U.S. patent application number 17/554861 was filed with the patent office on 2022-06-16 for methods for nucleic acid capture.
The applicant listed for this patent is ZYMO RESEARCH CORPORATION. Invention is credited to Jonathan A. CLAYPOOL, Xi-Yu JIA, Ryan KEMP, Marc E. VAN EDEN.
Application Number | 20220186207 17/554861 |
Document ID | / |
Family ID | 1000006168289 |
Filed Date | 2022-06-16 |
United States Patent
Application |
20220186207 |
Kind Code |
A1 |
KEMP; Ryan ; et al. |
June 16, 2022 |
METHODS FOR NUCLEIC ACID CAPTURE
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.
Inventors: |
KEMP; Ryan; (Irvine, CA)
; CLAYPOOL; Jonathan A.; (Irvine, CA) ; VAN EDEN;
Marc E.; (North Tustin, CA) ; JIA; Xi-Yu;
(Newport Beach, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ZYMO RESEARCH CORPORATION |
Irvine |
CA |
US |
|
|
Family ID: |
1000006168289 |
Appl. No.: |
17/554861 |
Filed: |
December 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15961995 |
Apr 25, 2018 |
11236325 |
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17554861 |
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14619037 |
Feb 10, 2015 |
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15961995 |
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61937824 |
Feb 10, 2014 |
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62013668 |
Jun 18, 2014 |
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62079358 |
Nov 13, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/1006
20130101 |
International
Class: |
C12N 15/10 20060101
C12N015/10 |
Claims
1-101. (canceled)
102. A method of isolating plasmid DNA, comprising: (a) obtaining a
sample comprising plasmid DNA; (b) capturing the plasmid DNA to a
mineral matrix with a phase separation solution comprising domiphen
bromide (DB) under conditions that allow for selective capture of
plasmid DNA versus RNA; (c) treating the mineral matrix and
captured plasmid DNA with a salt solution, thereby enhancing the
retention of the captured plasmid DNA; (d) washing the mineral
matrix and captured plasmid DNA with an organic wash solution; and
(e) eluting the plasmid DNA from the mineral matrix, thereby
isolating the plasmid DNA.
103. The method of claim 102, wherein the salt solution comprises
lithium salt, sodium salt, potassium salt, magnesium salt, calcium
salt, or combinations thereof.
104. The method of claim 102, wherein the organic wash solution
comprises an alcohol.
105. The method of claim 104, wherein the organic wash solution
comprises at least 60% ethanol or isopropanol.
106. The method of claim 102, wherein the phase separation solution
of (b) comprises 0.05% to 2% DB.
107. The method of claim 102, wherein the phase separation solution
of (b) comprises 0.05% to 1% DB and 0.05 M to 1.0 M lithium
chloride, sodium chloride, potassium chloride, lithium acetate,
sodium acetate, or potassium acetate.
108. The method of claim 102, wherein the phase separation solution
of (b) comprises a Tris-HCl buffer and 0.6 M to 1.05 M lithium
chloride.
109. The method of claim 102, wherein the obtaining a sample
comprising plasmid DNA comprises obtaining a bacterial cell lysate
by alkaline lysis.
110. The method of claim 109, wherein obtaining the bacterial cell
lysate comprises: (i) lysing cells comprising plasmid DNA with a
first solution thereby generating a lysate; and (ii) neutralizing
the lysate with a second solution thereby precipitating genomic DNA
and/or proteins.
111. The method of claim 110, further comprising (iii) clearing the
precipitate.
112. The method of claim 110, wherein the first solution comprises
sodium hydroxide and sodium dodecyl sulfate.
113. The method of claim 110, wherein the second solution comprises
potassium acetate and RNAse A.
114. The method of claim 113, wherein the potassium acetate is
present at a concentration of about 0.8 to about 3 M.
115. The method of claim 102, wherein the isolated plasmid DNA is
essentially free of endotoxin and/or PCR inhibitors.
116. A composition comprising: (i) domiphen bromide (DB); (ii) a
salt selected from the group consisting of NaCl, NaoAc, KCl, KoAc,
LiCl, LioAc, sodium formate, potassium formate, lithium formate,
calcium chloride, and magnesium chloride; and (iii) a mineral
matrix, comprising nucleic acid molecules captured thereto.
117. The composition according to claim 116, wherein the salt is
lithium chloride (LiCl).
118. The composition according to claim 117, further comprising a
Tris-HCl buffer.
119. The composition according to claim 118, comprising a final
concentration (M) of about 0.05 M to 1.05 M LiCl.
120. The composition according to claim 116, comprising a final
concentration (w/v) of about 0.2% to about 0.5% of DB.
121. The composition according to claim 116, wherein the mineral
matrix is a silica matrix and wherein the nucleic acid molecules
comprise plasmid DNA molecules.
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 15/961,995, filed Apr. 25, 2018, which a divisional of
U.S. patent application Ser. No. 14/619,037, filed Feb. 10, 2015,
now Abandoned, which claims the benefit of U.S. Provisional Patent
Application Nos. 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
1. Field of the Invention
[0002] The present invention relates generally to the field of
molecular biology. More particularly, it concerns nucleic acid
purification, particularly the isolation of DNA.
2. Description of Related Art
[0003] 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 downstream recombinant DNA manipulations. The sensitive
reactions commonly employed in molecular biology experiments of
reverse transcription, transcription, DNA and RNA sequencing,
polymerase chain reaction (PCR), restriction digests, ligation
reactions, end modifications, among other similar base modification
procedures require the DNA, or other nucleic acid molecules, be
essentially free from contaminants. It is also desirable to isolate
the nucleic acid in significant quantities to ensure a reliable
source of material with which to proceed to additional experiments.
In many instances there is a need to move a desired DNA, or
fragment thereof, through several manipulations to reach the
desired endpoint. 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.
[0004] Conventional procedures for isolating plasmid DNA, for
example, include harvesting the bacterial cells and obtaining the
plasmid DNA, or other target nucleic acid, in a pure form via
lysis, free from undesirable contaminating medium and cellular
constituents. This is typically called a cleared bacterial or
cellular lysate. The cell lysis may be performed in a variety of
ways including mechanical sonication or blending, enzymatic
digestion and also the traditional chemical means of alkaline
lysis. The alkaline lysis based protocols remain the basis for many
plasmid purification methods, though other procedures, such as the
boiling lysis, triton lysis, and polyethylene glycol protocols, are
also used (Bimboim and Dolly, 1979; Bimboim, 1983; Holmes and
Quigley, 1981; Clewell and Helinski, 1970; Lis and Schleif,
1975).
[0005] Approaches that coupled alkaline lysis to cesium chloride
gradient centrifugation and organic extraction with toxic and
caustic phenol/chloroform and alcohols have largely been replaced
by a variety of systems that use rapid and efficient
chromatographic methods. The observation that DNA bound
preferentially to ground glass or glass fiber disks in the presence
of high concentrations of sodium iodide or sodium perchlorate
allowed the development of new purification methodologies (Marko et
al., 1981; Vogelstein et al., 1979). The use of the chaotropic salt
solutions, such as guanidinium, iodide, perchlortate, and
trichloroacetate, coupled to forms of silica-based or other
chromatographic techniques, has resulted in a preferred methodology
for plasmid as well as general nucleic acid purification. Despite
these improvements and the development of numerous nucleic acid
purification systems there remains a need to develop improved
systems to satisfy demands for easier, faster protocols with
increased yield and reliability for high-level quantity
purification of plasmids and other nucleic acid materials.
SUMMARY OF THE INVENTION
[0006] 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.
[0007] In certain embodiments, there is provided method for phase
separating nucleic acids from a solution and capturing nucleic
acids with a mineral matrix for purification comprising (a)
contacting a nucleic acid-containing sample (e.g., a sample
comprising plasmid DNA) with a phase separation reagent comprising
a cationic surfactant of the embodiments and (b) capturing the
phase separated nucleic acid with the mineral matrix (e.g., a
silica-based matrix, such as borosilicate glass fiber). In some
aspects, the method further comprises one or more of the following
steps: c) treating the captured nucleic acid with a salt solution
(e.g., thereby increasing the retention of nucleic acid); (d)
washing the captured nucleic acid with an organic wash solution
(e.g., thereby purifying the captured nucleic acid); and/or (e)
eluting the captured nucleic acid from the mineral matrix, thereby
isolating the nucleic acid. Thus, methods of the embodiments,
involve the mixing of a number of reagents as part of the
purification protocol. Through-out the application concentrations
or various reagent constituents are, in some cases, listed for
simplicity as those in the original solution (prior to any mixing).
However, concentration of solution components are also provided as
"a final concentration" (e.g., in w/v, v/v or M), which as used
herein refers to the concentration of the component at the time of
nucleic acid capture to a mineral matrix (e.g., just prior to a
wash step).
[0008] Thus, in one embodiment there is provided a reagent for use
in purification of nucleic acids, such as plasmid DNA, the reagent
comprising a cationic surfactant of Formula I:
##STR00001## [0009] wherein: [0010] X.sub.1 is nitrogen,
phosphorus, N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18), provided that when X.sub.1 is
N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18), then R.sub.2, R.sub.3, and R.sub.4
are absent and when R.sub.2, R.sub.3, and R.sub.4 are absent, then
X.sub.1 is N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18); [0011] R.sub.1 is
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), a substituted version of any of
these groups or --Y.sub.1--Z--Y.sub.2; [0012] Y.sub.1 is
alkandiyl.sub.(C.ltoreq.6), alkendiyl.sub.(C.ltoreq.6),
alkyndiyl.sub.(C.ltoreq.6), arenediyl.sub.(C.ltoreq.6),
alkoxydiyl.sub.(C.ltoreq.6), or a substituted version of any of
these groups; [0013] Z is --O--, --NH--, --S--, --C(O)O--,
--OC(O)--, --NHC(O)--; or --C(O)NH--; [0014] Y.sub.2 is
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), or a substituted version of any of
these groups; [0015] R.sub.2, R.sub.3, and R.sub.4 are each
independently alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), a substituted version of any of
these groups or --Y.sub.3--Z--Y.sub.4; [0016] Y.sub.3 is
alkandiyl.sub.(C.ltoreq.6), alkendiyl.sub.(C.ltoreq.6),
alkyndiyl.sub.(C.ltoreq.6), arenediyl.sub.(C.ltoreq.6), or a
substituted version of any of these groups; [0017] Z is --O--,
--NH--, --S--, --C(O)O--, --OC(O)--, --NHC(O)--; or --C(O)NH--;
[0018] Y.sub.4 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
substituted version of any of these groups; and [0019] A is
bromide, chloride, iodide, phosphate, sulfate, acetate, formate,
propionate, oxalate, or succinate,
[0020] provided that when R.sub.2, R.sub.3 and R.sub.4 are
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), or a halo substituted version of
any of these groups and X.sub.1 is nitrogen or phosphorus, then
R.sub.1 is --Y.sub.1--Z--Y.sub.2. For example, in some aspects, a
phase separation reagent of the embodiments comprises about 0.1% to
about 10% of a cationic surfactant of Formula I (e.g., 0.15% to
about 10.0%, 0.15% to about 5.0%, 0.5% to about 2.0%, or 0.8% to
about 1.2%). Thus, in some aspects, a cationic surfactant of
Formula I is present in a final concentration (w/v) of 0.025% to
about 2.5% (e.g., 0.0375% to about 2.5.0%, 0.0375% to about 1.25%,
0.125% to about 0.5%, or 0.2% to about 0.3%). In still further
aspects, a phase separation reagent comprises a salt selected from
the group consisting of NaCl, NaoAc, KCl, KoAc, LiCl, LioAc, sodium
formate, potassium formate, lithium formate, calcium chloride,
magnesium chloride and a mixture thereof. In yet further aspects, a
phase separation reagent further comprises a pH buffering reagent,
such as a Tris or Bis-Tris or KoAc. Thus, in some aspects, a phase
separation reagent comprises a pH between about 3.5 and about 9.0,
between about 5.0 and 8.0, between about 6.0 and 8.0; between about
6.5 and 7.5 or between about 6.7 and 7.3 (e.g., between 6.8 and
7.3).
[0021] In some aspects, a surfactant of Formula I is further
defined as:
##STR00002## [0022] wherein: X.sub.1 is nitrogen or phosphorus;
R.sub.1 is --Y.sub.1--Z--Y.sub.2; Y.sub.1 is
alkandiyl.sub.(C.ltoreq.6), alkendiyl.sub.(C.ltoreq.6),
alkyndiyl.sub.(C.ltoreq.6), arenediyl.sub.(C.ltoreq.6),
alkoxydiyl.sub.(C.ltoreq.6), or a substituted version of any of
these groups; Z is --O--, --NH--, --S--, --C(O)O--, --OC(O)--,
--NHC(O)--; or --C(O)NH--; Y.sub.2 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
substituted version of any of these groups; R.sub.2, R.sub.3, and
R.sub.4 are each independently alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), a
substituted version of any of these groups or
--Y.sub.3--Z--Y.sub.4; Y.sub.3 is alkandiyl.sub.(C.ltoreq.6),
alkendiyl.sub.(C.ltoreq.6), alkyndiyl.sub.(C.ltoreq.6),
arenediyl.sub.(C.ltoreq.6), or a substituted version of any of
these groups; Z is --O--, --NH--, --S--, --C(O)O--, --OC(O)--,
--NHC(O)--; or --C(O)NH--; Y.sub.4 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
substituted version of any of these groups; and A is bromide,
chloride, iodide, phosphate, sulfate, acetate, formate, propionate,
oxalate, or succinate. In still further aspects, a cationic
surfactant of Formula I is defined as:
[0022] ##STR00003## [0023] wherein: X.sub.1 is nitrogen or
phosphorus; R.sub.1 is --Y.sub.1--Z--Y.sub.2; Y.sub.1 is
alkandiyl.sub.(C.ltoreq.6) or substituted
alkandiyl.sub.(C.ltoreq.6); Z is --O--; Y.sub.2 is
alkyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), or a substituted version of any of these
groups; R.sub.2, R.sub.3, and R.sub.4 are each independently
alkyl.sub.(C.ltoreq.30) or substituted alkyl.sub.(C.ltoreq.30); and
A is bromide, chloride, iodide, phosphate, sulfate, acetate,
formate, propionate, oxalate, or succinate. In still further
aspects, a cationic surfactant of Formula I is defined as:
[0023] ##STR00004## [0024] wherein X.sub.1 is
N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18); R.sub.1 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), a
substituted version of any of these groups or
--Y.sub.1--Z--Y.sub.2; Y.sub.1 is alkandiyl.sub.(C.ltoreq.6),
alkendiyl.sub.(C.ltoreq.6), alkyndiyl.sub.(C.ltoreq.6),
arenediyl.sub.(C.ltoreq.6), or a substituted version of any of
these groups; Z is --O--, --NH--, --S--, --C(O)O--, --OC(O)--,
--NHC(O)--; or --C(O)NH--; Y.sub.2 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
substituted version of any of these groups; and A is bromide,
chloride, iodide, phosphate, sulfate, acetate, formate, propionate,
oxalate, or succinate.
[0025] Thus, in some aspects, a cationic surfactant according to
Formula I comprises wherein a position A, which is bromide or
chloride; R.sub.1 is --Y.sub.1--Z--Y.sub.2; R.sub.2 which is
alkyl.sub.(C.ltoreq.30) (e.g., methyl); R.sub.3 which is
alkyl.sub.(C.ltoreq.30) (e.g., methyl); and/or R.sub.4 which is
alkyl.sub.(C.ltoreq.30) (e.g., methyl). In certain aspects, R.sub.1
is alkyl.sub.(C.ltoreq.30), alkyl.sub.(C.ltoreq.20),
alkyl.sub.(C.ltoreq.10), alkyl.sub.(C.ltoreq.4), decane,
hexadecane, aryl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.20),
aryl.sub.(C.ltoreq.10), benzyl, ethyl or methyl. In still further
aspects, R.sub.2 is alkyl.sub.(C.ltoreq.20),
alkyl.sub.(C.ltoreq.10), alkyl.sub.(C.ltoreq.4), ethyl or methyl.
In yet further aspects, R.sub.3 is methyl, ethyl, benzyl,
aralkyl.sub.(C.ltoreq.30), alkyl.sub.(C.ltoreq.20),
alkyl.sub.(C.ltoreq.10), alkyl.sub.(C.ltoreq.4) or dodecane. In yet
still further aspects, R.sub.4 is alkyl.sub.(C.ltoreq.20),
alkyl.sub.(C.ltoreq.10), alkyl.sub.(C.ltoreq.4), ethyl or methyl.
In still further aspects, X.sub.1 is pyridinium,
4-carbamoylpyridinium, 1-decylimidazolium,
1-decyl-2-methyl-imidazolium, N-heteroaryl.sub.(C.ltoreq.18) or
substituted N-heteroaryl.sub.(C.ltoreq.18).
[0026] In some further aspects, the R.sub.1 position of a cationic
surfactant of Formula I is --Y.sub.1--Z--Y.sub.2, and Y.sub.1 is
alkandiyl.sub.(C.ltoreq.6) (e.g., --CH.sub.2CH.sub.2-- or
--CH.sub.2--), alkoxydiyl.sub.(C.ltoreq.6) (e.g.,
--CH.sub.2CH.sub.2OCH.sub.2CH.sub.2-- or --CH.sub.2OCH.sub.2--); Z
is --O--, --OC(O)-- or --NHC(O)--; and Y.sub.2 is
aryl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.20),
aryl.sub.(C.ltoreq.10), phenyl,
(2,4,4-trimethylpentan-2-yl)-benzene, dodecane, 9-decene or
tridecane.
[0027] Thus, in some specific embodiments a cationic surfactant of
Formula I is defined as:
##STR00005## ##STR00006##
[0028] In certain very specific embodiments the cationic surfactant
is domiphen bromide ((Dodecyldimethyl-2-phenoxyethyl)ammonium
bromide); DB), myristoylcholine chloride,
benzyldimethyl(2-dodecyloxyethyl)-ammonium chloride, benzethonium
chloride, cetylpyridinium chloride, dodecylpyridinium chloride,
1-decyl-3-methylimidazolium chloride,
1,3-didecyl-2-methylimidazolium chloride, lauryl choline chloride,
1-dodecyl-2-methyl-3-benzimidazolium chloride,
4-carboyl-1-N-hexadecyl pyridinium chloride,
N-ethyl-N,N-dimethyl-2-(10-undecenoylamino)ethanaminium bromide, or
benzethonium chloride.
[0029] In further embodiment there is provided a nucleic acid
binding mixture comprising: (a) an aqueous solution comprising (i)
0.5-20% of a phase separating agent of formula I and (ii) 0.05 M to
1.0 M salt, such as a lithium salt, sodium salt, potassium salt,
magnesium salt, or calcium salt, and combinations thereof (e.g.,
LiCl or NaCl); (b) a mineral matrix of the embodiments; and (c) at
least a first nucleic acid molecule (e.g., DNA). In some aspects,
the binding mixture comprises 0.5-20% or 1-10% DB. In further
aspects, the binding mixture comprises 0.05 M to 1.0 M LiCl, and
optionally comprises KOAc. In certain aspects, the binding mixture
comprises 0.5 M to 1.0 M NaCl, and optionally comprises KOAc. Thus,
in a preferred embodiment, a binding mixture comprises (a) an
aqueous solution comprising (i) 0.5-5% DB and (ii) 0.05 M to 1.0 M
LiCl; (b) a mineral matrix of the embodiments; and (c) at least a
first plasmid DNA molecule. In yet a further preferred embodiment,
a binding mixture comprises (a) an aqueous solution comprising (i)
1-10% DB and (ii) 0.5 M to 1.0 M NaCl; (b) a mineral matrix of the
embodiments; and (c) at least a genomic DNA molecule.
