the NID solutions decreased the NID extraction time down to 5 min for all NIDs studied (Figure 5, lanes 10-14). Other osmolytes tested (LiCI, LiAc, KAc, EDTA, glucose, NH₄Ac, and NH₄HCO₃) were effective but differed in their propensity to extract chromosomal DNA and precipitate DNA in aqueous isopropanol solutions (data not shown). These data demonstrated that osmolytes and elevated temperatures enhanced the activities of a variety of NIDs, allowing rapid and efficient extraction of plasmid DNA in a single solution format.

Example 9: NIP miniprep plasmid DNA is a robust substrate for digestion, ligation, and sequencing

[0064] We next compared performance of the pl_TM330 plasmid DNA obtained by either NID or alkali miniprep in digestion, ligation, and sequencing reactions.

[0065] The high copy number pl_TM330 plasmid was extracted by either alkali or NIDs protocols. XL1-Blue cells harboring pl_TM330 in 1.5 ml. LB cultures were used for isolation. After alcohol precipitation, alkali DNA pellets were rinsed with 0.5 ml. ethanol and dissolved in 40 μΙ_ TE buffer, but NID DNA pellets were directly dissolved in 40 μΙ_ TE buffer. The extracted DNA was then digested with the restriction endonuclease Sacl, and the products were separated in a gel. The plasmid used for these experiments contained 2 Sacl sites separated by approximately 400 bp, and appearance of this band was analyzed by densitometry. As shown in Figure 6A, 9 μΙ_ aliquots of native DNA of two independently isolated samples were loaded in lanes 1 , 2 (alkali, A) and lanes 3, 4 (NID, N). Densitometry analysis was reported for lanes 1-4. 9 μΙ_ DNA was also used for every DNA restriction and ligation reaction in 1 1 μΙ_ total volume. Restriction digests with the indicated amount of restriction enzyme for the indicated length of incubation at 37°C are shown in lanes 5, 7, 9, 11 for the alkali method and lanes 6, 8, 10, 12 for the NID protocol in Figure 6A. Arrow indicates "irreversibly denatured" DNA. Arrowhead indicates the 400 bp band, and densitometry analysis of this band is reported for lanes 5-12. DNA restriction reactions were carried out in NEB1 buffer. The NID and alkali minipreps produced similar amounts and molecular forms of DNA, except for some "irreversibly denatured" (fast migrating, single stranded) DNA forms in the alkali method (Figure 6A, lanes 1 ,2 vs. 3,4, arrow). At two concentrations (0.5 vs. 1 U) and incubation times (0.5 vs. 1 hour), the endonuclease Sacl digested NID miniprep DNA more efficiently than alkali miniprep DNA, as determined by densitometry quantification of the 400 bp band (Figure 6A, lanes 5-12, arrowhead). NID miniprep DNA also performed significantly better in ligation reactions.

[0066] As shown in Figure 6B, plasmid samples digested using 2 U of Sacl for 1 hour (lanes 1 , 4) were ligated using 0.1 Weiss unit T4 DNA ligase at 15°C for 30 minutes (lanes 2, 5). Ligation products were then re-digested using 2 U Sacl for 1 hour (lanes 3, 6). DNA restriction and ligation reactions were carried out in NEB1 buffer, but 1 mM ATP was added for the ligation reactions. Arrowhead indicates 400 bp band, B= cut sample, L= ligated sample, and D= re-cut sample. Lane 2 shows the intermediate ligation products of the 400 bp DNA fragment in lane 1. The major ligation product had a higher molecular weight and intermediate products were virtually absent compared to alkali isolation (Figure 6B, lanes 2, 5). Ligated NID DNA was completely re-cut using Sacl (Figure 6B, lanes 3, 6).