[0030] In yet a further embodiment there is provided a method of
isolating DNA, comprising (a) obtaining a sample comprising DNA;
(b) capturing the DNA to a mineral matrix with a phase separation
solution comprising domiphen bromide (DB) and a salt (e.g., a
lithium salt, sodium salt, potassium salt, magnesium salt, calcium
salt or a mixtures thereof); (c) washing the mineral matrix and
captured DNA with a wash solution; and (d) eluting the plasmid DNA,
thereby isolating the DNA. For example, a sample for use according
to the embodiments can be a cell lysate, such as a mammalian or
bacterial cell lysate. Optionally, a method comprises treating the
captured DNA with a salt solution (after the capturing), thereby
increasing the retention of the captured DNA. For example, the salt
solution can comprise a lithium salt, sodium salt, potassium salt,
magnesium salt, calcium salt or a mixtures thereof. In some
aspects, (b) capturing DNA to a mineral matrix is in the presence
of 0.05% to 1% DB. In further aspects, the wash solution can be an
organic was solution (e.g., a solution comprising phenol) or a
solution comprising an alcohol (e.g., Ethanol (EtOH) and/or
isopropanol). For example, a wash solution of the embodiments may
comprise at least about 75%, 80%, 85%, 90% or 95% alcohol, such as
a lower alcohol (e.g., EtOH). In certain preferred aspects, an
isolated DNA produced by the methods of the embodiments is
essentially free of RNA, endotoxin and/or PCR inhibitors. In
further aspects, a mineral matrix for use herein is comprised in
column, such a spin column adapted for use in centrifuge or
microcentrifuge. In further aspects, obtaining a sample comprising
DNA comprises obtaining a bacterial cell lysate by alkaline lysis.
For example, such a method can comprise (i) lysing cells comprising
nucleic acid including DNA with a basic solution thereby generating
a lysate; (ii) neutralizing the lysate with an acidic solution
thereby precipitating a genomic DNA fraction and proteins; and
(iii) clearing the precipitate. In certain aspects, capturing
plasmid DNA to a mineral matrix with a phase separation solution
(b) and clearing the precipitate (iii) are performed concurrently
(or essentially simultaneously).
[0031] In certain aspects, a method of the embodiments is defined
as a method of selectively isolating genomic DNA. For example, in
some aspects, such a method comprises (a) obtaining a sample
comprising genomic DNA; (b) capturing the genomic DNA to a mineral
matrix with a phase separation solution comprising domiphen bromide
(DB) and a salt (e.g., a lithium salt, sodium salt, potassium salt,
magnesium salt, calcium salt or a mixtures thereof); (c) washing
the mineral matrix and captured genomic DNA with a wash solution;
and (d) eluting the genomic DNA, thereby isolating the genomic DNA.
Optionally, a method comprises treating the captured DNA with a
salt solution (after the capturing), thereby increasing the
retention of the captured DNA. For example, the salt solution can
comprise a lithium salt, sodium salt, potassium salt, magnesium
salt, calcium salt or a mixture thereof. In some cases, selectively
isolating genomic DNA, comprises capturing the genomic DNA to a
mineral matrix in the presence of DB and a sodium salt, such as
sodium chloride. For example, in some cases, capturing the genomic
DNA to a mineral matrix is in the presence of 0.05% to 1% DB and
0.5 M to 1.0 M NaCl. In further aspects, the capturing the genomic
DNA to a mineral matrix is in the presence of DB, NaCl and KOAc. In
still further aspects, the isolated genomic DNA is essentially free
of plasmid DNA and/or RNA.
[0032] In yet further aspects, a method of the embodiments is
defined as a method of selectively isolating plasmid DNA. For
example, in some aspects, such a method comprises (a) obtaining a
sample comprising plasmid DNA; (b) capturing the plasmid DNA to a
mineral matrix with a phase separation solution comprising domiphen
bromide (DB) and a salt (e.g., a lithium salt, sodium salt,
potassium salt, magnesium salt, calcium salt or a mixtures
thereof); (c) washing the mineral matrix and captured plasmid DNA
with a wash solution; and (d) eluting the plasmid DNA, thereby
isolating the plasmid DNA. Optionally, a method comprises treating
the captured DNA with a salt solution (after the capturing),
thereby increasing the retention of the captured DNA. For example,
the salt solution can comprise a lithium salt, sodium salt,
potassium salt, magnesium salt, calcium salt or a mixtures thereof.
In some cases, selectively isolating plasmid DNA, comprises
capturing the plasmid DNA to a mineral matrix in the presence of DB
and a lithium salt, such as lithium chloride. For example, in some
aspects, capturing the plasmid DNA to a mineral matrix is in the
presence of 0.05% to 1% DB and 0.05 M to 1.0 M LiCi. In further
aspects, the capturing the genomic DNA to a mineral matrix is in
the presence of DB, LiCl and KOAc. In still further aspects, the
isolated plasmid DNA is essentially free of genomic DNA and/or
RNA.
[0033] In still a further embodiment, there is provided method for
selectively condensing plasmid DNA and capturing the DNA to a
mineral matrix for purification comprising (a) contacting a
plasmid-containing sample (e.g., a bacterial lysate) with a phase
separation reagent comprising a cationic surfactant of Formula I
(e.g., DB) and (b) capturing the phase separated nucleic acid to
the mineral matrix (e.g., a silica-based matrix, such as
borosilicate glass fiber) in the presence of an effective amount of
a salt selected from the group consisting of NaCl, NaoAc, KCl,
KoAc, LiCl, LioAc, sodium formate, potassium formate, lithium
formate, calcium chloride, and magnesium chloride, thereby
selectively capturing plasmid DNA with the mineral matrix. For
example, in some aspects, the salt solution comprises a lithium
salt, e.g., LiCl present at a desired final concentration. In
certain aspects, capturing the nucleic acid with the mineral matrix
is in the presence of a final concentration (M) of about
0.025M-0.5M, about 0.125M-0.5M, about 0.15 M-0.375 M, about 0.25
M-0.375 M, about 0.3 M-0.4 M, about 0.2 M-0.4 M, or about 0.375
M-0.5 M LiCl and a final concentration (w/v) of about 0.05% to 5%
or 10%, e.g., 0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about
0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about
0.25-0.5% of the cationic surfactant (e.g., DB). In certain
preferred aspects, capturing the nucleic acid on mineral matrix is
in the presence of a final concentration of LiCl of between about
0.05 M and 1.05 M; about 0.1 M and 0.65 M or about 0.1 M and 0.5 M.
For example, in some aspects, the salt solution comprises a sodium
salt, e.g., NaCl present at a desired final concentration. In
certain aspects, capturing the nucleic acid with the mineral matrix
is in the presence of a final concentration (M) of about
0.025M-0.5M, about 0.125M-0.5M, about 0.15 M-0.375 M, about 0.2
M-0.4 M, or about 0.375 M-0.5 M LiCl and a final concentration
(w/v) of about 0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about
0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about
0.25-0.5% of the cationic surfactant (e.g., DB). In certain
preferred aspects, capturing the nucleic acid on mineral matrix is
in the presence of a final concentration of NaCl of between about
0.05 M and 1.05 M; about 0.1 M and 0.65 M or about 0.1 M and 0.5 M.
For example, in some aspects, the salt solution comprises a
potassium salt, e.g., KoAc present at a desired final
concentration. In certain aspects, capturing the nucleic acid with
the mineral matrix is in the presence of a final concentration (M)
of about 0.025M-0.5M, about 0.125M-0.5M, about 0.15 M-0.375 M,
about 0.2 M-0.4 M, or about 0.375 M-0.5 M KoAc and a final
concentration (w/v) of about 0.05-1%, about 0.1-0.8%, about
0.2-0.5%, about 0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%, about
0.25%-0.4%, about 0.25-0.5% of the cationic surfactant (e.g., DB).
In certain preferred aspects, capturing the nucleic acid on mineral
matrix is in the presence of a final concentration of LiCl, NaCl,
or Potassium Acetate of between about 0.05 M and 1.05 M; about 0.1
M and 0.65 M or about 0.1 M and 0.5 M (e.g., between about 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and about 1.0 M). In some
aspects, a method of the embodiments further comprises adjusting
the pH (e.g., using buffers such as Sodium Acetate, Tris HCl,
Bis-Tris, Bis-Tris Propane and others). In some cases, adjusting pH
can be used in controlling the selective binding properties of a
cationic surfactant contemplated herein.
[0034] In a further embodiment, there is provided method for
selectively condensing large DNA (e.g., genomic DNA) and capturing
the DNA to a mineral matrix for purification comprising (a)
contacting a nucleic acid containing sample (e.g., a bacterial or
mammalian cell lysate) with a phase separation reagent comprising a
cationic surfactant of Formula I (e.g., DB) and (b) capturing the
phase separated nucleic acid to the mineral matrix (e.g., a
silica-based matrix, such as borosilicate glass fiber) in the
presence of an effective amount of a salt selected from the group
consisting of NaCl, NaoAc, KCl, KoAc, LiCl, LioAc, sodium formate,
potassium formate, lithium formate, calcium chloride, and magnesium
chloride, thereby selectively capturing large DNA with the mineral
matrix. For example, in some aspects, the salt solution comprises a
lithium salt, e.g., LiCl present at a desired final concentration.
In certain aspects, capturing the nucleic acid with the mineral
matrix is in the presence of a final concentration (M) of about
0.05 M-1.0 M, about 0.25 M-0.9 M, about 0.5 M-0.7 M, about 0.55
M-0.7 M, or about 0.6 M-0.7 M LiCl and a final concentration (w/v)
of about 0.05 to 5% or 10%, e.g., about 0.05-1%, about 0.1-0.8%,
about 0.2-0.5%, about 0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%,
about 0.25%-0.4%, about 0.25-0.5% of the cationic surfactant (e.g.,
DB). For example, in some aspects, the salt solution comprises a
sodium salt, e.g., NaCl present at a desired final concentration.
In certain aspects, capturing the nucleic acid with the mineral
matrix is in the presence of a final concentration (M) of about
0.05 M-1.0 M, about 0.25 M-0.9 M, about 0.5 M-0.7 M, about 0.55
M-0.7 M, or about 0.6 M-0.7 M NaCl and a final concentration (w/v)
of about 0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about 0.1-0.25%,
about 0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about 0.25-0.5%
of the cationic surfactant (e.g., DB). In certain aspects,
capturing the nucleic acid with the mineral matrix is in the
presence of a final concentration (M) of about 0.025M-0.5M, about
0.125M-0.5M, about 0.15 M-0.375 M, about 0.2 M-0.4 M, or about
0.375 M-0.5 M KoAc and a final concentration (w/v) of about
0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about 0.1-0.25%, about
0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about 0.25-0.5% of the
cationic surfactant (e.g., DB). In certain preferred aspects,
capturing the nucleic acid on mineral matrix is in the presence of
a final concentration of LiCl, NaCl, or Potassium Acetate of
between about 0.05 M and 1.05 M; about 0.1 M and 0.65 M or about
0.1 M and 0.5 M (e.g., between about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9 and about 1.0 M). In certain preferred aspects,
capturing the nucleic acid on mineral matrix is in the presence of
a final concentration of NaCl, LiCl, or KoAc of between about 0.5 M
and 0.8 M; about 0.5 M and 0.7 M or about 0.6 M and 0.7 M. As used
herein "large DNA" refers to DNA segments longer than about 2 kb, 5
kb or 25 kb, such as genomic DNA. In some aspects, a method of the
embodiments further comprises adjusting the pH (e.g., using buffers
such as Sodium Acetate, Tris HCl, Bis-Tris, Bis-Tris Propane and
others). In some cases, adjusting pH can be used in controlling the
selective binding properties of a cationic surfactant contemplated
herein.
[0035] In some aspects, a phase separation reagent for use
according the embodiments comprises a final concentration (w/v)
about 0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about 0.1-0.25%,
about 0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about 0.25-0.5%
of a cationic surfactant of Formula I. In further aspects, a phase
separation reagent and/or a salt solution for use according to the
embodiments further comprises a pH buffer, such as a Tris or
Bis-Tris buffer. Thus, in some aspects, a phase separation reagent
and/or salt solution comprises a pH between about 6.0 and about
8.0.
[0036] In further aspects, a phase separation reagent (or a salt
solution of step c of the instant methods) comprises a salt
selected from the group consisting of NaCl, NaoAc, KCl, KoAc, LiCl,
LioAc, sodium formate, potassium formate, lithium formate, calcium
chloride, and magnesium chloride. Thus, in some aspects, a phase
separation reagent or a salt solution for use according to the
embodiments comprises a final concentration (M) of about
0.025M-1.05M, about 0.025M-0.5M, about 0.125M-0.5M, about
0.125M-0.6M, about 0.25M-0.375M, about 0.3M-0.4M, or about
0.375M-0.5M lithium chloride or sodium chloride or potassium
acetate. In still further aspects, a method of the embodiments
comprises contacting a nucleic acid to a mineral matrix in the
presence of compound of a cationic surfactant of formula I and a
lithium chloride salt. In still further aspects, a phase separation
reagent and/or salt solution comprises both lithium chloride and
sodium chloride. In still further aspects, a phase separation
reagent and/or salt solution comprises both lithium chloride and
potassium acetate.
[0037] Some aspects of the embodiments concern contacting a nucleic
acid-containing sample with a phase separation reagent. In some
cases the sample is contacted with the phase separation reagent at
different ratios. For example, a ratio of between about 10:1 and
1:10; 6:1 and about 1:2; or about 4:1, 3:1, 2:1 or 1:1
sample:reagent represent different embodiments. Other
sample:reagent ratios would be apparent to one skilled in the art
and represent additional embodiments where the final concentration
of each constituents present in the solution remains the same no
matter the ratio of the components that are added in, including the
cationic surfactants disclosed herein, prior to bringing the sample
in contact with a "Solid Support Carrier" to isolate nucleic acids.
In further aspects, eluting a nucleic acid comprises eluting using
water or a Tris buffered solution.
[0038] 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 certain aspects, resuspending cells in a first solution
comprises resuspending cells in a buffered solution comprising a
chelator, such a Tris buffered solution comprising EDTA. In still
further aspects, a second solution (for use in lysing step
according to the embodiments) comprises sodium hydroxide, sodium
dodecyl sulfate. In some cases, a lysing in step (b) is performed
for less than about 10 minutes, such as for 1-2, 1-5 or 1-8
minutes. In further aspects, a third solution (for use in
neutralizing the sample) comprises potassium acetate (e.g., in
a=concentration of about 0.1-3M, about 0.1-2M, about 0.1-1M, about
0.1M to 0.3M, or about 0.25M) and that may optionally contain RNAse
A as this may also be present in the resuspension buffer (P1). In
some cases, a method of the embodiments further comprises clearing
the precipitate after the neutralizing step (step (c)), such as by
filtration using cellulose paper, silica, or glass fiber or by
centrifugation. In still further aspects, the first solution,
second solution, third solution, and/or the phase separation
reagent comprise a dye. In preferred aspects, the first solution,
second solution, third solution, and/or the phase separation
reagent comprise dyes having different colors.
[0040] In certain aspects, a method of the embodiments comprises a
phase separation reagent comprising a cationic surfactant or an
ionic liquid. For example, the phase separation reagent cation can
be domiphen bromide, myristoylcholine chloride,
benzyldimethyl(2-dodecyloxyethyl)-ammonium chloride, benzethonium
chloride, cetylpyridinium chloride, dodecylpyridinium chloride,
1-decyl-3-methylimidazolium chloride,
1,3-didecyl-2-methylimidazolium chloride, lauryl choline chloride,
1-dodecyl-2-methyl-3-benzimidazolium chloride,
4-carboyl-1-N-hexadecyl pyridinium chloride,
N-ethyl-N,N-dimethyl-2-(10-undecenoylamino)ethanaminium bromide, or
benzethonium chloride. In preferred aspects, the phase separation
reagent is an ammonium cationic surfactant. In further preferred
aspects, the cationic surfactant is domiphen bromide. In still
further aspects, a phase separation reagent comprises an ammonium
cationic surfactant and an anionic surfactant. For example, the
anionic surfactant can be present in a final concentration of
between about 0.001 and 1% or between about 0.001 and 0.1%.
[0041] 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.
[0042] It is also recognized that specific embodiments of this
invention can be adapted for isolation of any nucleic acid from a
variety of sources. For example, nucleic acids may be isolated from
cultured cells, bacteria, yeast, blood, solid tissues, plant
tissues, sputum, lymph fluid, Cerebrospinal fluid (CSF), urine or
serum samples. In some aspects, a sample for nucleic acid isolation
is a bacterial culture, a fungal culture, a urine sample or a serum
sample.
[0043] In still a further embodiment there is provided a kit for
nucleic acid (e.g., genomic DNA, plasmid, and RNA) purification
comprising a phase separation reagent comprising a cationic
surfactant of Formula I. Such a kit may further comprise a mineral
matrix, a first suspension solution, a second lysis solution, a
third neutralization solution, a phase separation solution, a
treatment solution, a wash solution, an elution solution, columns
(or plates), a reservoir, a filter (e.g., a syringe filter),
collection tubes (or plates), and/or instructions for using the
kit.
[0044] In still yet a further embodiment there is provided a
composition comprising (i) a cationic surfactant of Formula I; (ii)
a salt selected from the group consisting of NaCl, NaoAc, KCl,
KoAc, LiCl, LioAc, sodium formate, potassium formate, lithium
formate, calcium chloride, and magnesium chloride; and (iii) a
mineral matrix, comprising nucleic acid molecules (e.g., genomic
DNA or plasmid DNA molecules) captured thereto. For example, the
prepared sample composition may comprise final concentrations
immediately prior to contacting the solution with a solid support
carrier of about 0.05-1%, about 0.1-0.8%, about 0.2-0.5%, about
0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%, about 0.25%-0.4%, about
0.25-0.5% final concentration (w/v) of a cationic surfactant of
Formula I (e.g., DB). In further aspects, the composition comprises
about 0.05 M-1.0 M, about 0.2 M-0.9M about 0.25 M-0.8 M, about 0.5
M-0.7 M, about 0.55 M-0.7 M, or about 0.6 M-0.7 M about 0.1 M,
about 0.2 M about 0.3 M about 0.4 M, about 0.5 M about 0.6 M about
0.7 M about 0.8 M, about 0.9 M about 1 M salt, such a lithium salt
(e.g., lithium chloride), final concentrations (M). In still
further aspects, the mineral matrix of the composition is a
silica-based matrix, such as borosilicate glass fiber. In further
aspects, the composition comprises about 0.05 M-1.0 M, about 0.2
M-0.9M about 0.25 M-0.8 M, about 0.5 M-0.7 M, about 0.55 M-0.7 M,
or about 0.6 M-0.7 M about 0.1 M, about 0.2 M about 0.3 M about 0.4
M, about 0.5 M about 0.6 M about 0.7 M about 0.8 M, about 0.9 M
about 1 M salt, such a sodium salt (e.g., sodium chloride), final
concentrations (M). In still further aspects, the mineral matrix of
the composition is a silica-based matrix, such as borosilicate
glass fiber. In further aspects, the composition comprises about
0.05 M-1.0 M, about 0.2 M-0.9M about 0.25 M-0.8 M, about 0.5 M-0.7
M, about 0.55 M-0.7 M, or about 0.6 M-0.7 M about 0.1 M, about 0.2
M about 0.3 M about 0.4 M, about 0.5 M about 0.6 M about 0.7 M
about 0.8 M, about 0.9 M about 1 M salt, such a potassium salt
(e.g., potassium acetate), final concentrations (M). In still
further aspects, the mineral matrix of the composition is a
silica-based matrix, such as borosilicate glass fiber. In further
aspects, the composition comprises a mixture of lithium salts such
as lithium chloride, sodium salts such as sodium chloride, and
potassium salts such as potassium acetate. The composition
comprises about 0.05 M-1.0 M, about 0.2 M-0.9M about 0.25 M-0.8 M,
about 0.5 M-0.7 M, about 0.55 M-0.7 M, or about 0.6 M-0.7 M about
0.1 M, about 0.2 M about 0.3 M about 0.4 M, about 0.5 M about 0.6 M
about 0.7 M about 0.8 M, about 0.9 M about 1 M salt, or some
denomination of each of the salts in combination or denomination
described. In some aspects, a method of the embodiments further
comprises adjusting the pH (e.g., using buffers such as Sodium
Acetate, Tris HCl, Bis-Tris, Bis-Tris Propane and others).
[0045] In a further embodiment, there is provided a method for
purification of DNA (e.g., genomic DNA) from an adhesion resin. For
example, in some aspects, methods of the embodiments can be used to
isolate genomic DNA from a soluble tape that is used to lift cells
from a surface (e.g., from a fingerprint).
[0046] As used herein the phrase "elution profile" refers to the
proportion of nucleic acid eluted from a binding matrix in a first
elution versus successive elutions (e.g., a second and third
elution). Methods of the embodiments preferable produce elution
profile wherein the majority proportion of the DNA (e.g., greater
than 50%, 60%, 70%, 80% or 90%) bound to a matrix is eluted in a
first elution relative to a second (or second and third)
elution.
[0047] As used herein, "essentially free," in terms of a specified
component, is used herein to mean that none of the specified
component has been purposefully formulated into a composition
and/or is present only as a contaminant or in trace amounts. The
total amount of the specified component resulting from any
unintended contamination of a composition is therefore well below
0.05%, preferably below 0.01%. Most preferred is a composition in
which no amount of the specified component can be detected with
standard analytical methods.
[0048] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one.
[0049] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternatives are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or." As used herein "another" may mean at least a second or
more.
[0050] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0051] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0052] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0053] FIGS. 1A-B--Titration of NaCl with
1-decyl-3-methylimidazolium chloride. Agarose gel electrophoresis
of a solution of pDNA/degraded RNA purified using a sodium chloride
titration with 1-decyl-3-methylimidazolium chloride. Each lane is
labeled with the concentration of NaCl (M). L=1 kb ladder.
pDNA=plasmid DNA. (A) Five minute run time (RNA check). (B)
Forty-five minute run time.
[0054] FIG. 2--Titration of NaCl with
1,3-didecyl-2-methylimidazolium chloride. Agarose gel
electrophoresis of a solution of pDNA/degraded RNA purified using a
sodium chloride titration with 1,3-didecyl-2-methylimidazolium
chloride. Each lane is labeled with the concentration of NaCl (M).
L=1 kb ladder. pDNA=plasmid DNA. RNA=degraded RNA. Gels were
electrophoresed for 5 minutes as an RNA check.
[0055] FIGS. 3A-D--Titration of NaCl with cetylpyridinium bromide.
Agarose gel electrophoresis of nucleic acid samples purified from
bacterial culture using a sodium chloride titration of various
cetylpyridinium bromide (CPB) concentrations. Each lane is labeled
with the concentration of NaCl (M). L=1 kb ladder. pDNA=plasmid
DNA. RNA=degraded RNA. Gels were electrophoresed for 5 minutes as
an RNA check. (A) 1% CPB. (B) 0.75% CPB. (C) 0.5% CPB. (D) 0.25%
CPB.
[0056] FIGS. 4A-B--Titration of NaCl with decylpyridinium chloride.
Agarose gel electrophoresis of nucleic acid samples purified from
bacterial culture using a sodium chloride titration of
decylpyridinium chloride. Each lane is labeled with the
concentration of NaCl (M). L=1 kb ladder. pDNA=plasmid DNA.
RNA=degraded RNA. (A) Five minute run time (RNA check). (B)
Forty-five minute run time.
[0057] FIGS. 5A-B & 5C-E--Titration of NaCl and LiCi with
domiphen bromide using nucleic acids in solution. Agarose gel
electrophoresis of a solution of pDNA/degraded RNA purified using
either a sodium chloride titration or a lithium chloride titration
of domiphen bromide (DB). Each lane is labeled with the
concentration of NaCl (M) or LiCl (M). *=Control. L=1 kb ladder.
pDNA=plasmid DNA. (A and B) Five minute run time (RNA check). (C-E)
Forty-five minute run time.
[0058] FIGS. 6A-B; 6C-D; and 6E--Titration of LiCi with domiphen
bromide using bacterial cultures. (A-D) Agarose gel electrophoresis
of nucleic acid samples purified from bacterial culture using a
lithium chloride titration of DB. Each lane is labeled with the
concentration of LiCl (M). *=Control Plasmid DNA. L=1 kb ladder.
pDNA=plasmid DNA. RNA=degraded RNA. (A and C) Five minute run time
(RNA check). (B and D) Forty-five minute run time. (E) Agarose gel
image depicting the quantities of plasmid DNA isolated after using
varying concentrations of LiCl (M) in the first wash buffer. Each
lane is labeled with the concentration of LiCl (M).
[0059] FIGS. 7A-B; 7C-D; and 7E-F--Evaluation of various salts and
their effects on total recovery and selectivity of nucleic acids
recovered. Agarose gel electrophoresis of nucleic acid samples
purified from bacterial culture using various salt titrations of
DB. Each lane is labeled with the concentration of salt (M). L=1 kb
ladder. pDNA=plasmid DNA. RNA=degraded RNA. (A, C, and E) Five
minute run time (RNA check). (B, D, and F) Forty-five minute run
time. (C and D) * indicate supplementation with 0.5 M LiCl.
[0060] FIGS. 8A-B and 8C-D--Effect of pH on capture capacity and
RNA elimination while using 1 M KoAc using DB. Agarose gel
electrophoresis of nucleic acid samples purified from bacterial
culture using LiCl titration of DB at various pH values and
N-Lauroylsarcosine sodium salt concentrations. L=1 kb ladder.
pDNA=plasmid DNA. RNA=degraded RNA. Circled concentrations indicate
the "window" in which the small nucleic acid fragments were
selectively removed. (A and B) Each lane is labeled with the
concentration of LiCl. (C and D) Each lane is labeled with the
concentration of N-Lauroylsarcosine. (A and C) Five minute run time
(RNA check). (B and D) Forty-five minute run time.
[0061] FIGS. 9A-B--Evaluation of various salts, pH, and
N-Lauroylsarcosine sodium salt to determine an optimal condition
for capturing DNA using DB. Agarose gel electrophoresis of nucleic
acid samples purified from bacterial culture using various LiCl
concentrations, various pH values, and various N-Lauroylsarcosine
concentrations. L=1 kb ladder. BL=blank. pDNA=plasmid DNA. Each
lane is labeled with a sample ID (see, Table 2). (A) Five minute
run time (RNA check). (B) Forty-five minute run time.
[0062] FIGS. 10A-B--Determination of capture conditions. Agarose
gel electrophoresis of nucleic acid samples purified from bacterial
culture using various pH values and LiCl concentrations. L=1 kb
ladder. pDNA=plasmid DNA. 2x indicates that two preps were loaded
through the same column. 1 h indicates a 1 hour incubation in the
phase separation buffer prior to loading onto the column. Each lane
is labeled with a sample ID (see, Table 3). (A) Five minute run
time. (B) Forty-five minute run time.
[0063] FIG. 11--Matrix porosity vs. nucleic acid recovery.
Neutralized bacterial lysate prepared from 35 mL of overnight
culture was loaded onto a spin column containing borosilicate glass
fiber with a nominal particle retention rating of either 1.0 .mu.m
(Porex Grade B), 1.6 .mu.m (Porex Grade A), or 2.7 km (Porex Grade
D) using a CTAB solution.
[0064] FIG. 12--Capture of Plasmid. Neutralized bacterial lysate
prepared from 50 mL of overnight culture was centrifuged to examine
if a precipitate (ppt) formed after addition of a P4 buffer (left
Experimental with ppt, right control (no ppt).
[0065] FIG. 13--Capture of Plasmid Gel. Approximately 10 .mu.m of
elution from Experimental and controls from Example 11 were run on
an agarose gel to visualize the DNA (1M: 1 kb Marker, 2-3
Experimental, 4-5: Control).
[0066] FIGS. 14A-B--Evaluation of Tris-HCl Buffer. Substitution of
Bis-Tris for Tris-HCl shifts optimal LiCl concentration of Domiphen
Bromide based phase separation buffer. About 10 .mu.l of elution
was visualized following 20 min (A) to check for undesirable RNA
contamination (RNA Check) and 60 min (B) in a full length run to
examine plasmid isolation in agarose gel electrophoresis. In each
FIG. 12A-B the first two lanes are controls with Bis-Tris as the
buffer component. LiCl concentrations of 0.5M, 0.75M, 1.0M, 1.15M,
and 1.25M are shown to titrate the preferred LiCl concentration for
plasmid capture. The Marker (M) is a 1 kb DNA Marker.
[0067] FIGS. 15A-B--Evaluation of various cationic surfactants for
nucleic acid isolation. Agarose gel electrophoresis of nucleic acid
samples purified from bacterial culture using various NaCl
concentrations and various cationic surfactants with a forty-five
minute run time. L=1 kb ladder. Each group of lanes is labeled with
the cationic surfactant and then within each group the different
NaCl concentrations are indicated.
[0068] FIGS. 16-17--Genomic DNA Isolation using Domiphen Bromide.
Agarose gel electrophoresis of nucleic acid samples purified from
HeLa cell culture using various NaCl (FIG. 16) and LiCl (FIG. 17)
concentrations with a forty-five minute run time. L=1 kb ladder.
Two samples at each concentration indicated are shown.
[0069] FIG. 18--Binding the Surfactant-Nucleic Acid Complex to
Magnetic Beads using Domiphen Bromide. Agarose gel electrophoresis
of nucleic acid samples purified from yeast culture using
magnetic-silica particles as the solid phase carrier in various
NaCl concentrations with a forty-five minute run time. L=1 kb
ladder.
[0070] FIGS. 19A-B--Titration of buffer, pH, and NaCl to optimize
Genomic DNA binding using Domiphen Bromide. Agarose gel
electrophoresis of nucleic acid samples purified from bacterial
culture using various NaCl concentrations and pH values. L=1 kb
ladder. Con=Control.
[0071] FIG. 20A-B-- Treatment solution was evaluated with
increasing concentrations of sodium chloride. Agarose gel
electrophoresis of nucleic acid samples was performed from purified
from E. coli culture using 0, 0.1, 0.2, 0.5, 0.7 M NaCl. Each lane
is labeled with the concentration of NaCl (M). L=1 kb ladder. (A)
Five minute run time (RNA check). (B) Forty-five minute run
time.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0072] Embodiments of the present invention provides an efficient,
safe and inexpensive method for the isolation and/or cleaning of
nucleic acids such as genomic DNA or plasmid DNA using a phase
separation reagent such as domiphen bromide (DB) with high
selectivity (e.g. capture of DNA versus RNA) and tunability (e.g.
controlling capture of large plasmids over other sized species).
The use of such surfactants allows for the ability to isolate
nucleic acids in high purity at high concentrations using
inexpensive materials, such as glass fiber membranes. Furthermore,
the use of DB in particular increased the capture capacity of
silica by nearly 17 fold as compared to using chaotropic
salt-driven binding. This high recovery of plasmid per milligram of
mineral matrix over other nucleic acids such as RNA allows for high
selectivity and the production of excellent elution profiles and
highly concentrated plasmid. High plasmid recovery in also
unexpectedly enhanced or boosted with DB by treatment of captured
plasmid with salt solutions. Further with DB the elution profile
can be fortuitously tuned or controlled to isolate preferentially
large DNA species such as genomic DNA or plasmid DNA. Such DB
mediated elution can be defined as the fraction of DNA released
each time a low salt solution or water is added to the matrix which
is then separated by convenient methods such as centrifugation.
Other phase separation reagents failed to display similar
selectivity or tunability for nucleic acid capture compared to DB.
A desired elution profile is one that reflects a 100% release of
nucleic acids, preferentially plasmid DNA, from the mineral matrix
in a single elution however generally an 80-90% release of the
nucleic acids on the first elution is highly desirable.
Approximately, 80-90% is a representative elution profile is
attained using the cationic surfactants disclosed herein.
[0073] Certain embodiments of the present invention allow for the
concentration of nucleic acids from large volumes of liquid by
capture by primarily precipitation with different phase separation
reagents, from samples including, but not limited to sputum, lymph
fluid, cerebrospinal fluid (CSF), urine, serum, sweat, various
aspirates, and other liquid biological sources, without the need
for excessively large volumes of ethanol, isopropanol, or other
commonly used DNA precipitating agents.
[0074] Embodiments of the present invention as provide a method of
rapidly purifying nucleic acids and selectively genomic DNA and
plasmid over smaller degraded nucleic acids such as RNase A
digested RNA in a genomic or plasmid preparation. The present
method can be performed at the same speed as chaotropic salt-based
methods, achieves recoveries similar to that of anion exchange
based chromatography and purity greater than both of these methods
directly from a spin column. This is especially critical when
dealing with large sample sizes such as bacterial cultures of 100
ml, 1 L, or larger, since the amount of silica required using
chaotropic salt driven binding methods requires excessively large
quantities of mineral matrix. In addition this method does not
suffer from the extremely slow processing times of anion exchange
methods that can require up to 3 hours to purify DNA.
[0075] Mineral matrices that could be implemented with embodiments
of the present invention include a porous or non/porous carrier
composed of metal oxides and or mix metal oxides. These metal
oxides include materials commonly used in the art such as
silicon-oxygen based compounds. Borosilicate or silicate in the
form of glass fibers, silica gel, zeolites, or diatomaceous earth
are most commonly used due their inexpensive and non-toxic
properties, however other minerals such as aluminum oxide, titanium
dioxide, zirconium dioxide or a mixture thereof could be used in
different embodiments.
[0076] In addition cellulose based resins and or other membranes
that can exclude based on size could be used in various embodiments
of the invention. Other commonly used filtration techniques and
matrix compositions or resins could also be used, e.g. ion
exchange.
[0077] Alkaline lysis embodiments of the present invention may
incorporate the use of dyes in the purification buffers for visual
monitoring of the steps for preparing the bacterial lysate
filtrate. See, U.S. Pat. No. 7,754,873, incorporated herein by
reference.
[0078] Nucleic acids specifically genomic DNA can be isolated from
microbial fermentation and/or eukaryotic cellular cultures or
biological body fluids (e.g. sputum, lymph fluid, cerebrospinal
fluid (CSF), urine, serum, sweat, various aspirates, and other
liquid biological sources) and solid tissues. Nucleic acids
specifically plasmid DNA can be isolated from microbial
fermentation. The plasmid DNA can be preferentially isolated from
Escherichia coli (E. coli) strains that are used to produce such
material for molecular biology manipulations. It is recognized that
other prokaryotic bacterial or eukaryotic species can also be used
as vehicles for the purification of nucleic acids and plasmid DNA.
The nucleic acid to be purified is typically DNA especially, or
genomic DNA or plasmid DNA and like vectors of a variety of sizes,
but could also be RNA, in alternative embodiments. In the case of
plasmid DNA and like vectors it may or may not contain foreign DNA
sequences, though generally will for most applications.
[0079] The cellular culture can be grown in a variety of culture
mediums that can be modified to alter or regulate replication of
the plasmid DNA, RNA, or other nucleic acid molecules. The cells
are harvested by centrifugation and the culture media removed to
provide a cell pellet. In a preferred embodiment the nucleic acid
that is isolated is plasmid DNA that can be of a variety of sizes
with specific control elements that either comprises heterologous
DNA or synthetic sequences that are commonly known in the art.
[0080] The isolation of plasmid DNA is a preferred embodiment of
the present invention. All steps of the preferred embodiment of the
present invention may be carried out at room temperature, about
15-30.degree. C. Isolation of plasmid DNA is well known in the art.
A preferred method of plasmid isolation comprises modified mild
alkaline lysis of host cells containing a plasmid, sodium hydroxide
(NaOH) and sodium dodecyl sulphate (SDS), NaOH/SDS, denaturation,
and precipitation of unwanted cellular macromolecular components as
an insoluble precipitate, coupled to column-based silica, or other
chromatography or purification methods. Isolation buffers based on
alkaline lysis protocols are well known in the art and variations
of compositions are contemplated as embodiments of the present
invention that are compatible with various commercially available
chromatographic columns and technologies. Alkaline lysis procedures
generally use sodium acetate, potassium acetate, as well as a
variety of other salts, including chaotropic salts. Ribonuclease
RNAase A is commonly added to degrade contaminating RNA from the
lysate. The clarification of the lysate can be performed by
centrifugation or filtration methods both of which are known in the
art. The plasmid is pure, typically with an OD260/280 ratio above
1.8. The plasmid DNA is suitably pure for use in the most sensitive
experiments.
[0081] Yeast species (e.g. Saccharomyces cerevisiae), fungi
species, other microorganisms, human (Homo sapiens) liquid tissue
(e.g. sputum, lymph fluid, cerebrospinal fluid (CSF), urine, serum,
sweat, various aspirates, and other liquid biological sources)
solid tissue, or tissue from a variety of species commonly used in
diagnostic, research or clinical laboratories are contemplated as
compatible with this purification procedure as sources of DNA and
are all alternative embodiments of the present invention.
Procedures for handling and preparing samples from these various
species are well known in the art and are reported in the
scientific literature.
I. General Protocol for Isolating Genomic DNA
[0082] Certain embodiments of the invention provide a method of
selectively isolating genomic DNA from a sample, such as a cell
lysate. For example, in some aspects, such a method comprises (a)
obtaining a sample comprising genomic DNA; (b) capturing the
genomic DNA to a mineral matrix with a phase separation solution
comprising DB and a salt (e.g., a potassium and/or sodium salt);
(c) washing the mineral matrix and captured genomic DNA with a wash
solution; and (d) eluting the genomic DNA, thereby isolating the
genomic DNA. A sample, for use according to the embodiments may be
any sample that comprises genomic DNA. In some cases, the sample
can be a sample from mammalian cells, such as a tissue or cell
sample. For example, the sample can a fingerprint residue (e.g., in
an adhesive matrix), hair or hair follicle, urine, fecal matter,
mucus membrane secretion (e.g., saliva) or a blood sample. Methods
for lysis of such samples are well known in the art and can be used
in conjunction with the DNA isolation methods disclosed herein.
[0083] Thus, in some aspects, there is provided method for
selectively condensing large DNA (e.g., genomic DNA, artificial
chromosomes, cosmids, plasmid) and capturing the DNA to a mineral
matrix for purification comprising (a) contacting a nucleic acid
containing sample (e.g., a bacterial or mammalian cell lysate) with
a phase separation reagent comprising a cationic surfactant of
Formula I (e.g., DB) and (b) capturing the phase separated nucleic
acid to the mineral matrix (e.g., a silica-based matrix, such as
borosilicate glass fiber) in the presence of an effective amount of
a salt selected from the group consisting of lithium salts, sodium
salts, potassium salts, magnesium salts, calcium salts and mixtures
thereof (e.g., NaCl, NaoAc, KCl, KoAc, LiCl, LioAc, sodium formate,
potassium formate, lithium formate, calcium chloride, and magnesium
chloride), thereby selectively capturing large DNA with the mineral
matrix. For example, in some aspects, the salt solution comprises a
lithium salt, e.g., LiCl present at a desired final concentration.
In certain aspects, capturing the nucleic acid with the mineral
matrix is in the presence of a final concentration (M) of about
0.05 M-1.05 M, about 0.25 M-0.9 M, about 0.5 M-0.7 M, about 0.55
M-0.7 M, or about 0.6 M-0.7 M LiCl and a final concentration (w/v)
of about 0.05 to 5% or 10%, e.g., about 0.05-1%, about 0.1-0.8%,
about 0.2-0.5%, about 0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%,
about 0.25%-0.4%, about 0.25-0.5% of the cationic surfactant (e.g.,
DB). In some aspects, the salt solution comprises a sodium salt,
e.g., NaCl present at a desired final concentration. In certain
aspects, capturing the nucleic acid with the mineral matrix is in
the presence of a final concentration (M) of about 0.05 M-1.0 M,
about 0.25 M-0.9 M, about 0.5 M-0.7 M, about 0.55 M-0.7 M, or about
0.6 M-0.7 M NaCl and a final concentration (w/v) of about 0.05-1%,
about 0.1-0.8%, about 0.2-0.5%, about 0.1-0.25%, about 0.2-0.3%,
about 0.25-0.3%, about 0.25%-0.4%, about 0.25-0.5% of the cationic
surfactant (e.g., DB). In certain aspects, capturing the nucleic
acid with the mineral matrix is in the presence of a final
concentration (M) of about 0.025M-0.5M, about 0.125M-0.5M, about
0.15 M-0.375 M, about 0.2 M-0.4 M, or about 0.375 M-0.5 M KoAc and
a final concentration (w/v) of about 0.05-1%, about 0.1-0.8%, about
0.2-0.5%, about 0.1-0.25%, about 0.2-0.3%, about 0.25-0.3%, about
0.25%-0.4%, about 0.25-0.5% of the cationic surfactant (e.g., DB).