[0067] Isolation of low copy number plasmids requires higher bacterial culture volumes, which might result in increased DNA impurities. Thus, as shown in Figure 6C, we isolated pEL04, a low copy number plasmid, using the NID procedure and then assessed digestion. DH5a cells harboring the low copy number plasmid pEL04, which contains 2 Kpnl sites separated by approximately 1 .5 Kb. NI D plasmid isolation was performed as reported in Example 1 , except that lysozyme concentration in the extraction buffer was 50 μg/mL. 9 μί DNA in 1 1 [iL total reaction volume were used for digestions. Referring to Figure 6C, Lane 1 : native DNA (N); Lane 2: DNA digestion by 1 .5u Kpnl ; Lane 3: 3u Kpnl; Lane 4: 5u Kpnl; and Lane 5: 1 Kb Plus DNA Ladder (Invitrogen). The incubation times were 1 hour. Densitometry analysis of the expected 1 .5 Kb digestion product is reported in lanes 2-4. We isolated DNA from 2 mL cultures (vs. 1.5 mL in high copy number plasmids), and dissolved the DNA in 20 μί TE buffer (vs. 40 \L in high copy number plasmids). As the amount of DNA was enough for only two digestions, we combined DNA from three independent samples. We used the salt-sensitive restriction endonuclease Kpnl, with expected digestion products of 1 .5 and 3.5 Kb. We found that 3 units of Kpnl were sufficient to achieve almost complete digestion (Figure 6C). These data showed that the N ID procedure can be used to isolate DNA from low copy number plasmids.

[0068] Crude lysates of alkali minipreps cannot be used for downstream applications because of their low DNA concentration, SDS content, and acidic pH. NID minipreps are free of these shortcomings, leading us to examine whether NID crude lysates could be directly used in downstream applications, such as digestion and sequencing reactions. We assessed how NID plasmid DNA from crude lysates (i.e., N ID miniprep, steps 1 -4) performed in digestion reactions compared to isopropanol precipitated-NID plasmid DNA (i.e., NID miniprep, steps 1 -6). 2 mL bacterial cultures containing plasmid B254 were processed according to the NI D miniprep plasmid isolation procedure with (complete procedure, CP) or without (crude lysate, CL) isopropanol precipitation. 50 mM MgS0₄ was added to the CL to chelate 50 mM EDTA in the extraction buffer. All CL samples were digested in 15 μΙ_ NEB 1 buffer (no salt), and 1 μΙ_ CL was used for digestions. CP samples were digested in the specific NEB buffers recommended for the restriction endonuclease, and 0.5 μΙ_ DNA was used for every reaction. Samples were incubated with 5U of each enzyme at 37°C for 1 hr. The reaction products of CL digestion were loaded in odd-numbered lanes, while digestion products of CP DNA were loaded in even- numbered lanes. All tested restriction endonucleases except Xhol digested crude lysate DNA (Figure 7). DNA molecular forms were similar when NID crude lysates were immediately precipitated with isopropanol or stored overnight at room temperature (data not shown). The possible effects of storage were tested because covalently closed circular (CCC) plasmid DNA derived from NID-based procedures can be relaxed by heat and/or storage in EDTA containing solutions.

[0069] We also found that NID miniprep plasmid DNA was a robust template in sequencing reactions without requiring any additional purification. NID crude lysates of XL1-Blue cells harboring B256 plasmid were prepared as described in Example 1 but using 5 mM EDTA in the extraction buffer. Lysate EDTA was chelated with 5 mM MgS0₄ before the sequencing reaction. 12 μΙ_ primer/crude lysate mix containing 4 pmoles M13f primer was diluted by adding 15 μΙ_ water. 6 μΙ_ of this diluted mix and 4 μΙ_ of Big Dye mix made up the sequencing reaction mixes. NID crude lysates could also be used in sequencing reactions as 1 μΙ_ crude lysate generated reliable sequencing data with trimmed length 783 bases. Increasing the amount of crude lysate to 3 μΙ_ improved the trimmed length slightly to 804 bases, but 4 μΙ_ crude lysates reduced it significantly to 523 bases. Taken together, these data provide evidence that NID minipreps can be reduced to a one-step procedure (i.e., isopropanol precipitation is not required). NID miniprep plasmid DNA was highly suited for a variety of common downstream applications, and outperformed standard alkali miniprep DNA in digestion and ligation reactions, possibly because of higher purity.