In certain preferred aspects, capturing the nucleic acid on mineral
matrix is in the presence of a final concentration of LiCl, NaCl,
or Potassium Acetate of between about 0.05 M and 1.05 M; about 0.1
M and 0.65 M or about 0.1 M and 0.5 M (e.g., between about 0.1,
0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and about 1.0 M). In further
preferred aspects, capturing the nucleic acid on mineral matrix is
in the presence of a final concentration of NaCl, LiCl, or KOAc of
between about 0.5 M and 0.8 M; about 0.5 M and 0.7 M or about 0.6 M
and 0.7 M. As used herein "large DNA" refers to DNA segments longer
than about 2 kb, 5 kb, 10 kb, 20 kb or 25 kb, such as genomic and
plasmidDNA or other forms such as artificial chromosomes, cosmids
and the like. In some aspects, a method of the embodiments further
comprises adjusting the pH (e.g., using buffers such as Sodium
Acetate, Tris HCl, Bis-Tris, Bis-Tris Propane and others). In some
cases, adjusting pH can be used in controlling the selective
binding properties of a cationic surfactant contemplated
herein.
[0084] In preferred aspects, selectively isolating genomic DNA from
a sample, comprises capturing the genomic DNA to a mineral matrix
in the presence of DB and a sodium salt, such as sodium chloride.
For example, in some cases, capturing the genomic DNA to a mineral
matrix is in the presence of 0.05% to 1% DB and 0.05 M to 1.0 M
NaCl. In further aspects, the capturing the genomic DNA to a
mineral matrix is in the presence of DB, NaCl, LiCl and KOAc. In
still further aspects, the isolated genomic DNA is essentially free
of endotoxins, and PCR inhibitors.
II. General Protocol for Isolating Plasmid DNA
[0085] Methods of nucleic acid isolation using chaotropic salts
comprise (a) alkaline lysis of the starting material, (b)
neutralization/precipitation and subsequent capture of the nucleic
acids with a solid phase mineral matrix such as with a glass or
silica membrane, which is located on a solid support in a
centrifuge column; (c solid support carrier with a salt solution
that promotes retention of nucleic acids on the mineral or glass
matrix, (d) washing of the captured nucleic acids with an organic
solvent such as lower alcohols (i.e., C1-5 alcohols); and (e)
elution of the nucleic acids with water or a buffer of low ionic
strength.
[0086] In a preferred embodiment below, the volume of each solution
added in steps a-d are equal in volume. Other concentrations/volume
ratios could be readily made by those skilled in the art, and
represent alternative embodiment. Methods of nucleic acid isolation
using the present invention comprise (a) suspension of pelleted
bacterial culture in TE buffer (50 mM Tris-Cl, pH 8.0, 10 mM EDTA)
(Solution P1). (b) Lysis of cells/sample for a specified amount of
time depending on the scale of the prep using a common lysis
buffer, e.g., Solution P2 (200 mM NaOH, 1% SDS). Preferably, the
incubation lasts from about 1-5 minutes. (c) Neutralization of the
alkaline lysis solution and precipitation of genomic DNA and
proteins using a common neutralization buffer, (Solution P3) that
contains about 1-3 M potassium acetate (KoAc) and about 200
.mu.g/mL RNAse A. Clearance of the precipitated genomic DNA and
proteins using a filtration method, such as Whatman cellulose
paper, silica, glass fiber, membranes or other commonly used
filters. (d) Addition of a phase separation solution (Buffer P4),
comprising a phase separating agent of the embodiments. In one
preferred embodiment, Solution P4 comprises 0.1-10% (e.g., 1%)
domiphen bromide, 0.2 M-2.0 M (e.g., 1.1 M) Tris-HCl, pH 6.0-8.0
(e.g., 7.2), 0.1 M-1.5 M (e.g., 0.8M) lithium chloride and,
optionally, 0.1-1.5 M KOAc. The solution containing the cleared
lysate and the phase separation buffer is mixed and loaded onto a
mineral matrix, e.g., silica or glass fiber. The sample is washed
with a salt solution buffered with Tris and protected from heavy
metals by EDTA. One preferred embodiment for the wash solution
comprises 0.1 M-1.0 M (e.g., 0.45 M) NaCl/5 mM-100 mM (e.g., 40 mM)
Tris pH 7-9.5 (e.g., 8.5)/0.05 mM-1.5 mM (e.g., 0.5 mM) EDTA;
however, a range of types and concentrations of salts, such as
sodium chloride, lithium chloride, or potassium chloride, etc, can
be used. The sample is washed with an organic wash that solubilizes
the surfactants but forces the nucleic acid to remain precipitated
on the mineral matrix. One preferred embodiment for the organic
solvent wash solution is greater than 80% (e.g., about 95%)
ethanol. Elution of the nucleic acid from the glass or mineral
matrix can be done using water or any standard low salt solution,
such as TE.
[0087] Although the volume of each buffer changes depending on the
volume of bacterial culture used, the volume of each buffer
relative to the others is equal in the context of the experiments
disclosed herein. For example: 5 mL P1, 5 mL P2, 5 mL P3, and 5 mL
P4 was used when 30 ml of bacterial culture was the sample source.
However, this is not an absolute requirement of the method, as
those skilled in the art could easily adjust the ratios and
concentrations of the buffers with routine experimentation. It
should be noted that all concentrations are listed in many cases
for simplicity as those in the original solution or buffer, not by
the final concentration after mixing the four solutions. In other
cases the same solutions or buffers may be indicated as final
concentrations (w/v, v/v or M) to illustrate preferred
compositions. For example, a concentration of 2 M NaCl in Buffer P4
translates to a 0.5 M NaCl final concentration. In a preferred
embodiment a phase separation solution comprising starting
concentrations of about 0.1-10% (e.g., 1%) domiphen bromide, about
0.2 M-2.0 M (e.g., 1.1 M) Tris-HCl, pH 6.0-8.0 (e.g., 7.2), 0.1
M-1.5 M (e.g., 0.8M) lithium chloride which is added to a cleared
lysate where the cleared lysate is the supernatant collected post
filtration of the precipitated debris. This translates to a phase
separation solution comprising a final concentration comprising
about 0.025-2.5% (e.g., 0.25%) domiphen bromide (w/v), about) 0.05
M-0.5M (e.g., 0.275M) Tris-HCl, pH pH 6.0-8.0 (e.g., 7.2), and
about 0.025 M-0.375M (e.g., 0.2M) lithium chloride. This phase
separation solution allows the capture/retention of Domiphen
bromide--Nucleic Acid (DB-NA) complex onto a glass or mineral
carrier. This phase separation solution has the unique ability of
selectively capturing nucleic acids based upon size. The phase
separation solution can be tuned by changing concentrations such
that size selection of nucleic acids can be accomplished. The phase
separation solution also shows greatly enhanced recovery as
compared to chaotropic salts methods. In addition use of DB-NA
methodology prevents problems of guandinium contamination which is
inhibitory to many downstream applications and notoriously
difficult to completely remove.
[0088] In a preferred embodiment following the step of capturing
nucleic acids with a surfactant such as DB to a glass or mineral
matrix further comprises treating the captured nucleic acid with a
salt solution which increases retention of nucleic acid on the
mineral silica matrix or membrane. This treatment with the salt
solution comprises, for example, treatment with a solution
comprising 0.1-1.0 M salt, such as NaCl. In some aspects, the salt
solution further comprises a pH buffer and/or a chelator. An
exemplary salt solution comprises 0.1-1.0 M (e.g., 0.45 M) NaCl, 5
mM-100 mM (e.g., 40 mM) Tris pH 7.0-9.5 (e.g., 8.5) and 0.05 mM-1.5
mM (e.g., 0.5 mM) EDTA. The treatment results in the added
retention of nucleic acid on the mineral silica matrix or membrane
which dramatically enhances the recovery of nucleic acid. The
treatment with the salt buffer is also pertinent to the cleaning of
the sample by means of removing weakly bound contaminating
molecules that were not removed by the selective capture step.
Unlike CTAB-NA-mineral matrix complexes, DB-NA-mineral matrix
complexes when treated with a salt solution do not aid in the
removal of smaller nucleic acids such as degraded RNA (0113348).
However, a salt treatment of the DB-NA-Mineral Matrix has the
unexpected effect of dramatically enhancing or boosting retention
and therefore recovery of nucleic acid. Further, when using DB,
removal of potentially undesirable nucleic acids (e.g.: washing
away of small contaminating nucleic acids such as degraded RNA) at
this step is unexpectedly achieved indirectly by the high degree of
selectivity gained in the capture step effectively removing such
unwanted nucleic acid species.
[0089] The treatment with the salt buffer is followed by a wash,
with a wash solution comprising an organic solvent, such as lower
alcohols. If the matrix is not washed with an appropriate organic
solvent that removes the detergent then little or no DNA will be
removed from the column during the elution step. Without being
bound to a particular theory the organic solvent wash solutions are
thought to force the nucleic acids to remain precipitated and/or
captured to the matrix, while the surfactants are solvated and
removed. The alcohol content must be sufficiently high enough or
some fraction of the nucleic acids will be lost during the wash
step. Common wash buffers known in the art containing .gtoreq.70%
ethanol could be implemented. However, the preferred wash solutions
for the removal of the cationic surfactants disclosed herein
comprise about 95% ethanol, or about 90% isopropanol, or about 90%
butanol, or about 75% Ethanol/17% Isopropanol/8% water/0.4 mM Tris
pH 8.5, 0.004 mM EDTA. The higher alcohol content in these
solutions was found to greatly improve retention and therefore
recovery of the isolated nucleic acid. In other systems, especially
those that use chaotropic salts such as guanidinium thiocyanate
require a lower percentage of alcohol in order to ensure that the
salts are efficiently removed, which can cause some loss of
captured nucleic acids, thereby decreasing overall recovery. These
salts are considered harmful to most downstream applications and
therefore the user must be particularly careful to prevent salt
contamination.
[0090] In addition, the protocol is suitable to scale up from small
cultures for use in large scale methods for use with mid to large
sized cultures, e.g. from about 0.1 mL to several liters or
industrial sized cultures using fermentation equipment known in the
art, all of which are embodiments of the present invention.
[0091] In the following examples the ratios disclosed are
preferred, however, this is not an absolute requirement of the
method, as those skilled in the art could easily adjust the ratios
and concentrations of the buffers with routine experimentation to
generate fully functional embodiments.
[0092] In one example, 1-5 mL of pelleted bacterial culture may be
resuspended in 300 .mu.L of Buffer P1. To this, 300 .mu.L each of
Buffer P2, Buffer P3, and Buffer P4 may be added during the course
of the protocol. In this example, the treatment to increase yield
is performed with 400 .mu.L of salt buffer solution and the wash is
performed twice with 400 .mu.L of the organic wash solution.
[0093] In another example, 30-50 mL of pelleted bacterial culture
may be resuspended in 5 mL of Buffer P1. To this, 5 mL each of
Buffer P2, Buffer P3, and Buffer P4 may be added during the course
of the protocol. In this example, the treatment to increase yield
is performed with 800 .mu.L of salt buffer solution and the
critical wash is performed with 800 .mu.L of the organic solvent
wash solution.
[0094] In yet another example, 100-200 mL of pelleted bacterial
culture may be resuspended in 15 mL of Buffer P1. To this, 15 mL
each of Buffer P2, Buffer P3, and Buffer P4 may be added during the
course of the protocol. In this example, the treatment to increase
yield is performed with 5 mL of salt buffer solution and the
critical wash is performed with 5 mL of the organic solvent wash
solution.
[0095] In yet another example, 1-2 L of pelleted bacterial culture
may be resuspended in 150 mL of Buffer P1. To this, 15 mL each of
Buffer P2, Buffer P3, and Buffer P4 may be added during the course
of the protocol. In this example, the treatment to increase yield
is performed with about 40 to 50 mL of salt buffer solution and the
critical wash is performed with about 80 to 100 mL of the organic
solvent wash solution.
[0096] Modified Alkaline Lysis Protocol
[0097] In further aspects a modified alkaline lysis protocol may be
used as disclosed in U.S. Pat. No. 7,867,751, incorporated herein
by reference. Thus, in some aspects, there is provided a method for
isolating plasmid DNA from bacteria by alkaline lysis, the method
comprising the steps of: (a) providing a bacterial suspension
comprising bacteria having plasmid DNA; (b) adding a modified P2
reagent directly to the bacterial suspension, wherein the P2
reagent comprises an alcohol to reduce SDS precipitation; (c)
adding a modified P3 reagent to the bacterial suspension to produce
an alkaline lysate, wherein the modified reagent comprises a
chaotropic agent; (d) removing cell debris from the alkaline lysate
by filtration or centrifugation to obtain a lysate filtrate; (e)
contacting the lysate filtrate with a phase separating agent of the
embodiments (e.g., DB) and, optionally, a salt in presence of a
mineral matrix thereby capturing DNA to the matrix; (e) washing the
plasmid DNA bound to the matrix; and (f) eluting the plasmid
DNA.
[0098] The cell debris may be removed by centrifugation and
transferring the cleared lysate (lysate filtrate) to a DNA capture
device or similar device having a DNA binding matrix. The cell
debris may also be removed using a filtration apparatus and
transferring the lysate filtrate to a DNA capture device or similar
device having a DNA binding matrix.
[0099] In some aspects, step (d) and (e) are performed
concurrently. For example, the filtering can be in the presence of
a phase separating agent (and optionally a salt of the
embodiments). In this case, the alkaline lysate is passed through a
lysate filtration device having a (i) filtering medium and (ii) a
DNA binding matrix. For example, the filtering and capturing can be
performed using a single centrifugation or pressure step. The
lysate filtration device and DNA capture device may be discrete
components or a single assembly, i.e., a combined DNA isolation
apparatus.
[0100] One example of how this method could be performed is through
the use of concentrated or otherwise modified lysis and
neutralization solutions with buffers (e.g. binding buffer) of the
present invention. One skilled in the art would recognize that by
adjusting the concentration or form (semi-solid, dry solids) of
reagents in the modified or unmodified P2 (lysis solution),
modified or unmodified P3 (neutralization solution), or P4 (phase
separation solution) have been contemplated and would be considered
routine optimization. Alternative contemplated embodiments include
separating the lysis solution into two solutions wherein the sodium
hydroxide is added separately from the SDS. In some cases the
neutralization solution may or may not contain guanidine salts. In
some cases the modified P2 may or may not contain alcohol to
improve solubility of SDS.
[0101] Further provided are modified P2 and P3 reagents for use in
methods of alkaline lysis. For example, the modified P2 reagent may
be a liquid comprising alcohol (to reduce SDS precipitation).
Examples of suitable alcohols include isopropanol, 1-propanol, and
ethanol. In some case a modified P3 reagent is a liquid comprising
a chaotropic agent, and, in some cases, potassium chloride. In
still further aspects, a modified P3 reagent may comprise a phase
separating agent of the embodiments. In further aspects, the P3
reagent is a solid, comprising a solid acid. Again, in the case of
a solid P3, the reagent may comprise a phase separating agent of
the embodiments. Alkaline lysis may be performed using liquid,
solid, or immobilized P2 or P3 reagents.
III. Phase Separating Agents and Chemical Definitions
[0102] In certain aspects, the phase separation reagents for use
according to the embodiments are heterocycles containing a cationic
nitrogen or phosphorous or tetra substituted cationic nitrogen or
phosphorous molecule with a long alkyl chain a minimum of 8 carbons
long. However, the length of the primary hydrocarbon chain only
contributes to the total hydrophobicity of the molecule why the
total hydrocarbon content is equally important due to its effect on
how the heterocyclic or quaternary ammonium/phosphonium ions
interact with the biological molecules. Without being bound to a
particular the mechanism by which these molecules act to bind
nucleic acids to a mineral matrix is thought to rely on their
ability to form micelles with the nucleic acids in aqueous systems.
Therefore, based on this theory the only requirement is that the
hydrophobic interactions are favorable enough to facilitate micelle
formation. A 6 carbon chain with an aromatic residue or two
attached may be able to facilitate micelles formation, for example
benzethonium chloride. Functional groups, such as ethers, esters,
amides, ketones, aldehydes, and halogens, ultimately should not
affect the functionality of these molecules so long as micelles can
still form, however they can provide previously un-described and
surprising beneficial characteristics as in the case of the
preferred molecule--domiphen bromide of enhances selectivity and
tunability for nucleic acid isolation.
[0103] The results disclosed herein show that each surfactant has
unique properties that cannot be predicted. That is to say, that
the behavior of each phase separation reagent based upon each
unique functional group(s) cannot be explicitly predicted by the
literature currently available. The preferred surfactant, domiphen
bromide, meets the requirements described herein and due to the
phenoxyethyl functional group the molecule acquired the ability to
select for nucleic acids based upon size. The binding functionality
of DB also imparted a significant boost in recovery of DNA when a
salt solution was used as a treatment prior to washing with an
organic solvent. Such increased recovery of nucleic acids and in
particular DNA is not observed for other phase separation agents
such as cationic surfactants (U.S. Pat. App. 2008/0113348 and U.S.
Pat. No. 8,679,744).
[0104] Accordingly, in some aspects, a cationic surfactant
comprises the general of Formula I:
##STR00007##
[0105] wherein: X.sub.1 is nitrogen, phosphorus,
N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18), provided that when X.sub.1 is
N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18), then R.sub.2, R.sub.3, and R.sub.4
are absent and when R.sub.2, R.sub.3, and R.sub.4 are absent, then
X.sub.1 is N-heteroaryl.sub.(C.ltoreq.18) or substituted
N-heteroaryl.sub.(C.ltoreq.18); R.sub.1 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), a
substituted version of any of these groups or
--Y.sub.1--Z--Y.sub.2; Y.sub.1 is alkandiyl.sub.(C.ltoreq.6),
alkendiyl.sub.(C.ltoreq.6), alkyndiyl.sub.(C.ltoreq.6),
arenediyl.sub.(C.ltoreq.6), alkoxydiyl.sub.(C.ltoreq.6), or a
substituted version of any of these groups; Z is --O--, --NH--,
--S--, --C(O)O--, --OC(O)--, --NHC(O)--; or --C(O)NH--; Y.sub.2 is
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), or a substituted version of any of
these groups; R.sub.2, R.sub.3, and R.sub.4 are each independently
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), a substituted version of any of
these groups or --Y.sub.3--Z--Y.sub.4; Y.sub.3 is
alkandiyl.sub.(C.ltoreq.6), alkendiyl.sub.(C.ltoreq.6),
alkyndiyl.sub.(C.ltoreq.6), arenediyl.sub.(C.ltoreq.6), or a
substituted version of any of these groups; Z is --O--, --NH--,
--S--, --C(O)O--, --OC(O)--, --NHC(O)--; or --C(O)NH--; Y.sub.4 is
alkyl.sub.(C.ltoreq.30), alkenyl.sub.(C.ltoreq.30),
alkynyl.sub.(C.ltoreq.30), aryl.sub.(C.ltoreq.30),
aralkyl.sub.(C.ltoreq.30), heteroaryl.sub.(C.ltoreq.30),
heteroaralkyl.sub.(C.ltoreq.30), or a substituted version of any of
these groups; and A is an inorganic or organic anion such as
fluoride, chloride, bromide, iodide, phosphate, sulfite, sulfate,
hydrogen sulfate, thiosulfate, perchlorate, chlorite, carbonate,
bicarbonate, phosphate, nitrate, nitrite, acetate, formate,
propionate, oxalate, or succinate, malanate, borate, cyanate,
thiocyanate, hydroxide or other common anions known in the art such
as benzoate, salicylate, or p-toluenesulfonate; provided that when
R.sub.2, R.sub.3 and R.sub.4 are alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
halo substituted version of any of these groups and X.sub.1 is
nitrogen or phosphorus, then R.sub.1 is --Y.sub.1--Z--Y.sub.2. In
some aspects, a cationic surfactant comprises a structure according
to formula I wherein if X.sub.1 is a nitrogen or phosphorous and if
R.sub.1 is not a substituted N-heteroaryl or a N-heteroaryl, then,
Y.sub.1 is alkandiyl.sub.(C.ltoreq.6), alkendiyl.sub.(C.ltoreq.6),
alkandiyl.sub.(C.ltoreq.6), arenediyl.sub.(C.ltoreq.6),
alkoxydiyl.sub.(C.ltoreq.6), or a substituted version of any of
these groups; Z is --O--, --NH--, --S--, --C(O)O--, --OC(O)--,
--NHC(O)--; or --C(O)NH--; and Y.sub.2 is alkyl.sub.(C.ltoreq.30),
alkenyl.sub.(C.ltoreq.30), alkynyl.sub.(C.ltoreq.30),
aryl.sub.(C.ltoreq.30), aralkyl.sub.(C.ltoreq.30),
heteroaryl.sub.(C.ltoreq.30), heteroaralkyl.sub.(C.ltoreq.30), or a
substituted version of any of these groups.
[0106] Preferably, the cationic detergent is an ammonium cationic
surfactant with functional groups that tether the alkyl chains,
saturated or unsaturated, rings or aryl groups to the central
nitrogen by means of an ether, thioether, ester, or amide. However,
all cationic detergents that involve a heterocyclic ring structure,
such as imidazole or pyridine, are contemplated as alternative
embodiments of the invention.
[0107] When used in the context of a chemical group: "hydrogen"
means --H; "hydroxy" means --OH; "oxo" means=O; "carbonyl" means
--C(.dbd.O)--; "carboxy" means --C(.dbd.O)OH (also written as
--COOH or --CO.sub.2H); "halo" means independently --F, --Cl, --Br
or --I; "amino" means --NH.sub.2; "hydroxyamino" means --NHOH;
"nitro" means --NO.sub.2; imino means=NH; "cyano" means --CN;
"isocyanate" means --N.dbd.C.dbd.O; "azido" means --N.sub.3; in a
monovalent context "phosphate" means --OP(O)(OH).sub.2 or a
deprotonated form thereof; in a divalent context "phosphate" means
--OP(O)(OH)O-- or a deprotonated form thereof, "mercapto" means
--SH; and "thio" means=S; "sulfonyl" means --S(O).sub.2--; and
"sulfinyl" means --S(O)--.
[0108] In the context of chemical formulas, the symbol "--" means a
single bond, ".dbd." means a double bond, and ".ident." means
triple bond. The symbol "" represents an optional bond, which if
present is either single or double. The symbol "" represents a
single bond or a double bond. Thus, for example, the structure
##STR00008##
includes the structures
##STR00009##
As will be understood by a person of skill in the art, no one such
ring atom forms part of more than one double bond. The symbol "",
when drawn perpendicularly across a bond indicates a point of
attachment of the group. It is noted that the point of attachment
is typically only identified in this manner for larger groups in
order to assist the reader in unambiguously identifying a point of
attachment. The symbol "" means a single bond where the group
attached to the thick end of the wedge is "out of the page." The
symbol "" means a single bond where the group attached to the thick
end of the wedge is "into the page". The symbol "" means a single
bond where the conformation (e.g., either R or S) or the geometry
is undefined (e.g., either E or Z). Similarly, the covalent bond
symbol "--", when connecting stereogenic atom, does not indicate
any preferred stereochemistry, it does cover all stereoisomers,
including the "" and "" forms.
[0109] Any undefined valency on an atom of a structure shown in
this application implicitly represents a hydrogen atom bonded to
the atom. A bold dot on a carbon atom indicates that the hydrogen
attached to that carbon is oriented out of the plane of the paper.
When a group "R" is depicted as a "floating group" on a ring
system, for example, in the formula:
##STR00010##
then R may replace any hydrogen atom attached to any of the ring
atoms, including a depicted, implied, or expressly defined
hydrogen, so long as a stable structure is formed. When a group "R"
is depicted as a "floating group" on a fused ring system, as for
example in the formula:
##STR00011##
then R may replace any hydrogen attached to any of the ring atoms
of either of the fused rings unless specified otherwise.
Replaceable hydrogens include depicted hydrogens (e.g., the
hydrogen attached to the nitrogen in the formula above), implied
hydrogens (e.g., a hydrogen of the formula above that is not shown
but understood to be present), expressly defined hydrogens, and
optional hydrogens whose presence depends on the identity of a ring
atom (e.g., a hydrogen attached to group X, when X equals --CH--),
so long as a stable structure is formed. In the example depicted, R
may reside on either the 5-membered or the 6-membered ring of the
fused ring system. In the formula above, the subscript letter "y"
immediately following the group "R" enclosed in parentheses,
represents a numeric variable. Unless specified otherwise, this
variable can be 0, 1, 2, or any integer greater than 2, only
limited by the maximum number of replaceable hydrogen atoms of the
ring or ring system.
[0110] For the groups and classes below, the following
parenthetical subscripts further define the group/class as follows:
"(Cn)" defines the exact number (n) of carbon atoms in the
group/class. "(C.ltoreq.n)" defines the maximum number (n) of
carbon atoms that can be in the group/class, with the minimum
number as small as possible for the group in question, e.g., it is
understood that the minimum number of carbon atoms in the group
"alkenyl.sub.(C.ltoreq.8)" or the class "alkene.sub.(C.ltoreq.8)"
is two. For example, "alkoxy.sub.(C.ltoreq.10)" designates those
alkoxy groups having from 1 to 10 carbon atoms (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, or 10, or any range derivable therein (e.g., 3 to 10
carbon atoms). (Cn-n') defines both the minimum (n) and maximum
number (n') of carbon atoms in the group. Similarly,
"alkyl.sub.(C2-10)" designates those alkyl groups having from 2 to
10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range
derivable therein (e.g., 3 to 10 carbon atoms)).
[0111] The term "saturated" as used herein means the compound or
group so modified has no carbon-carbon double and no carbon-carbon
triple bonds, except as noted below. The term does not preclude
carbon-heteroatom multiple bonds, for example carbon oxygen double
bonds or a carbon nitrogen double bonds. Moreover, it does not
preclude a carbon-carbon double bond that may occur as part of
keto-enol tautomerism or imine/enamine tautomerism.
[0112] The term "aliphatic" when used without the "substituted"
modifier signifies that the compound/group so modified is an
acyclic or cyclic, but non-aromatic hydrocarbon compound or group.
In aliphatic compounds/groups, the carbon atoms can be joined
together in straight chains, branched chains, or non-aromatic rings
(alicyclic). Aliphatic compounds/groups can be saturated, that is
joined by single bonds (alkanes/alkyl), or unsaturated, with one or
more double bonds (alkenes/alkenyl) or with one or more triple
bonds (alkynes/alkynyl). Where the term "aliphatic" is used without
the "substituted" modifier, then only carbon and hydrogen atoms are
present. When the term is used with the "substituted" modifier one
or more hydrogen atom has been independently replaced by --OH, --F,
--Cl, --Br, --I, --NH.sub.2, --NO.sub.2, --CO.sub.2H,
--CO.sub.2CH.sub.3, --CN, --SH, --OCH.sub.3, --OCH.sub.2CH.sub.3,
--C(O)CH.sub.3, --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--N(CH.sub.3).sub.2, --C(O)NH.sub.2, --OC(O)CH.sub.3, or
--S(O).sub.2NH.sub.2.
[0113] The term "alkyl" when used without the "substituted"
modifier refers to a monovalent saturated aliphatic group with a
carbon atom as the point of attachment, a linear or branched,
cyclo, cyclic or acyclic structure, and no atoms other than carbon
and hydrogen. Thus, as used herein cycloalkyl is a subset of alkyl.
The groups --CH.sub.3 (Me), --CH.sub.2CH.sub.3 (Et),
--CH.sub.2CH.sub.2CH.sub.3 (n-Pr or propyl),
--CH(CH.sub.3).sub.2(i-Pr, .sup.iPr or isopropyl),
--CH(CH.sub.2).sub.2(cyclopropyl),
--CH.sub.2CH.sub.2CH.sub.2CH.sub.3 (n-Bu),
--CH(CH.sub.3)CH.sub.2CH.sub.3 (sec-butyl),
--CH.sub.2CH(CH.sub.3).sub.2(isobutyl), --C(CH.sub.3).sub.3
(tert-butyl, t-butyl, t-Bu or .sup.tBu),
--CH.sub.2C(CH.sub.3).sub.3(neo-pentyl), cyclobutyl, cyclopentyl,
cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl
groups. The term "alkanediyl" when used without the "substituted"
modifier refers to a divalent saturated aliphatic group, with one
or two saturated carbon atom(s) as the point(s) of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, no
carbon-carbon double or triple bonds, and no atoms other than
carbon and hydrogen. The groups, --CH.sub.2-- (methylene),
--CH.sub.2CH.sub.2--, --CH.sub.2C(CH.sub.3).sub.2CH.sub.2--,
--CH.sub.2CH.sub.2CH.sub.2--, and
##STR00012##
are non-limiting examples of alkanediyl groups. The term
"alkylidene" when used without the "substituted" modifier refers to
the divalent group .dbd.CRR' in which R and R' are independently
hydrogen, alkyl, or R and R' are taken together to represent an
alkanediyl having at least two carbon atoms. Non-limiting examples
of alkylidene groups include: .dbd.CH.sub.2,
.dbd.CH(CH.sub.2CH.sub.3), and .dbd.C(CH.sub.3).sub.2. When any of
these terms is used with the "substituted" modifier one or more
hydrogen atom has been independently replaced by --OH, --F, --Cl,
--Br, --I, --NH.sub.2, --NO.sub.2, --CO.sub.2H, --CO.sub.2CH.sub.3,
--CN, --SH, --OCH.sub.3, --OCH.sub.2CH.sub.3, --C(O)CH.sub.3,
--NHCH.sub.3, --NHCH.sub.2CH.sub.3, --N(CH.sub.3).sub.2,
--C(O)NH.sub.2, --OC(O)CH.sub.3, or --S(O).sub.2NH.sub.2. The
following groups are non-limiting examples of substituted alkyl
groups: --CH.sub.2OH, --CH.sub.2Cl, --CF.sub.3, --CH.sub.2CN,
--CH.sub.2C(O)OH, --CH.sub.2C(O)OCH.sub.3, --CH.sub.2C(O)NH.sub.2,
--CH.sub.2C(O)CH.sub.3, --CH.sub.2OCH.sub.3,
--CH.sub.2OC(O)CH.sub.3, --CH.sub.2NH.sub.2,
--CH.sub.2N(CH.sub.3).sub.2, and --CH.sub.2CH.sub.2Cl. The term
"haloalkyl" is a subset of substituted alkyl, in which one or more
hydrogen atoms has been substituted with a halo group and no other
atoms aside from carbon, hydrogen and halogen are present. The
group, --CH.sub.2Cl is a non-limiting example of a haloalkyl. An
"alkane" refers to the compound H--R, wherein R is alkyl. The term
"fluoroalkyl" is a subset of substituted alkyl, in which one or
more hydrogen has been substituted with a fluoro group and no other
atoms aside from carbon, hydrogen and fluorine are present. The
groups, --CH.sub.2F, --CF.sub.3, and --CH.sub.2CF.sub.3 are
non-limiting examples of fluoroalkyl groups. An "alkane" refers to
the compound H--R, wherein R is alkyl.
[0114] The term "alkenyl" when used without the "substituted"
modifier refers to an monovalent unsaturated aliphatic group with a
carbon atom as the point of attachment, a linear or branched,
cyclo, cyclic or acyclic structure, at least one nonaromatic
carbon-carbon double bond, no carbon-carbon triple bonds, and no
atoms other than carbon and hydrogen. Non-limiting examples of
alkenyl groups include: --CH.dbd.CH.sub.2 (vinyl),
--CH.dbd.CHCH.sub.3, --CH.dbd.CHCH.sub.2CH.sub.3,
--CH.sub.2CH.dbd.CH.sub.2 (allyl), --CH.sub.2CH.dbd.CHCH.sub.3, and
--CH.dbd.CH--C.sub.6H.sub.5. The term "alkenediyl" when used
without the "substituted" modifier refers to a divalent unsaturated
aliphatic group, with two carbon atoms as points of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, at least
one nonaromatic carbon-carbon double bond, no carbon-carbon triple
bonds, and no atoms other than carbon and hydrogen. The groups,
--CH.dbd.CH--, --CH.dbd.C(CH.sub.3)CH.sub.2--,
--CH.dbd.CHCH.sub.2--, and
##STR00013##
are non-limiting examples of alkenediyl groups. When these terms
are used with the "substituted" modifier one or more hydrogen atom
has been independently replaced by --OH, --F, --Cl, --Br, --I,
--NH.sub.2, --NO.sub.2, --CO.sub.2H, --CO.sub.2CH.sub.3, --CN,
--SH, --OCH.sub.3, --OCH.sub.2CH.sub.3, --C(O)CH.sub.3,
--NHCH.sub.3, --NHCH.sub.2CH.sub.3, --N(CH.sub.3).sub.2,
--C(O)NH.sub.2, --OC(O)CH.sub.3, or --S(O).sub.2NH.sub.2. The
groups, --CH.dbd.CHF, --CH.dbd.CHCl and --CH.dbd.CHBr, are
non-limiting examples of substituted alkenyl groups. An "alkene"
refers to the compound H--R, wherein R is alkenyl.
[0115] The term "alkynyl" when used without the "substituted"
modifier refers to an monovalent unsaturated aliphatic group with a
carbon atom as the point of attachment, a linear or branched,
cyclo, cyclic or acyclic structure, at least one carbon-carbon
triple bond, and no atoms other than carbon and hydrogen. As used
herein, the term alkynyl does not preclude the presence of one or
more non-aromatic carbon-carbon double bonds. The groups,
--C.ident.CH, --C.ident.CCH.sub.3, and --CH.sub.2C.ident.CCH.sub.3,
are non-limiting examples of alkynyl groups. When alkynyl is used
with the "substituted" modifier one or more hydrogen atom has been
independently replaced by --OH, --F, --Cl, --Br, --I, --NH.sub.2,
--NO.sub.2, --CO.sub.2H, --CO.sub.2CH.sub.3, --CN, --SH,
--OCH.sub.3, --OCH.sub.2CH.sub.3, --C(O)CH.sub.3, --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --N(CH.sub.3).sub.2, --C(O)NH.sub.2,
--OC(O)CH.sub.3, or --S(O).sub.2NH.sub.2. An "alkyne" refers to the
compound H--R, wherein R is alkynyl.
[0116] The term "aryl" when used without the "substituted" modifier
refers to a monovalent unsaturated aromatic group with an aromatic
carbon atom as the point of attachment, said carbon atom forming
part of a one or more six-membered aromatic ring structure, wherein
the ring atoms are all carbon, and wherein the group consists of no
atoms other than carbon and hydrogen. If more than one ring is
present, the rings may be fused or unfused. As used herein, the
term does not preclude the presence of one or more alkyl group
(carbon number limitation permitting) attached to the first
aromatic ring or any additional aromatic ring present. Non-limiting
examples of aryl groups include phenyl (Ph), methylphenyl,
(dimethyl)phenyl, --C.sub.6H.sub.4CH.sub.2CH.sub.3 (ethylphenyl),
naphthyl, and the monovalent group derived from biphenyl. The term
"arenediyl" when used without the "substituted" modifier refers to
a divalent aromatic group with two aromatic carbon atoms as points
of attachment, said carbon atoms forming part of one or more
six-membered aromatic ring structure(s) wherein the ring atoms are
all carbon, and wherein the monovalent group consists of no atoms
other than carbon and hydrogen. As used herein, the term does not
preclude the presence of one or more alkyl group (carbon number
limitation permitting) attached to the first aromatic ring or any
additional aromatic ring present. If more than one ring is present,
the rings may be fused or unfused. Non-limiting examples of
arenediyl groups include:
##STR00014##
When these terms are used with the "substituted" modifier one or
more hydrogen atom has been independently replaced by --OH, --F,
--Cl, --Br, --I, --NH.sub.2, --NO.sub.2, --CO.sub.2H,
--CO.sub.2CH.sub.3, --CN, --SH, --OCH.sub.3, --OCH.sub.2CH.sub.3,
--C(O)CH.sub.3, --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--N(CH.sub.3).sub.2, --C(O)NH.sub.2, --OC(O)CH.sub.3, or
--S(O).sub.2NH.sub.2. An "arene" refers to the compound H--R,
wherein R is aryl. Benzene and toluene are non-limiting examples of
arenes.
[0117] The term "aralkyl" when used without the "substituted"
modifier refers to the monovalent group -alkanediyl-aryl, in which
the terms alkanediyl and aryl are each used in a manner consistent
with the definitions provided above. Non-limiting examples of
aralkyls are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When
the term is used with the "substituted" modifier one or more
hydrogen atom from the alkanediyl and/or the aryl has been
independently replaced by --OH, --F, --Cl, --Br, --I, --NH.sub.2,
--NO.sub.2, --CO.sub.2H, --CO.sub.2CH.sub.3, --CN, --SH,
--OCH.sub.3, --OCH.sub.2CH.sub.3, --C(O)CH.sub.3, --NHCH.sub.3,
--NHCH.sub.2CH.sub.3, --N(CH.sub.3).sub.2, --C(O)NH.sub.2,
--OC(O)CH.sub.3, or --S(O).sub.2NH.sub.2. Non-limiting examples of
substituted aralkyls are: (3-chlorophenyl)-methyl, and
2-chloro-2-phenyl-eth-1-yl.
[0118] The term "heteroaryl" when used without the "substituted"
modifier refers to a monovalent aromatic group with an aromatic
carbon atom or nitrogen atom as the point of attachment, said
carbon atom or nitrogen atom forming part of one or more aromatic
ring structures wherein at least one of the ring atoms is nitrogen,
oxygen or sulfur, and wherein the heteroaryl group consists of no
atoms other than carbon, hydrogen, aromatic nitrogen, aromatic
oxygen and aromatic sulfur. As used herein, the term does not
preclude the presence of one or more alkyl, aryl, and/or aralkyl
groups (carbon number limitation permitting) attached to the
aromatic ring or aromatic ring system. If more than one ring is
present, the rings may be fused or unfused. Non-limiting examples
of heteroaryl groups include furanyl, imidazolyl, indolyl,
indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl,
phenylpyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl,
quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl,
thiazolyl, thienyl, and triazolyl. The term "N-heteroaryl" refers
to a heteroaryl group with a nitrogen atom as the point of
attachment. The term "heteroarenediyl" when used without the
"substituted" modifier refers to an divalent aromatic group, with
two aromatic carbon atoms, two aromatic nitrogen atoms, or one
aromatic carbon atom and one aromatic nitrogen atom as the two
points of attachment, said atoms forming part of one or more
aromatic ring structure(s) wherein at least one of the ring atoms
is nitrogen, oxygen or sulfur, and wherein the divalent group
consists of no atoms other than carbon, hydrogen, aromatic
nitrogen, aromatic oxygen and aromatic sulfur. As used herein, the
term does not preclude the presence of one or more alkyl, aryl,
and/or aralkyl groups (carbon number limitation permitting)
attached to the aromatic ring or aromatic ring system. If more than
one ring is present, the rings may be fused or unfused.
Non-limiting examples of heteroarenediyl groups include:
##STR00015##
When these terms are used with the "substituted" modifier one or
more hydrogen atom has been independently replaced by --OH, --F,
--Cl, --Br, --I, --NH.sub.2, --NO.sub.2, --CO.sub.2H,
--CO.sub.2CH.sub.3, --CN, --SH, --OCH.sub.3, --OCH.sub.2CH.sub.3,
--C(O)CH.sub.3, --NHCH.sub.3, --NHCH.sub.2CH.sub.3,
--N(CH.sub.3).sub.2, --C(O)NH.sub.2, --OC(O)CH.sub.3, or
--S(O).sub.2NH.sub.2.