Example 10: NID minipreps are time-efficient compared to the alkali miniprep

[0070] Alkali minipreps known in the art require three solutions for completion of plasmid DNA isolation, but NID minipreps as described herein require only one. To confirm that NID minipreps were more time-efficient compared to alkali minipreps, we compared completion time with up to thirty samples of alkali and NID minipreps. Both procedures were performed without rinsing DNA pellets with 70% ethanol. Distriman repetitive pipette (Gilson, Inc., Middleton, Wl) was used to dispense solutions. Initiation of next step of procedure was not allowed until ongoing step was completed. The total procedure time was the sum of operational time and the preparation/working equipment times, such as incubators and centrifuges. The time required to complete one miniprep was similar, although the NID miniprep required relatively less operator- dependent time (Table 5). As the number of samples increased, the time advantage of the NID miniprep increased significantly for both the total and operator-dependent times. For thirty minipreps, the total time was 78 min for alkali, but only 59 min for NID minipreps, while the operator-dependent time was 58 min for alkali vs. 34 min for NIDs (Table 5). In addition, the ratio of the operator-dependent time to the total time was 74% with alkali vs. 58% with NID minipreps (Table 5).

Table 5. Time requirements of alkali vs. NID plasmid minipreps.

[0071] Claiming a time advantage of a procedure over previous ones is often difficult because it is hard to evaluate the data and extrapolate the conclusions to any sample number. To address this issue, we developed a linear regression mathematical model. When the same operator completed both the alkali and NID procedure, the total procedures times were dependent variables as shown in Table 5 (linear correlation coefficient, r, 0.988, implying that T_ai_k= aTni_d+5, where a and b are constants). Thus, the ratio of the variables

a+b/nk (see Materials and Methods in Example 1 ). When the number of samples (n) was high, 5/n/ ~0, and T_ai_k/T_nicra, a constant. This suggested that although alkali and NID operational times can change based on the operator, their ratio tended to be invariant. Using the operator- dependent times in Table 5, k was calculated as 2.03 ± 0.06 min (coefficient of determination, r² =0.995) and 1.20 ± 0.05 min (r²=0.991 ) for the alkali and NID minipreps, respectively. Thus, at high sample numbers (when operator-dependent time can replace total time), NID minipreps were 69% more time-efficient compared to alkali minipreps (2.03/1.20 - 1 x 100). Overall, these data demonstrated that NID minipreps saved significant time compared to alkali minipreps, and suggested that NID minipreps are particularly suitable for high-throughput applications.

Example 11 : NID minipreps effectively isolate bacteriophage lambda particles

[0072] To further assess the NID procedure, we attempted to extract bacteriophage lambda particles. Lambda prophage of UB61 cells can be induced by heat shock. The phage particles accumulate in host bacteria without lysing them because of a mutation in lambda gene S. Cell lysis and release of phage particles can be achieved by briefly incubating the cells with chloroform (Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: A Laboratory Manual. New York Cold Spring Harbour Lab. Press).

[0073] Lambda phage DNA was then isolated using NID-based extraction. Referring to Figure 8, the DNA samples loaded in lanes 1-5 were generated by varying only the composition of the phage particle extraction buffer. The particles were precipitated and the lambda DNA was extracted from all samples using the same procedure as described in Example 1. In Figure 8, for the sample in Lane 1 , bacteria were incubated in the isotonic component of the lambda NID extraction buffer, i.e. 5% sucrose and 50 mM Tris pH 8, and 10 μί DNA were loaded in the gel. Note minimal leakage of phage particles in this lane. For the sample in Lane 2 the same buffer as lane 1 was used, but with the addition of 50 mM CaCI₂, and 10 μί DNA were loaded. Note partial suppression of phage particles leakage. For the sample in lanes 3 and 4, all components of the lambda NID extraction buffer were added except lysozyme and Tween 80, respectively, and 3 μί DNA were loaded. For the sample in Lane 5, bacteria were incubated with the complete lambda NID extraction buffer, and 2 μί DNA were loaded. For the sample in Lane 6, cells were lysed with chloroform (full lysis sample), and 0.5 μί DNA were loaded on the gel. For the sample in Lanes 7 and 8, Hind III was used to digest the samples loaded in lanes 5 and 6, respectively. Densitometry analysis of the bands in lanes 1-6 was reported. Approximately 6 hours after heat induction of the lambda prophage and without addition of chloroform, UB-61 cells released some lambda particles in solutions of 5% sucrose and Tris (Figure 8, lane 1 ), which was defined as "leakage" of lambda particles. However, we found that 50 mM CaCI₂ in the extraction buffer decreased phage leakage (Figure 8, lane 2). Both lysozyme and Tween 80 were effective in phage isolation as in the absence of either one, extraction efficiency decreased markedly compared to the buffer containing all components (Figure 8, lanes 3-5). The NID method extracted about 50% less lambda DNA compared to full cell lysis (Figure 8, lanes 5-6, see DNA loading in legend). To confirm that the bands in lanes 4 and 5 are lambda DNA, we treated both samples with Hindi 11 to generate classical digestion products (Figure 8, lanes 7-8). These data demonstrated that NID minipreps are mild extraction procedures that can isolate various bacterial extra-chromosomal DNA elements.