[0119] The term "alkoxy" when used without the "substituted"
modifier refers to the group --OR, in which R is an alkyl, as that
term is defined above. Non-limiting examples of alkoxy groups
include: --OCH.sub.3 (methoxy), --OCH.sub.2CH.sub.3 (ethoxy),
--OCH.sub.2CH.sub.2CH.sub.3, --OCH(CH.sub.3).sub.2 (isopropoxy),
--O(CH.sub.3).sub.3(tert-butoxy), --OCH(CH.sub.2).sub.2,
--O-cyclopentyl, and --O-cyclohexyl. The terms "alkenyloxy",
"alkynyloxy", "aryloxy", "aralkoxy", "heteroaryloxy",
"heterocycloalkoxy", and "acyloxy", when used without the
"substituted" modifier, refers to groups, defined as --OR, in which
R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl,
and acyl, respectively. The term "alkoxydiyl" refers to the
divalent group --O-alkanediyl-, --O-alkanediyl-O--, or
-alkanediyl-O-alkanediyl-. The term "alkylthio" and "acylthio" when
used without the "substituted" modifier refers to the group --SR,
in which R is an alkyl and acyl, respectively. When any of these
terms is used with the "substituted" modifier one or more hydrogen
atom has been independently replaced by --OH, --F, --Cl, --Br, --I,
--NH.sub.2, --NO.sub.2, --CO.sub.2H, --CO.sub.2CH.sub.3, --CN,
--SH, --OCH.sub.3, --OCH.sub.2CH.sub.3, --C(O)CH.sub.3,
--NHCH.sub.3, --NHCH.sub.2CH.sub.3, --N(CH.sub.3).sub.2,
--C(O)NH.sub.2, --OC(O)CH.sub.3, or --S(O).sub.2NH.sub.2. The term
"alcohol" corresponds to an alkane, as defined above, wherein at
least one of the hydrogen atoms has been replaced with a hydroxy
group. Similarly, the term "ether" corresponds to an alkane, as
defined above wherein at least one of the hydrogen atoms has been
replaced with an alkoxy group.
IV. Relationship Between the Type of Salt and the Type of Detergent
Used
[0120] Variable results are observed for each unique cationic
detergent examined in embodiments of this invention indicating that
one cannot predict a particular phase separating agent (e.g.
cationic detergent) nucleic capture properties simply based upon
the fact that it contains a cation with a hydrophobic tail. Each
cationic detergent behaves differently as indicated by the results
provided in the Examples. Moreover, the cationic surfactants not
only interact differently with the nucleic acids, but they also are
strongly and uniquely affected by the salts and buffering
components added to the solution. Both the cation and the anion
play unique and unexpected roles in how the surfactants bind
nucleic acids (see, e.g. Example 6). A preferred molecule, domiphen
bromide, possesses the unique property of tunability. Tunability is
the ability to control of the size of the nucleic acids phased
separated by the surfactant-nucleic acid complex by varying
conditions including but not limited to salts, pH, and other
surfactants both ionic and non-ionic in the solution. This
tunability of domiphen bromide allowed for highly efficient removal
of small contaminating nucleic acids such as RNA. Domiphen bromide
(DB) also possessed the interesting property of providing for
enhanced (i.e. a boost) in recovery after a treatment with a salt
wash after capturing the DB-NA complex with a mineral matrix such
as glass fibers.
[0121] Specifically, acetates and citrates drastically change the
phase separation and capture properties of domiphen bromide. DB,
unlike CTAB, MTAB, cetylpyridinium bromide, etc., showed no
affinity for the small nucleic acids, such as the degraded RNA
commonly found in plasmid isolation that includes RNAse A. However,
in the presence of the potassium acetate, which is commonly used in
the neutralization buffer, small nucleic acids were bound and
recovered. Capturing such small RNA species is undesirable since
RNA is a contaminant for plasmid DNA isolation.
[0122] To rescue DB's ability to selectively bind the larger
nucleic acids, the conditions in the phase separation solution had
to be tuned, referred to herein as tunability. A reduced
concentration of potassium acetate in the neutralization buffer and
high concentrations of Bis-Tris or Tris in the phase separation
buffer was necessary (about 0.8 to about 1.1 M Bis-Tris or Tris)
along with an adjustment of the concentration of lithium chloride
to about 0.4-1.8 M. The original concentrations of potassium
acetate used varied in the range of about 1.0-3 M. This translates
to a final concentration (M) of about 0.1 M-0.45M LiCl and about
0.275M Bis-Tris or Tris and a range of about 0.25M-1M potassium
acetate used for the same solution. Reducing the potassium acetate
concentration alone was not as effective at rescuing DB's
selectivity. However, under the preferred conditions, the DB-based
phase separation buffer became more effective at selecting for
larger nucleic acids than CTAB-containing buffer. This highly
desirable and surprising rescue of high purity plasmid capture was
derived from DBs unique tunability which allows for selective
capture of nucleic acid species.
[0123] In the case of CTAB the buffer alone was not effective at
providing size selective capture of only the desired nucleic acid,
a salt wash was required for the effective removal of degraded RNA
(U.S. Pat. App. 2008/0113348 and U.S. Pat. No. 8,679,744). However,
the first wash buffer, which uses salts to remove weakly
interacting nucleic acids, was not sufficient in the DB-based
system to remove the degraded RNA as no removal was observed (See
Example 18). Instead a treatment solution containing salts such as
sodium chloride or lithium chloride in the range of 0.1-0.7 M (e.g.
0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, and 0.7M) enhanced the
retention of nucleic acids acting as a significant boost to
recovery yields. This is contrary to the popular surfactant CTAB
and similar cationic surfactants as disclosed by Thorsten Singer,
where it is indicated that a special wash buffer is required to
remove contaminating RNA to obtain the additional selectivity
required to clean the sample. DB attains this selectivity for
nucleic acid capture solely within the phase separation buffer and
does not require a wash to remove contaminating RNA. The selective
phase separation/capture properties for DB effectively removes
unwanted nucleic acid species (i.e. degraded RNA in a plasmid
preparation). Such RNA species are simply not captured to the
matrix.
[0124] In the case of Tris-HCl, substitution of Tris-HCl for
Bis-Tris (control) for shifts the preferred LiCl concentration of
Domiphen Bromide capture pf plasmid DNA. Preferred LiCl
concentration of between about 0.6 M to about 1.05M with about
0.7M, about 0.8 M, about 0.9M and about 1.0M LiCl being preferred
with a Tris-HCL based capture buffer.
V. Definitions
[0125] As used herein, the term "Capture" means both adsorption and
absorption and further includes but is not limited to trapping of
phase separated nucleic acid-cationic surfactant complexes by a
Solid Support Carrier (e.g., a mineral matrix).
[0126] As used herein, the term "heterologous" refers to a
combination of elements not naturally occurring. For example,
heterologous DNA refers to DNA not naturally located in the cell,
or in a chromosomal site of the cell. Preferably, the heterologous
DNA includes a gene foreign to the cell. For example, the present
invention includes chimeric DNA molecules that comprise a DNA
sequence and a heterologous DNA sequence which is not part of the
DNA sequence. A heterologous expression regulatory element is such
an element that is operatively associated with a different gene
than the one it is operatively associated with in nature. In the
context of the present invention, a gene encoding a protein of
interest is heterologous to the vector DNA in which it is inserted
for cloning or expression, and it is heterologous to a host cell
containing such a vector, in which it is expressed.
[0127] As used herein, the term "isolated" means that the
referenced material is removed from the environment in which it is
normally found. Thus, an isolated biological material can be free
of cellular components, i.e., components of the cells in which the
material is found or produced. Isolated nucleic acid molecules
include, for example, genomic DNA, a PCR product, RNA including an
isolated mRNA, a cDNA, or a restriction fragment. Isolated nucleic
acid molecules also include, for example, sequences inserted into
plasmids, cosmids, artificial chromosomes, and the like. An
isolated nucleic acid molecule is preferably excised from the
genome in which it may be found, and more preferably is no longer
joined to non-regulatory sequences, non-coding sequences, or to
other genes located upstream or downstream of the nucleic acid
molecule when found within the genome. An isolated protein may be
associated with other proteins or nucleic acids, or both, with
which it associates in the cell, or with cellular membranes if it
is a membrane-associated protein.
[0128] As used herein, "Plasmid" includes but is not limited to
plasmids, cosmids, phage vectors, expression vectors, viral
vectors, yeast shuttle vectors, and yeast artificial chromosomes.
Reference to Plasmids includes constructs or vectors of the present
invention that may comprise DNA sequences that facilitate the
cloning and propagation of the DNA including but not limited to DNA
other than that of the host organism (i.e. often heterologous DNA).
A large number of plasmids vectors, including bacterial and fungal
shuttle vectors, have been described for replication and/or
expression in a variety of eukaryotic and prokaryotic host cells
and are encompassed by this definition.
[0129] As used herein, "Selectivity" means the ability to bind
nucleic acids based on size anf type of the nucleic acid, for
example by binding genomic DNA or plasmid DNAs, preferentially over
RNA and or degraded RNA using phase separation reagents.
[0130] As used herein, "Tunability" means the ability to control
the size selection of the phase separation reagent by varying
conditions including but not limited to salts, pH, and other
surfactants both ionic and non-ionic in the solution.
[0131] As used herein, "Solid Support Carrier" means mineral
matrices that could be implemented with embodiments of the present
invention and include porous or non/porous carrier composed of
metal oxides and mix metal oxides. Such metal oxides include
materials commonly used in the art such as silicon-oxygen based
compounds. Borosilicate or silicate in the form of glass fibers,
silica gel, zeolites, or diatomaceous earth are most commonly used
due their inexpensive and non-toxic properties, however other
minerals such as aluminum oxide, titanium dioxide, zirconium
dioxide or mixtures thereof are included in this term as
alternative embodiments.
[0132] As used herein, "Prepared Sample" means a sample prior to
the isolation of nucleic acid therefrom (e.g. Plasmid via a solid
support carrier) comprising the final concentration of each
constituent present, including but not limited to cationic
surfactants, salt solutions, buffers or other constituents known in
the art or disclosed herein. The final concentration of each
constituent in a Prepared Sample can be measured by any standard
analytical technique including commonly used metrics including but
not limited to molarity (M), molality (m), volume to volume (v/v)
percentage, weight to volume (w/v) percentage, and weight to weight
(w/w) percentage.
VI. Examples
[0133] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--Titration of NaCl with 1-decyl-3-methylimidazolium
chloride (DMIC)
[0134] 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 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% 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 .mu.l 0.5 M NaCl/80 mM Tris pH 8.5/0.5 mM EDTA and
subsequently washed with 700 .mu.l 95% ethanol. Finally, the
nucleic acids were eluted from the glass fiber matrix using a
microcentrifuge. Each reaction was visualized by agarose gel
electrophoresis at 5 min as an RNA check and again at 45 min to
evaluate the full-length run (FIGS. 1A-B). Under these conditions,
the DMIC selectively captured the larger DNA and removed the
degraded RNA.
Example 2--Titration of NaCl with 1,3-didecyl-2-methylimidazolium
chloride (DDMIC)
[0136] 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.
[0137] 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, the solution was washed with 700 .mu.l 0.5 M NaCl/80
mM Tris pH 8.5/0.5 mM EDTA and then washed with 700 .mu.l 95%
ethanol. Finally, the nucleic acid was eluted from the glass fiber
matrix. Each reaction was visualized by agarose gel electrophoresis
at 5 min to check for the presence of degraded RNA (FIG. 2).
[0138] 1,3-Didecyl-2-methylimidazolium chloride successfully
captured plasmid DNA; however, it did not selectively remove
degraded RNA as NaCl was titrated into the buffer. After 1.75 M
NaCl, this solution began to phase separate before being adding to
the the sample. Therefore, 1.75 M NaCl was as high as this buffer
could be titrated using NaCl. Other salts could potentially be used
to further tune the selectivity of this phase separation reagent,
but for the purpose of this initial examination only NaCl was
investigated.
Example 3--Titration of NaCl with Cetylpyridinium Bromide (CPB)
[0139] 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 5 ml
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 .mu.l 0.5 M
NaCl/80 mM Tris pH 8.5/0.5 mM EDTA and then washed with 700 .mu.l
of 95% ethanol. Finally, the captured nucleic acid was eluted. Each
reaction was visualized by agarose gel electrophoresis at 5 min to
check for the presence of degraded RNA (FIGS. 3A-D).
[0140] By varying the w/v % of CPB from 0.25%-1% while titrating
NaCl from 0-1.75 M, it was found that the degraded RNA found below
the lowest band of the 1 kb ladder was not completely removed using
NaCl alone. Cetylpyridinium surfactants have the unique property of
having very little tunability or selectivity even in the presence
of many different salts, surfactants or buffer conditions. These
molecules tend to form Surfactant-NA complexes very strongly with
all sizes of nucleic acids despite the conditions and for this
reason these molecules may be interesting for the precipitation or
capture of cell free DNAs, microRNAs, or other smaller nucleic
acids to a mineral matrix.
Example 4--Titration of NaCl with Decylpyridinium Chloride
(DPC)
[0141] A solution of decylpyridinium chloride (DPC) 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, JM109 cells transformed with pGEM were 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 a 5 ml P3 buffer that
contained 1.5 M potassium acetate about pH 4.9 and 200 .mu.g/ml
RNAse A. The precipitated cellular debris were cleared by using a
glass fiber filter. 5 ml P4 buffer (1% DPC with titration of 0-2 M
NaCl) was added and mixed thoroughly prior to loading the solution
onto a glass fiber matrix. The matrix was washed with 700 .mu.l 0.5
M NaCl/80 mM Tris pH 8.5/0.5 mM EDTA and then washed with 700 .mu.l
95% ethanol. Finally, the captured nucleic acid was eluted. Each
reaction was visualized by agarose gel electrophoresis at 5 min as
to check for the presence of degraded RNA again at 45 min to
evaluate overall quality (FIGS. 4A-B).
[0142] DPC behaved similarly to cetylpyridinium bromide in that it
does not appear to remove degraded RNA with a simple salt
titration; however it has exceptionally improved solubility. The
pyridinium salts as a class appear to efficiently provide for the
recovery of nucleic acids with very limited or no ability to select
for size. This indicates that the utility of each phase separation
reagent is highly dependent on the goals of the purification and
that these reagents do not follow the trends at all that have been
observed for binding and other properties some of the commonly used
cationic surfactants (e.g. CTAB).
Example 5--Titration of NaCl and LiCi with Domiphen Bromide
(DB)
[0143] Nucleic acids in solution. A solution of plasmid DNA (pDNA)
and degraded RNA was used to test the ability of domiphen bromide
(DB) 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 RNA used was RNA digested with RNAse
A, which runs as a smear below the 1 kb ladder on an agarose
gel.
[0144] 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% DB with titration of 0-3M NaCl and 0-3.75M LiCl) 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 .mu.l 0.5 M NaCl/80 mM Tris pH 8.5/0.5 mM EDTA and
finally washed with 700 .mu.l 95% ethanol. Finally, the nucleic
acid was eluted from the glass fiber matrix. Each reaction was
visualized by agarose gel electrophoresis at 5 min to check for the
presence of degraded RNA and again at 45 min to evaluate the
overall quality.
[0145] The results from these experiments using DB to isolate
plasmid DNA (FIGS. 5A-E) indicated that DB very effectively
precipitates and selectively binds large nucleic acids to the glass
fiber matrix. The results showed high recovery and greater
selectivity and tunability by isolating preferred sized large
nucleic acids (i.e. plasmids) over small nucleic acids (i.e. RNA
species) compared to an optimized system that uses CTAB. DB also
showed capture of large sized nucleic acids (plasmid) was linearly
affected by increasing salt concentration. These results indicate
that alkyl surfactants such as CTAB do not behave similarly to the
functionalized or heterocyclic phase separation reagents discussed
herein. It was highly unexpected that the phase separation reagents
showed unique and distinct differences in nucleic acid capture
characteristics depending on the particular reagent chosen. The
behavior of each phase separation reagent cannot be predicted based
upon the information currently available due to structure alone.
For example some surfactants such as cetlypyridinium bromide show
little selectivity and capture all nucleic acids exceptionally
well.
[0146] Notably, the amount of pDNA recovered increased with
increasing concentrations of salt (both NaCl and LiCl) indicating
the DB-NA complex was enhanced by the ionic strength of the
solution. When sodium chloride was titrated into the phase
separation buffer indicated in the publication (US2008/01223348
A1), increased concentrations of salt actually decreased total
capture of nucleic acids. Domiphen bromide-NA complexes were
weakened only after ionic strength of the solution disrupted the
interaction in its entirety.
[0147] Bacterial culture. A solution of DB was evaluated with
increasing concentrations of lithium chloride to determine how DB
behaves in the context of a plasmid isolation using alkaline lysis.
For this, JM109 cells transformed with pGEM were grown overnight
for 16 hours. The cells contained within 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 a 5 ml P3 buffer
that contained 1.5 M potassium acetate at about pH 4.9 and 200
.mu.g/ml RNAse A. The precipitated cellular debris was cleared by
using a glass fiber filter. 5 ml P4 buffer (1% DB with titration of
either 1-2.75 M LiCl and subsequently narrowly with 1.55-1.7M LiCl)
was added prior to loading the solution onto a glass fiber matrix.
The matrix was washed with 700 .mu.l 0.65 M LiCl and then washed
with 700 .mu.l 95% ethanol. Finally, the captured nucleic acid was
eluted. Each reaction was visualized by agarose gel electrophoresis
at 5 min as to check for the presence of RNA and again at 45 min to
evaluate the full-length run (FIGS. 6A-D). See Table 1 for the
quantification of plasmid DNA recovered for the titration of
1.55-1.7M LiCl. The range of LiCl concentration present in the
phase separation buffer translates to final concentration (M) range
of about 0.3875M-0.425M LiCl in the prepared sample.
[0148] Under the conditions of typical alkaline lysis, isolation of
plasmid DNA from bacterial culture is very different than that of
pure water or a TE solution. After clearing the cell debris, the
solution that remains contains primarily potassium acetate, pH 5.5,
DNA, degraded RNA, and residual proteins or other debris that do
not precipitate after the SDS precipitation. Interestingly, nucleic
acid recovery was enhanced using this system as compared to a
simplified example previously discussed. However DB appeared to
have lost its ability to selectively bind large nucleic acids over
small nucleic acids such as degraded RNA. Upon, addition of lithium
chloride the selectivity of the system was partly restored where
plasmid DNA was preferentially captured over degraded RNA. However
it appeared that lithium chloride alone in the presence of a
cleared lysate was not sufficient to prevent the smaller fragments
of degraded RNA from being copurified without the further tuning of
the phase separation buffer, as small amounts of degraded RNA
appeared to be retained. Lithium chloride facilitated enrichment of
plasmid DNA over degraded RNA, but it did not restore the selective
properties observed when using DB. Furthermore, as if the sample
was not processed immediately after the addition of DB the DB-RNA
interactions became more pronounced indicating the interaction was
not completely inhibited under these conditions. (Data not shown).
Without being bound to a particular theory it was believed that the
potassium acetate caused the dramatic change in the capture profile
of domiphen bromide (See example 6).
TABLE-US-00001 TABLE 1 Domiphen bromide/LiCl titration from cleared
lysate Treatment Group [pDNA] (ng/.mu.L) Avg. [pDNA] Std Dev 1.5M
LiCl 591.8 663.1 100.83 1.5M LiCl 734.4 1.55M LiCl 761.95 676.77
120.47 1.55M LiCl 591.58 1.6M LiCl 542.89 556.39 19.08 1.6M LiCl
569.88 1.65M LiCl 445.47 514.17 97.16 1.65M LiCl 582.87 1.7M LiCl
699.85 575.43 175.96 1.7M LiCl 451.01 1.75M LiCl 393 387.15 8.27
1.75M LiCl 381.3
[0149] Yield Boosting Treatment buffer optimization. An experiment
was performed to determine the preferred salt concentration in the
treatment solution with respect to increasing retention of plasmid
and thus overall yield. The salt concentration used strongly
affected the total plasmid recovered as indicated in FIG. 6E. Note
that at 0 M LiCl the intensity of the plasmid is greatly reduced
(lower yield), however upon the addition of even 0.1 M LiCl there
is a substantial boost in the recovery of plasmid. As the
concentration of salt in this treatment buffer was increased the
boost in retention and recovery of the captured nucleic acid
increased until at which point the ionic strength of the solution
disrupted the phase separation reagent nucleic acid complex and
washed away the nucleic acid
[0150] The presence of salt in this treatment greatly increased the
retention and recovery of plasmid in comparison to just a treatment
containing water only or skipping the treatment and only performing
the organic wash step. This titration indicated that 0.7 M LiCl
corresponded to the highest recovery of plasmid examined. However,
after 0.7 M LiCl the plasmid recovery was drastically reduced,
which is likely due to the disruption of the interaction between
the phosphate backbone of the nucleic acids and the phase
separation reagent. Common inorganic salts such as sodium chloride
could also be used to boost the retention and recovery of the
target nucleic acid(s) being isolated. The preferred range of salt
solution depends on the salt used however the range of salt
concentration that could be used lies between about 0.1M and about
1.0M LiCl, NaCl, or KCl. More specifically the preferred range is
between about 0.35 and about 0.55M LiCL, NaCl, or KCl.
[0151] The original intent of adding salts in the first treatment
solution was to remove molecules that weakly interact with the
domiphen bromide, however as one can see it had the unexpected
result of also greatly increasing the recovery of the plasmid DNA.
In contrast a similar solution applied to a nucleic acid/plasmid
isolation system with CTAB (Application US2008/0113348) was used to
remove RNA contamination however their results indicate no increase
in overall nucleic acid yield including genomic DNA and plasmid DNA
yield. In the CTAB system the recovery of total nucleic acids
decreased with increasing concentrations of salt. However we found
by using domiphen bromide as the phase separation/capture agent,
there was unexpectedly a dramatic increase in recovery of large
fragments that increased with increasing salt concentration in this
treatment step and no visible change in capture of small nucleic
acids such as degraded RNA. There was a point at which overall
recovery was diminished due to salt likely interfering with the
DB-Nucleic Acid-Borosilicate matrix interactions; however within
the range of about 0.1-0.7 M there was a significant improvement in
recovery versus no salt buffer solution treatment. The salt
treatment used in the DB system had the unique property of
increasing retention of nucleic acid thus boosting the yield in
addition to the originally intended contaminant removal, which
further exemplifies the unexpected nature of these phase separation
reagents especially functionalized and heterocyclic ammonium
surfactants.
Example 6--Evaluation of Various Salts for their Effect on Total
Recovery and Selectivity of Nucleic Acids Recovered Using Domiphen
Bromide (DB)
[0152] Various salts were evaluated to determine how the affect
total recovery and selectivity of domiphen bromide in the context
of plasmid preps based on alkaline lysis. For this experiment,
JM109 was transformed with pGEM and cultures were grown overnight
for about 16 hours. The cells contained within 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 a 5 ml
P3 buffer that contained 1.0 M potassium acetate pH 4.9 and 200
.mu.g/ml RNAse A. The precipitated cellular debris was cleared by
using a glass fiber filter. 5 ml P4, a 1% domiphen bromide solution
containing varying concentration of salts, was added to the cleared
lysate prior to loading the solution onto a glass fiber matrix. The
matrix was washed with 700 .mu.l 0.6 M LiCl and then washed with
700 .mu.l 95% ethanol. Finally, the captured nucleic acid was
eluted using water.
[0153] The following salts were investigated for their effects on
selectivity and total recovery: sodium citrate (0.25-1 M; FIGS.
7C-D), lithium bromide (0.5-1 M), lithium acetate (1-2.25 M; FIGS.
7A-B), magnesium chloride (0.1-1.25 M; FIGS. 7A-B), sodium formate
(1-2.25 M; FIGS. 7C-D), potassium acetate (1-2.25 M; FIGS. 7E-F),
potassium chloride (1-2.25 M; FIGS. 7E-F), and sodium acetate
(1-2.25 M; FIGS. 7E-F). Each reaction was visualized by agarose gel
electrophoresis at 5 min to check for the presence of degraded RNA
and again at 45 min to evaluate the full-length run.
[0154] The results indicated that potassium chloride, magnesium
chloride, and sodium formate were able to selectively recover
plasmid DNA. Lithium bromide caused the phase separation reagent,
domiphen bromide, to precipitate immediately and was therefore not
used.
[0155] The lithium acetate and sodium citrate did not increase the
selectivity of the phase separation reagent for large nucleic acids
as indicated by the presence of degraded RNA at concentrations up
to 2.25 M lithium acetate.
[0156] By qualitative examination of the gel electrophoresis
results the increased potassium acetate appeared to slowly mitigate
small nucleic acids from being recovered; however, it began to
cause the buffer to precipitate at concentrations higher than 2 M
KoAc and was therefore removed from consideration. Without being
bound to a particular theory it is believed that that slow decrease
in recovery of degraded RNA was based on the potassium cation as
potassium chloride facilitated removal of small fragments.
[0157] The above salts are functional in that they allowed capture
and some of the salts even enhanced capture (e.g., sodium acetate).
However, none of the salts added appeared to facilitate a large
degree of size selection, and therefore, were deemed suboptimal.
Both the cation and the anion in these salts directly affect what
and how much is captured. For instance, the acetate ion favored
capture of all nucleic acids, as indicated by the difference in
selectivity for large DNA between potassium chloride and potassium
acetate. Domiphen bromide in solution-based experiments that did
not contain potassium acetate showed no capture of small nucleic
acids, such as degraded RNA (See example 4).
Example 7--Evaluation of the Effect of pH and Salt Effect on
Capture Capacity and RNA Elimination while Using 1 M KoAc and
Domiphen Bromide (DB)
[0158] The purpose of the following experiment was to determine the
effect of pH on the capture of nucleic acids from a cleared lysate
using domiphen bromide (DB). The interest was to examine changes in
both total recovery and selective recovery of specific sized
nucleic acids.
[0159] For this experiment, JM109 was transformed with pGEM and
cultures were grown overnight for 16 hours. The cells contained
within 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 a 5 ml P3 buffer that contained 1.0 M
potassium acetate pH 4.9 and 200 .mu.g/ml RNAse A. The precipitated
cellular debris was cleared by using a glass fiber filter. 5 ml P4,
a 1% domiphen bromide solution containing varying concentration of
salts, Bis-Tris or Tris to control the pH, and or
N-Lauroylsarcosine sodium salt to see the effect of using an
anionic detergent, was added to the cleared lysate prior to loading
the solution onto a glass fiber matrix to capture the nucleic
acids. The matrix was washed with 700 .mu.l 0.6 M LiCl and then
washed with 700 .mu.l 95% ethanol. Finally, the captured nucleic
acid was eluted.
[0160] All the phase separation buffers were titrated with lithium
chloride to determine the effect of pH or N-Laurylsarcosine sodium
salt had on recovery or selective recovery of nucleic acids. Four
different buffers were tested: (1) 1.1 M Tris pH 8.5, 0.5-1.75 M
LiCl, 1% DB (FIGS. 8A-B); (2) 1.1 M Bis-Tris pH 7.0, 0.5-1.75 M
LiCl, 1% DB (FIGS. 8A-B); (3) 1.5-2.25 M LiCl, 1% DB (FIGS. 8A-B);
and (4) 1.5 M LiCl, 0.01-0.25% (w/v) N-Lauroylsarcosine, 1% DB
(FIGS. 8C-D). Each reaction was visualized by agarose gel
electrophoresis at 5 min to check for the presence of degraded RNA
and again at 45 min to evaluate the full-length run.
[0161] Without being bound to a particular theory it is thought
that the salts that composed the buffer played an equally important
role as the effect of pH on controlling DB's selectivity for
capture of large nucleic acids over small nucleic acids such as
degraded RNA. It is interesting that the effect of pH on DB's
ability to size select behaves exactly opposite to reports on CTAB
behavior ((U.S. Pat. App. 2008/0113348). This result was unexpected
and further demonstrates the unpredictable and highly tunable
nature of the functionalized and heterocyclic ammonium surfactants
such as DB.
[0162] Selective capture window. Without being bound to a
particular theory it is thought that the DB based cationic
surfactant-nucleic acid complex is facilitated to some extent by
the presence of salts and chemicals (e.g. buffers). The formation
of the micelle is facilitated by an increase in ionic strength of
the solution, however there is a point as which the salt
concentration begins to strongly interfere with the cationic
surfactant-nucleic acid complex. Which means that in the case of
the "tunable" surfactants, show this behavior, it was found that
there is a "window" of opportunity where one can select for the
nucleic acid size that is desirable, remove contaminants, and
maximize total recovery due to facilitating micelle formation. DB,
being a highly tunable surfactant allowed for selection to take
place under the conditions of salt/pH discussed in this experiment.
The results of this experiment indicate that adjusting the pH using
Bis-Tris and Tris did not dramatically change the total recovery of
nucleic acid; however, it did affect the selectivity. These changes
in pH and the addition of Bis-Tris/Tris salts affected the "window"
(FIGS. 8A-D, circled concentrations) in which the small nucleic
acid fragments were selectively removed. Note that the "window" in
which the degraded RNA is absent is much larger in the solutions
containing Tris and Bis-Tris as compared to the control solution
that contained varying concentrations of LiCl buffered by KoAc at
pH 4.9. This increase in the "window" of opportunity for selective
plasmid isolation is a key component to designing a kit for the
isolation of pure plasmid DNA. It should be noted that the effect
of pH from salts and chemical (e.g. buffers) is unpredictable since
Bis-Tris and Tris although similar are quite different molecules,
and both similarly affected the selectivity but at different salt
concentrations.
[0163] N-Lauroylsarcosine sodium salt also contributed to
selectivity in that it showed a propensity to remove smaller
nucleic acids; however, the system is very sensitive to small
changes in N-Lauroylsarcosine sodium salt concentration and
therefore would not be an ideal candidate for aiding selectivity.
It also appeared to enhance total recovery at 0.1% (w/v)
N-Lauroylsarcosine sodium salt. This translates to a final
concentration (w/v) of 0.025% (w/v) N-Lauroylsarcosine sodium salt.
The conditions that showed the most promise were titrated further
(see, Example 8).
[0164] There are two principle nucleic acid types that must be
removed when creating a plasmid isolation kit. The first is genomic
DNA, which is classically removed during the SDS/KoAc precipitation
step, and RNA, which is digested by RNAse A. However, the RNAse
does not completely remove the RNA, it simply digests it into
smaller fragments that range in size from about 1-100 bp.
Therefore, in order to create a kit that is free of nucleic acid
contaminants, the selective removal of small nucleic acids from the
plasmid prep is fundamental to its design.
[0165] Endotoxin removal. The wide range of selective capture
provided by this method for large nucleic acids preferentially over
small parallels another key feature of plasmid isolation
method--endotoxin removal. Other compounds that have weaker
affinity for the cationic surfactant could also be selectively
removed. This means that endotoxins are expected to be selectively
removed by the "Phase separation solution" if the proper ratio of
salts and the correct pH is achieved (see, circled concentrations).
Therefore, contamination could be problematic if the system is not
robustly buffered against slight changes in volumes due to user
handling or changes in volume of cleared lysate recovered post
filtration.
[0166] The plasmid pGEM was isolated from 100 ml of a JM109 E. coli
culture grown overnight and the average EU/.mu.g of plasmid DNA for
the preps was 0.985 when measured using the Pyrochrome Endotoxin
Specific Assay (Associates of Cape Cod, Inc). Compared to standard
chaotropic salt based methodology the phase separation solution is
approximately 1,200 times better at removing endotoxins. It
achieves approximately half the endotoxins of 2.times. CsCl
gradient centrifugation and about 9-10 times less endotoxins
compared to classic anion exchange methodology as estimated using
commercially available information for commercially available
products offered by QIAGEN.TM..
Example 8--Evaluation of Various Salts, pH, and N-Lauroylsarcosine
Sodium Salt to Determine Preferred Capture Conditions for DNA Using
Domiphen Bromide (DB)
[0167] For this experiment, JM109 was transformed with pGEM and
cultures were grown overnight for 16 hours. The cells contained
within 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 5 ml P3 buffer that contained 1.0 M
potassium acetate pH 4.9 and 200 .mu.g/ml RNAse A. The precipitated
cellular debris was cleared by using a glass fiber filter. 5 ml P4,
a 1% domiphen bromide solution containing varying concentrations of
salts, Tris or Bis-Tris, and N-Lauroylsarcosine sodium salt, was
added to the cleared lysate prior to loading the solution onto a
glass fiber matrix. The matrix was washed with 700 .mu.l 0.6 M LiCl
and then washed with 700 .mu.l 95% ethanol. Finally, the captured
nucleic acid was eluted. The various solutions are provided in
Table 1. Each reaction was visualized by agarose gel
electrophoresis at 5 min to check for the presence of RNA and again
at 45 min to evaluate the full-length run.
[0168] This experiment showed that all of the above solutions
provided a high degree of selectivity as indicated by the absence
of degraded RNA in the gels (FIGS. 9A-B), with comparable
recoveries of plasmid DNA (Table 2, bold values). 1% DB/2 M LiCl
provided the highest recovery; however, it has a narrow window of
selectivity and is therefore potentially more susceptible to
deviations in volumes and concentrations. The higher sensitivity to
user handling is not preferred despite the slightly higher
recovery. Bis-Tris pH 7.0 appears to provide the highest recovery
with reliability. Furthermore, (data not shown) as previously
discussed, increasing salt concentration alone appeared to remove
RNA degradation if processed immediately, however after short
incubations the DB-RNA complex began to form again and was
subsequently captured.
[0169] 1.1 M Bis-Tris pH 7.0/1.5 M lithium chloride/1% domiphen
bromide provided the second highest recovery at 709.1 ng/.mu.L,
which is only 30 ng/.mu.L less than the 1% DB/2 M LiCl; however, it
provided a larger window of selectivity as shown above. Currently
the preferred solution for phase separation contains between about
0.25%-4% domiphen bromide between about 0.4-2.25 LiCl, between
about 0-1.5 M Tris or Bis-Tris pH between about 7-8.5.
TABLE-US-00002 TABLE 2 Effect of pH, LiCl, and Sarcosine on capture
capacity and RNA elimination Molarity of LiCl pDNA (ng/.mu.L) Tris
pH 8.5 (1.1M) 1 557.2 1.25 596.5 Tris pH 8.5 (1.1M)/0.05% w/v
Sarcosine 1 427.1 1.25 510.7 Tris pH 8.5 (1.1M)/0.1% w/v Sarcosine
1 542.1 1.25 20.4 Bis-Tris pH 7.0 (1.1M) 1.25 403.1 1.5 709.1
Bis-Tris pH 7.0 (1.1M)/0.05% w/v Sarcosine 1.25 593.3 1.5 21.3
Bis-Tris pH 7.0 (1.1M)/0.1% w/v Sarcosine 1.25 106.2 1.5 8.3
Standard DB/LiCl only buffer 2 739.8 Standard DB/LiCl/0.05% w/v
Sarcosine 2 28.8 Standard DB/LiCl/0.1% w/v Sarcosine 2 N/A
Example 9--Determination of Preferred Capture Conditions
[0170] For this experiment, JM109 was transformed with pGEM asnd
cultures were grown overnight for 16 hours. The cells were
contained within 35 ml of culture 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 5 ml P3 buffer that contained 1.0 M
potassium acetate pH 4.9 and 200 .mu.g/ml RNAse A. The precipitated
cellular debris was cleared by using a glass fiber filter. 5 ml P4,
a 1% DB solution containing varying concentration of salts, Tris or
Bis-Tris, was added to the cleared lysate prior to loading the
solution onto a glass fiber matrix. The matrix was washed with 700
.mu.l 0.6 M LiCl and then washed with 700 .mu.l 95% ethanol.
Finally, the captured nucleic acid was eluted.
[0171] Three parameters evaluated using the three preferred phase
separation buffer solutions. The parameters (see, Table 3) tested
are as follows: total recovery from a standard prep, total recovery
if two preps were loaded through the same column (indicated as
2.times.), and lastly if a 1 hour incubation in the phase
separation buffer prior to loading it on the column affected
recovery. Each reaction was visualized by agarose gel
electrophoresis at 5 min to check for the presence of degraded RNA
and again at 45 min to evaluate the full-length run (FIGS.
10A-B).
[0172] 1.1 M Bis-Tris pH 7.0/1.5 M LiCl/1% DB outperformed the
other two candidates and achieved the highest recovery of 231 .mu.g
and displayed the most consistent results (Table 3). 1% DB/1.1 M
Bis-Tris pH 7.0/1.5 M LiCl was determined to be the preferred phase
separation buffer based upon its high degree of selectivity and
robustness in its recoveries. The recoveries were highly consistent
which indicates that the system is robust in its ability to
tolerate user handling. It is also highly selective in that the
recoveries listed in the table were free of contaminating degraded
RNA.
TABLE-US-00003 TABLE 3 Determination of capture conditions Total
Avg pDNA Std pDNA Phase Separation Buffer (ng/.mu.L) Dev (.mu.g)
1.1M Tris pH 8.5/1.25M LiCl/1% DB 544.7 68.0 108.9 1.1M Bis-Tris pH
7.0/1.5M LiCl/1% DB 603.0 1.1 120.6 2M LiCl/1% DB 635.6 42.5 127.1
1.1M Tris pH 8.5/1.25M LiCl/1% DB 917.8 160.8 183.6 1.1M Bis-Tris
pH 7.0/1.5M LiCl/1% DB 1158.2 176.4 231.6 2M LiCl/1% DB 976.9 129.5
195.4 1.1M Tris pH 8.5/1.25M LiCl/1% DB 584.1 262.3 116.8 1.1M
Bis-Tris pH 7.0/1.5M LiCl/1% DB 653.3 39.5 130.7 2M LiCl/1% DB
543.8 16.0 108.8
[0173] In order to further validate the preferred phase separation
buffer conditions, plasmid DNA was "spiked" into the cleared lysate
prior to adding the buffer. This was done to determine the maximum
capture capacity using the 3 ply Alhstrom 141 (15 mg matrix) on the
Zymo Spin V column. A stock solution of 542 ng/.mu.L was used to
add 100, 200, or 400 .mu.L of pDNA solution. This solution was then
mixed with 5 mL of phase separation buffer and loaded onto the
column. Total recovery was measured using a Nanodrop.TM. (Table
4).
TABLE-US-00004 TABLE 4 Determining Recovery Volume of Avg Fraction
pGEM Added pDNA pDNA of pDNA (ul) (ng/ul) (ng/ul) Recovered 100
606.60 668.40 0.72 100' 730.20 200 856.70 898.50 0.75 200' 940.30
400 1468.90 1488.60 0.86 400' 1508.30
Example 10--Matrix Porosity Vs. Nucleic Acid Recovery
[0174] The following experiment used neutralized bacterial lysate
prepared from 35 mL of overnight culture that was loaded onto spin
columns containing borosilicate glass fiber with a nominal particle
retention rating of either 1.0 .mu.m (Porex Grade B), 1.6 .mu.m
(Porex Grade A), or 2.7 .mu.m (Porex Grade D) using a previously
reported CTAB buffer. There was an enormous difference in the
capture capacity between each of the grades of glass used that
strongly correlates to the nominal particle retention rating (FIG.
11). This is counter intuitive as one would logically reason that a
denser less porous matrix would be more effective at capture and
collection of the CTAB/nucleic acid complex. The mostly likely
reason for the slightly higher capture between the Porex Grade B
and the Porex Grade A glass fibers is because the grade B had an
additional 1-2 mg of matrix, thereby providing a slightly larger
surface area for the CTAB/DNA complex to bind to. Despite the
slight difference in recovery between Grade B and Grade A, the
Grade D clearly demonstrated an unexpected significantly higher
yield.
Example 11--Capture of Plasmid
[0175] Approximately 50 ml of culture containing JM109 transformed
with pGEM plasmid was used for each preparation. About 8 ml of P1
was used to resuspend the cells. About 8 ml of P2 was used to lyse
the cells for about 3 minutes. About 8 ml of P3 was used to
neutralize the lysis buffer and precipitate debris such as protein
and genomic DNA. The solution was cleared of precipitate by passing
the solution through a filter. About 8 ml of P4 was added and mixed
thoroughly.
[0176] Two of the samples (controls) were processed by directly
loading them onto the silica filter followed by about 5 ml of a
first wash and two 5 ml second washes.
[0177] Two other samples were centrifuged at 4,000.times.g for 15
minutes (experimental). A precipitate appeared at the bottom of
these experimental samples following this centrifugation (FIG. 12,
left). The supernatant was poured onto the silica filter and washed
using the regiment described above. Both samples were eluted using
200 .mu.lof 10 mM Tris pH 8.5 and 0.1 mM EDTA.
[0178] The plasmid DNA is selectively precipitated by the addition
of the domiphen bromide under the conditions described above as
indicated by the formation of a precipitate for the experimental
sample but not for the controls (FIG. 12, left and right arrows
respectively). Further confirmation that the precipitate does in
fact contain plasmid DNA was determined by processing the
supernatant of the samples post centrifugation (Table 5).
[0179] The samples that showed a supernatant contained
approximately 10% of the DNA that the control groups contained
indicating the precipitate contained a majority of the DNA.