Example 12: Effects of different plasmid isolation procedures on E. coli cells

[0074] We reasoned that the efficiency of various isolation methods might be dependent on their effects on E. coli cells. Thus, we assessed E. coli cell morphology and plasmid DNA molecular forms after the NID procedure and the following approaches: isolation using Brij-58 according to Godson and Sinsheimer (Godson GN, Sinsheimer RL. Biochim. Biophys. Acta. 1967, 149, 476-488), isolation using Brij-58 and DOX according to Clewell and Helinski (Clewell DB, Helinski DR. Biochemistry 1970, 9, 4428-4440), isolation using Brij-58 and DOX in highly hypertonic salt-sucrose solutions according to Summerton et al. (Summerton J, Atkins T, Bestwick R. Anal. Biochem. 1983, 133, 79-84), and the classical alkali method according to Birnboim and Doly (Birnboim HC, Doly J. Nucleic Acids Res. 1979, 7, 1513-1523). Because the first 3 methods did not provide a miniprep protocol, we scaled-down these procedures to generate 150 μΙ_ cell lysates, matching the volume of crude extract of our NID approach.

[0075] Plasmids were isolated from 2 ml. XL1-Blue cultures containing multicopy plasmid B254, a pBluescript-based plasmid. In Figure 9, the sample in Lane 1 is 1 Kb DNA ladder. For the sample in Lane 2, the isolation was according to Godson and Sinsheimer using Brij-58, and 15 [iL DNA were loaded. Panel A shows corresponding E.coli cell morphology. For the sample in Lane 3, isolation was according to Clewell and Helinski using Brij-58 and DOX. In this and all following lanes 3 μί DNA were loaded. Panel B shows corresponding E.coli cell morphology. For the sample in Lane 4, isolation was according to Summerton et al. using Brij-58 and DOX in highly hypertonic salt-sucrose solutions. Panel C shows corresponding E.coli cell morphology. For the sample in Lane 5, alkali isolation was according to Birnboim and Doly. Panel D shows corresponding E.coli cell morphology. For the sample in Lane 6, isolatedion was by NID isolation. Panel E shows corresponding E.coli cell morphology. Densitometry analysis of plasmid DNA isolated with the different methods was reported.

[0076] Bacteria extracted in Brij-58 maintained a rod-like appearance (Figure 9, panel A), but the amounts of fast and slow-migrating plasmid DNA were low (Figure 9, lane 2). When Brij-58 and DOX were used, numerous bacteria maintained a rod-like appearance but some were disrupted (Figure 9, panel B), although the levels of fast and slow-migrating plasmid DNA remained low (Figure 9, lane 3). Isolation using Brij-58 and DOX in hypertonic salt-sucrose solutions led to a more significant degree of bacterial cell disruption (Figure 9, panel C) and higher amounts of fast-migrating plasmid DNA, but slow-migrating DNA remained low (Figure 9, lane 4). The alkali method led to complete disintegration of bacteria (Figure 9, panel D) and the levels of both fast and slow-migrating plasmid DNA were high (Figure 9, lane 5). The NID miniprep led to an intermediate degree of bacterial disruption and protoplast-like cell morphology (Figure 9, panel E). Levels of both fast and slow-migrating plasmid DNA were high (Figure 9, lane 6). To facilitate comparison of the different methods, we summarized their key features in table format (Table 6). These data revealed how different plasmid DNA isolation methods affect E. coli cells, and provided evidence for the connection between the bacterial cell morphology induced by a specific procedure and its efficiency.

Table 6. Main features of various bacterial extra-chromosomal element isolation procedures.

Claims

CLAIMS We claim:

1. A method for extracting a polynucleotide comprising contacting a cell or cell suspension with a composition comprising a non-ionic detergent (NID) under conditions that allow disruption of the cell membrane and extraction of the polynucleotide.

2. The method of claim 1 , wherein the composition further comprises at least one enzymatic cell lysis agent or at least one chemical cell lysis agent.

3. The method of claim 1 or 2, wherein the composition further a composition comprising an osmolyte selected from a sugar, an amino acid, a polyol, a methylamine, a methylsolfonium compound, and a salt, or a combination thereof.

4. The method of any one of claims 1-3, further comprising centrifuging the composition to isolate the cell lysate comprising the polynucleotide.

5. A method for purifying a polynucleotide comprising:

(1 ) contacting a solution containing the polynucleotide with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition;

(2) contacting the solution resulting from step (1 ) with a composition comprising a non-ionic detergent (NID);

(3) adding a hydrophobic halogenated hydrocarbon to the solution resulting from step (2); and

(4) centrifuging the solution resulting from step (3) to obtain a first aqueous phase comprising the polynucleotide.

6. The method of claim 5, further comprising: (5) repeating steps (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from step (4) to obtain a second aqueous phase comprising the polynucleotide.

7. The method of claim 6, further comprising:

(6) saturating the second aqueous phase comprising the polynucleotide resulting from step (5) with a hydrophobic halogenated hydrocarbon;

(7) centrifuging the solution resulting from step (6) to form a supernatant and pellet; and

(8) collecting the supernatant comprising the polynucleotide.

8. The method of any one of claims 5-7, further comprising:

(a) reconstituting the polynucleotide from step (5) or (8) in a composition comprising LiCI;

(b) precipitating the polynucleotide from the solution resulting from step (a) with isopropanol; and

(c) centrifuging the isopropanol solution from (b) to pellet the polynucleotide and dissolving the polynucleotide pellet in a buffer.

9. A method for extracting a lipopolysaccharide from a solution comprising a polynucleotide, the method comprising:

(1 ) contacting the solution with a composition comprising a Hofmeister series salt (HSS) and a buffer that maintains the pH at about 8-10 in the composition;

(2) contacting the solution resulting from step (1 ) with a composition comprising a non-ionic detergent (NID);

(3) adding a hydrophobic halogenated hydrocarbon to the solution resulting from step (2);

(4) centrifuging the solution resulting from step (3) to obtain a first aqueous phase comprising the polynucleotide; and

(5) repeating steps (2), (3), and (4) on the first aqueous phase comprising the polynucleotide resulting from step (4) to obtain a second aqueous phase comprising the polynucleotide.

10. The method of claim 9, further comprising:

(6) saturating the second aqueous phase comprising the polynucleotide resulting from step (5) with a hydrophobic halogenated hydrocarbon;

(7) centrifuging the solution resulting from step (6) to form a supernatant and pellet; and

(8) collecting the supernatant comprising the polynucleotide.

11. The method of claim 9 or 10, further comprising:

(a) reconstituting the polynucleotide from step (5) or (8) in a composition comprising LiCI;

(b) isopropanol precipitating the polynucleotide from the solution resulting from step (a); and

(c) centrifuging the isopropanol solution from (b) to pellet the polynucleotide and dissolving the polynucleotide pellet in a buffer.

12. The method of claim 8 or 1 1 , wherein the polynucleotide pellet of step (c) is dissolved in a buffer about pH 8-10, and further comprising adding an equal volume of salt.

13. The method of any one of claims 5-12, further comprising heating at 70°C the solution resulting from step (3) before centrifuging in step (4).

14. The method of claim 7 or 10, further comprising heating the solution resulting from step (6) before centrifuging in step (7).

15. The method of any one of claims 5-8, wherein the solution containing the polynucleotide comprises a cell lysate.

16. The method of any one of claims 5-8, wherein the solution containing the polynucleotide comprises lipopolysaccharide.

17. The method of any one of the above claims, wherein the HSS is selected from a salt having: (a) an anion selected from group consisting of acetate, chloride, nitrate, bromide, chlorate, perchlorate, iodide, thiocyanate, and cyanate; or (b) a cation selected from the group consisting of potassium, sodium, lithium, magnesium, calcium, and guanidinium.

18. The method of any one of the above claims, wherein the HSS is selected from the group consisting of guanidinium chloride, LiCI, NaCI, NH₄CI, and LiAc.

19. The method of any one of the above claims, wherein the NID has an HLB of less than about 13 and a specific density greater than about 1.0.

20. The method of any one of the above claims, wherein the NID is selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert-octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene.

21. The method of any one of the above claims, wherein the NID has an HLB of less than about 13 and a specific density greater than about 1.0, and wherein the NID is selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert-octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene

22. The method of any one of the above claims, wherein the HSS comprises guanidinium and the NID is selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert- octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene.

23. The method of any one of claims 20-22, wherein the acyl polyoxyethylene sorbitan ester is a compound of the formula:

wherein n-i + n₂ + n₃ + n₄ = x, wherein x is an integer from 1 to 20, and R is Ci-C₂o alkyl.

24. The method of any one of claims 20-22, wherein the tert-octylphenyl polyoxyethylene ether is a compound of the formula:

wherein n is an integer from 5 to 8.

25. The method of any one of claims 20-22, wherein the alkyl polyoxyethylene is a compound of the formula:

R(OCH₂CH₂)xOH (Ml)

wherein x is an integer from 1 to 20, and R is Ci-C₂o alkyl.

26. The method of any one of claims 20-22, wherein the alkyl phenylpolyoxyethyl compound of the formula:

(IV) wherein x is an integer from 1 to 20.

27. The method of any one of the above claims, wherein the NID is added to a final concentration of about 0.1-10% by volume.

28. The method of any one of the above claims, wherein the HSS is present at a concentration of about 0.05 M to about 2 M in the composition.

29. The method of any one of the above claims, wherein the buffer comprises Tris-HCI.

30. The method of claim 6 or 9, wherein the solution resulting from step (5) comprises lipopolysaccharide in an amount less than about 10 EU/mL.

31. The method of claim 7 or 10, wherein the solution resulting from step (8) comprises lipopolysaccharide in an amount less than about 10 EU/mL.

32. The method of claim 8 or 1 1 , wherein the solution resulting from step (c) comprises lipopolysaccharide in an amount equal to or less than about 5 EU/mL.

33. The method of any one of the above claims, wherein the polynucleotide comprises plasmid DNA or phage DNA.

34. A kit for purifying a polynucleotide from a composition, the kit comprising:

(a) a composition comprising a HSS and a buffer that maintains the pH at about 8-10 in the composition; and

(b) a composition comprising a NID.

35. A kit for extracting a polynucleotide from a cell or cell suspension, the kit comprising:

(a) a composition comprising a NID; and

(b) optionally, a composition comprising a HSS and a buffer that maintains the pH at about 8-10 in the composition.

36. The kit of claim 34 or 35, further comprising instructions for use.

37. The kit of any one of claims 34-36, wherein the HSS is selected from the group consisting of guanidinium chloride, LiCI, NaCI, NH₄CI, and LiAc.

38. The kit of any one of claims 34-37, wherein the NID has an HLB of less than about 13 and a specific density greater than about 1.0.

39. The kit of any one of claims 34-38, wherein the NID is selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert-octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene.

40. The kit of any one of claims 34-39, wherein the NID has an HLB of less than about 13 and a specific density greater than about 1.0, and wherein the NID is selected from the group consisting of acyl polyoxyethylene sorbitan ester, tert-octylphenyl polyoxyethylene ether, alkyl polyoxyethylene, and alkyl phenylpolyoxyethylene.

41. The kit of claim 39 or 40, wherein the acyl polyoxyethylene sorbitan ester is a compound of the formula:

wherein n-i + n₂ + n₃ + n₄ = x, wherein x is an integer from 1 to 20, and R is Ci-C₂o alkyl.

42. The kit of claim 39 or 40, wherein the tert-octylphenyl polyoxyethylene ether is a compound of the formul

wherein n is an integer from 5 to 8.

43. The kit of claim 39 or 40, wherein the alkyl polyoxyethylene is a compound of the formula:

R(OCH₂CH₂)_xOH (III)

wherein x is an integer from 1 to 20, and R is Ci-C₂₀ alkyl.

44. The kit of claim 39 or 40, wherein the alkyl phenylpolyoxyethylene is a compound of the formula:

wherein x is an integer from 1 to 20.