Therefore it appears that the capture mechanism by which the
Domiphen bromide-DNA complex is retained is based on trapping it on
the silica membrane (Table 5).
TABLE-US-00005 TABLE 5 Domiphen bromide Precipitation of Nucleic
Acid. Sample (ng/ml) Experimental Control 1 64.0 353.3 2 38.0
315.6
Example 12--Further Evaluation of Tris-HCl and Bis-Tris Plasmid
Preparation Using DB
[0180] Substitution of Tris-HCl for Bis-Tris (control) for shifts
the preferred LiCl concentration of Domiphen Bromide capture of
plasmid DNA. The pGEM plasmid was purified from about 50 ml of
overnight JM109 E. coli culture in duplicate using approximately 1%
Domiphen Bromide, 1.40 M LiCl in 1.1 M Bis-Tris, pH 7.0 or
alternately in approximately 1% Domiphen Bromide in 1.1 M Tris-HCl,
pH 7.20 containing the LiCl concentrations shown. Samples were
processed essentially as described in Example 9. About 10 .mu.l of
elution was visualized following 20 min (A) to check for
undesirable RNA contamination (RNA Check) and 60 min (B) in a full
length run to examine plasmid isolation in agarose gel
electrophoresis. In each FIGS. 14A-B the first two lanes are
controls with Bis-Tris as the buffer component. LiCl concentrations
are shown to titrate the preferred LiCl concentration. The Marker
(M) is a 1 kb DNA Marker (Zymo Research Corp.) (FIG. 14). This
experiment shows a preferred LiCl concentration of between about
0.6 M to about 1.05M with about 0.7 M, about 0.8 M, about 0.9M and
about 1.0M LiCl being preferred with a Tris-HCL based capture
buffer.
Example 13--Genomic DNA Extraction Using Novel Phase Separation
Reagents
[0181] Various phase separation reagents were used to isolate
nucleic acids from E. coli as a model for genomic DNA and RNA
isolation to examine the widely unpredictable characteristics of
such phase separation reagents to determine if genomic DNA could be
efficiently isolated by the following protocol. The protocol for
the ZR Fungal/Bacterial DNA MiniPrep was followed according to the
manufacture suggested protocol to isolate DNA and as a measure of
extraction efficiency. Approximately, 1 ml of bacterial culture was
used for each experiment. Samples were centrifuged at 900.times.g
for 1.5 minutes to pellet the cells, supernatants were discarded,
the pellets were washed with 200 .mu.l 1.times. PBS, centrifuged
again at 900.times.g for 1.5 minutes to pellet the cells,
supernatants discarded, and then the cells were resuspended using
200 .mu.l of a solution containing 10 mM Tris HCl pH 8.5 and 0.1 mM
EDTA prior to processing. The samples were pooled to ensure
homogeneity during processing and subsequently lysed using 0.5 mm
high-density beads (Zymo, ZR BeadBashing.TM. Kit) vortexed at 6.5
m/s in Sodium Chloride (NaCl) solutions of increasing final
concentrations (see Table 6). The final concentration of NaCl in
the samples tested ranged from about 0.5 M to about 1.0 M.
Different cationic surfactants were added to a final concentration
of 0.25%. The sample was loaded onto a commercially available Zymo
Spin III-P column centrifuged at 10,000.times.g and subsequently
washed with 700 .mu.l 0.6 M LiCl and 700 .mu.l and 95% ethanol
(.times.2). The ZR Fungal/Bacterial DNA MiniPrep Kit was used as a
control to for extraction efficiency according to the manufacture
suggested protocol. The sample was by pelleting and bead bashing
steps described as above utilizing the manufactures supplied Lysis
Solution and adding three volumes of Genomic Lysis Buffer to the
lysate. The sample was loaded onto a Zymo-Spin IIC column
centrifuged at 16,000.times.g and subsequently washed with 200
.mu.l DNA Pre-Wash Buffer and 500 .mu.l g-DNA Wash Buffer. All
samples were eluted in 100 .mu.l DNA Elution Buffer (Zymo Research
Corp.) after 5 min incubation at room temp.
TABLE-US-00006 TABLE 6 Evaluation of the extraction efficiency of
different cationic detergents. Concentration Yield (ng/.mu.l)
(.mu.g) 0.5M NaCl 0.25% Lauroylcholine chloride 40.3 4.03 hydrate
0.7M NaCl 0.25% Lauroylcholine chloride 1.8 0.18 hydrate 0.9M NaCl
0.25% Lauroylcholine chloride 1.4 0.14 hydrate 1M NaCl 0.25%
Lauroylcholine chloride 0 0 hydrate 0.5M NaCl 0.25% 1,3-dideoyl-2-
229.3 22.93 methylimidazolium chloride 0.7M NaCl 0.25%
1,3-dideoyl-2- 101.5 10.15 methylimidazolium chloride 0.9M NaCl
0.25% 1,3-dideoyl-2- 8.4 0.84 methylimidazolium chloride 1M NaCl
0.25% 1,3-dideoyl-2- 6.5 0.65 methylimidazolium chloride 0.5M NaCl
0.25% octylimidazolium chloride 2.4 0.24 0.7M NaCl 0.25%
octylimidazolium chloride 1.7 0.17 0.9M NaCl 0.25% octylimidazolium
chloride 1.4 0.14 1M NaCl 0.25% octylimidazolium chloride 1.2 0.12
0.5M NaCl 0.25% 1-dodecyl-2-methyl-3- 42.2 4.22 benzylimidazolium
chloride 0.7M NaCl 0.25% 1-dodecyl-2-methyl-3- 76.2 7.62
benzylimidazolium chloride 0.9M NaCl 0.25% 1-dodecyl-2-methyl-3-
55.8 5.58 benzylimidazolium chloride 1M NaCl 0.25%
1-dodecyl-2-methyl-3- 6.9 0.69 benzylimidazolium chloride 0.5M NaCl
0.25% Domiphen Bromide 208.8 20.88 0.7M NaCl 0.25% Domiphen Bromide
158.1 15.81 0.9M NaCl 0.25% Domiphen Bromide 1.3 0.13 1M NaCl 0.25%
Domiphen Bromide 6.3 0.63 0.5M NaCl 0.25% cetylpyridinium chloride
133.3 13.33 0.7M NaCl 0.25% cetylpyridinium chloride 84.4 8.44 0.9M
NaCl 0.25% cetylpyridinium chloride 0.1 0.01 1M NaCl 0.25%
cetylpyridinium chloride 0 0 0.5M NaCl 0.25% benzethonium chloride
216 21.6 0.7M NaCl 0.25% benzethonium chloride 147.9 14.79 0.9M
NaCl 0.25% benzethonium chloride 5.3 0.53 1M NaCl 0.25%
benzethonium chloride 3.4 0.34 0.5M NaCl 0.25% 1-dodecyl pyridinium
11.6 1.16 chloride hydrate 0.7M NaCl 0.25% 1-dodecyl pyridinium 3.9
0.39 chloride hydrate 0.9M NaCl 0.25% 1-dodecyl pyridinium -0.1
-0.01 chloride hydrate 1M NaCl 0.25% 1-dodecyl pyridinium 0 0
chloride hydrate
[0182] The efficiency for DNA extraction and selectivity of the
cationic surfactants was evaluated via spectrophotometry and
visualized by agarose gel electrophoresis after 45 min (FIGS.
15A-B). Varying selectivity was observed for DNA and RNA for the
different cationic detergents tested. Laurylcholine chloride
hydrate showed nearly no binding of genomic DNA at the
concentrations of NaCl evaluated (e.g. about 0.5 M to about 1.0 M).
At about 0.5 M sodium chloride the RNA was selectively bound which
was a completely unexpected phenomenon. The surfactant 1,3
didecyl-2-methylimidazolium chloride demonstrated an ability to
selectively bind nucleic acids (tunability) as seen by the
selective binding of genomic DNA and RNA at 0.5 and 0.7 M NaCl
respectively (FIG. 15A). This trend is the opposite of what was
observed for domiphen bromide in the context of the plasmid DNA
isolation embodiments described in other embodiments where the
smaller nucleic acids were selectively removed as NaCl was added
under both near neutral and basic conditions. Under acidic
conditions the degraded RNA was not robustly and efficiently
removed. In the range of concentrations tested another surfactant
octylimidazolium did not bind nucleic acids. Still another
surfactant 1-dodecyl-2-methylimidazolium chloride showed the
ability to selectively remove large nucleic acids while retaining
RNA (tunability). Furthermore, DNA was selectively bound in
preference to RNA at lower concentrations of NaCl. Domiphen bromide
was not tunable using salt alone under the conditions used for
genomic DNA isolation. The surfactant Cetylpyridinium chloride
contrary to its performance for the plasmid DNA isolation
embodiments selectively removed large DNA molecules showing
tunability. The surfactant Benzethonium chloride behaved similarly
to Cetylpyridinium chloride. The surfactant 1-dodecyl pyridinium
chloride did not bind nucleic acids under any of the conditions
which were unexpected, as this molecule showed exemplary binding of
all sized nucleic acids under the conditions of the plasmid
preparation. These results show that genomic DNA can be efficiently
isolated with the described phase separation reagents but that each
individual phase separation reagent displays different
properties.
[0183] In addition to the isolation of genomic DNA from the cells,
the phase separation reagents also showed an ability to selectively
isolate RNA (example: cetylpyridinium chloride.) Furthermore, the
phase separation reagents all show varying capabilities to isolate
RNA. For example, under the conditions tested Lauroylcholine
chloride hydrate, 1,3-dideoyl-2-methylimidazolium chloride, and
1-dodecyl-2-methyl-3-benzylimidazolium chloride indicated an
ability to selectively isolate smaller RNA molecules in preference
to larger genomic DNA.
Example 14--Genomic DNA Isolation Using Domiphen Bromide
[0184] Domiphen Bromide was further examined for the selective
isolation of genomic DNA as well as RNA from human cells (HeLa
cells) by the following protocol. The protocol for the Quick-gDNA
MiniPrep was followed according to the manufacture suggested
protocol. Approximately, 1.5 million cells were resuspended using
200 .mu.l of a pH 8.5, 10 mM Tris 0.1 mM EDTA solution, pooled to
ensure homogenous mixtures, and bead bashed for 30 seconds using
0.5 mm high-density beads vortexed at 6.5 m/s in NaCl or LiCl
solutions of increasing final concentrations of about 0.5-0.8M (see
Tables 7 and 8). The Quick-gDNA MiniPrep was used as a control to
for extraction efficiency. The control samples were processed using
the manufacturer's provided lysis solution for bead bashing. Three
volumes of Genomic Lysis Buffer were added to the lysate. The
sample was loaded onto a Zymo-Spin IIC column centrifuged at
16,000.times.g and subsequently washed with 200 .mu.l DNA Pre-Wash
Buffer and 500 .mu.l g-DNA Wash Buffer. For experimental groups,
Domiphen bromide was added to a final concentration of about 0.25%.
The sample was loaded onto a column (Zymo Spin III-P column), and
centrifuged at 10,000.times.g, for 1 minute, washed with 700 .mu.l
0.6 M LiCl and centrifuged at .gtoreq.16,000.times.g, for 1 minute
more, and then twice washed with 700 .mu.l 95% ethanol and
centrifuged at .gtoreq.16,000.times.g, for 1 minute more. All
samples were eluted in 100 .mu.l DNA Elution Buffer (Zymo Research
Corp.) after 5 min incubation at room temperature (RT). Each sample
was also evaluated via spectrophotometry and visualized by agarose
gel electrophoresis at 5 min to check for the presence of degraded
RNA and again at 45 min to evaluate the full-length run (FIGS.
16-17).
TABLE-US-00007 TABLE 7 Determination of binding conditions using
NaCl. Average Standard Average Recovery Deviation Yield (ng/.mu.l)
(St. Dev.) (.mu.g) Control 31.60 0.85 3.16 0.57M Nacl 64.35 10.96
6.44 0.59M NaCl 48.80 20.08 4.88 0.61M NaCl 31.70 9.19 3.17 0.63M
NaCl 20.95 11.38 2.10 0.65M NaCl 13.55 8.70 1.36 0.68M NaCl 7.65
4.60 0.77
[0185] Table 8--Determination of Binding Conditions Using LiCl.
TABLE-US-00008 TABLE 8 Determination of binding conditions using
LiCl. Average Average Recovery Yield (ng/.mu.l) Stdev (.mu.g)
Control 15.70 1.56 1.57 0.5M LiCl 89.80 11.17 8.98 0.61M LiCl 97.70
4.38 9.77 0.7M LiCl 13.15 2.19 1.32 0.75M LiCl 2.10 1.13 0.21 0.8M
LiCl 0.45 0.21 0.05
[0186] Domiphen bromide displayed enrichment of isolated genomic
DNA when various salts such as sodium chloride and lithium chloride
were increased in concentration. For example, Domiphen bromide in
the presence of increasing the concentrations of NaCl demonstrated
the ability to selectively bind larger DNA while smaller nucleic
acids passed through the filter (FIG. 16). When Lithium chloride
(LiCl) was used it proved to be less selective showing decreased
ability to selectively bind different sized DNA fragments over the
conditions tested (FIG. 17) Indicating that the type of cation used
affects the selective power of Domiphen bromide.
Example 15--Binding the Surfactant-Nucleic Acid Complex to Magnetic
Beads
[0187] Domiphen Bromide was further evaluated for isolation of
genomic DNA as well as RNA using magnetic-silica particles as the
solid phase carrier. The following experiments used Saccharomyces
cerevisiae as the model organism. The protocol for the ZR
Fungal/Bacterial DNA MiniPrep was followed according to the
manufacture suggested protocol. Approximately, 50 mg wet weight of
yeast cells was resuspended using 200 .mu.l of a Tris-EDTA
solution, pooled to ensure homogenous mixtures, and lysed by
vortexing (bead bashed) using 0.5 mm high-density beads at 6.5 m/s
to lyse the yeast cells in NaCl solutions of increasing final
concentrations. For the control group, the manufacturer's provided
lysis solution was utilized for bead bashing. Three volumes of
Genomic Lysis Buffer were added to the lysate. The sample was
loaded onto a Zymo-Spin II column centrifuged at 16,000.times.g and
subsequently washed with 200 .mu.l DNA Pre-Wash Buffer and 500
.mu.l g-DNA Wash Buffer. For experimental groups, Domiphen Bromide
was added to a final concentration of about 0.25%. Approximately,
30 .mu.l of Magnetic Beads were added to each sample and were
washed with 700 .mu.l 0.6 M LiCl and 700 .mu.l 95% ethanol
(.times.2). All samples were eluted in 45 .mu.l DNA Elution after 5
min incubation at room temperature Buffer (Zymo Research Corp.).
Each sample was evaluated via spectrophotometry and visualized by
agarose gel electrophoresis after 45 min (FIG. 18).
TABLE-US-00009 TABLE 9 Compatibility of novel surfactants for
nucleic acid purification. Average Average Recovery Yield
(ng/.mu.l) Stdev (.mu.g) Column Control 81.70 1.56 8.17 MagBead -
0.06M NaCl 2233.70 79.05 223.37 MagBead - 0.11M NaCl 2329.05 32.88
232.91 MagBead - 0.45M NaCl 2048.00 60.81 204.80
[0188] This experiment demonstrated that the Domiphen
bromide-nucleic acid complex binds directly to magnetic-silica
beads. This was unexpected as precipitation was demonstrated in
embodiments utilizing centrifugation. Therefore, without being
bound to a particular theory it appears that the mode of capture is
unexpectedly of a dual action --both precipitation and binding.
Example 16--Titration of Buffer, pH, and NaCl to Optimize Genomic
DNA Binding Using Domiphen Bromide
[0189] Experiments using E. coli as the model system for genomic
DNA isolation were performed with Domiphen Bromide to assess the
effects of buffer composition and pH in order to better understand
isolation efficiencies, selectivity, and tunability. The protocol
for the ZR Fungal/Bacterial DNA MiniPrep was followed according to
the manufacture suggested protocol and approximately 1 ml of
bacterial culture was used for each isolation. Samples were
centrifuged to pellet the cells, supernatants were discarded, the
pellets were washed with 200 .mu.l 1.times. PBS, centrifuged,
supernatants discarded, and then the cells were resuspended using
200 .mu.l of a solution containing 10 mM Tris HCl pH 8.5 and 0.1 mM
EDTA prior to processing. The samples were pooled to ensure
homogeneity during processing and subsequently lysed (bead bashed)
using 0.5 mm high-density beads at 6.5 m/s in NaCl solutions of
increasing final concentrations. The ZR Fungal/Bacterial DNA
MiniPrep was used as a control to for extraction efficiency. For
the control group, the manufacturer's provided lysis solution was
utilized for bead bashing. Three volumes of Genomic Lysis Buffer
were added to the lysate. The sample was loaded onto a Zymo-Spin II
column centrifuged at 16,000.times.g and subsequently washed with
200 .mu.l DNA Pre-Wash Buffer and 500 .mu.l g-DNA Wash Buffer. For
experimental groups, the final concentration of NaCl in the samples
tested ranged from about 0.5 to about 1 M. Domiphen Bromide was
added to a final concentration of about 0.25% along with a Tris,
KoAC, or Bis-Tris based buffers at about 50 mM final concentration.
Domiphen Bromide was added to a final concentration of about 0.25%.
The sample was loaded onto a column (Zymo Spin III-P column), and
centrifuged at 10,000.times.g, for 1 minute, washed with 700 .mu.l
0.6 M LiCl and centrifuged at .gtoreq.16,000.times.g, for 1 minute
more, and then twice washed with 700 .mu.l 95% ethanol and
centrifuged at .gtoreq.16,000.times.g, for 1 minute more. All
samples were eluted in 100 .mu.l DNA Elution Buffer after a 5 min
incubation at room temp (Zymo Research Corp.). The efficiency and
selectivity of the cationic surfactants was evaluated via
spectrophotometry and visualized by agarose gel electrophoresis
after 45 min (FIGS. 19A-B).
TABLE-US-00010 TABLE 10 Genomic DNA binding in the presence of a
buffer. Average Average Recovery Yield (ng/.mu.l) Stdev (.mu.g) Con
Control 19.45 5.30 1.95 1 0.7 NaCl <50 mM 41.95 15.91 4.20 Tris
8.0 2 0.8M NaCl 1.65 1.06 0.17 50 mM KoAc pH 5.6 3 0.7M NaCl 26.10
7.64 2.61 50 mM KoAc pH 5.6 4 0.6M NaCl 67.15 6.29 6.72 50 mM KoAc
pH 5.6 5 0.5M NaCl 76.20 0.42 7.62 50 mM KoAc pH 5.6 6 0.8M NaCl
1.05 0.78 0.11 50 mM Bis-Tris pH 7.0 7 0.7M NaCl 20.65 4.31 2.07 50
mM Bis-Tris pH 7.0 8 0.6M NaCl 69.85 5.59 6.99 50 mM Bis-Tris pH
7.0 9 0.5M NaCl 79.15 5.16 7.92 50 mM Bis-Tris pH 7.0 10 0.8M NaCl
1.50 0.28 0.15 50 mM Tris pH 7.2 11 0.7M NaCl 11.65 0.64 1.17 50 mM
Tris pH 7.2 12 0.6M NaCl 71.10 8.06 7.11 50 mM Tris pH 7.2 13 0.5M
NaCl 78.05 3.61 7.81 50 mM Tris pH 7.2
[0190] Both acidic and neutral conditions appeared to enhance the
selectivity of nucleic acid binding using domiphen bromide with
increasing concentrations of sodium chloride (FIGS. 19A-B). As
concentration of sodium chloride increased smaller nucleic acids
such as RNA were selectively removed. At lower concentrations of
NaCl domiphen bromide facilitated binding of all nucleic acids.
This is contrary to what was observed using the popular surfactant
CTAB disclosed by Thorsten Singer (U.S. Pat. App. 2008/0113348) who
showed that acidic conditions favored binding of both large and
small fragments, meaning domiphen bromide has completely different
and unique characteristics that could not have been predicted based
upon the literature currently available.
Example 16--Isolation of DNA from an Adhesive
[0191] The objective of this experiment is to determine if water
soluble tape that could be used for lifting cells from a surface
for applications such forensics will inhibit DNA purification and
downstream applications. The tape evaluated was Scotch brand No.
5414 Water Soluble Wave Solder Tape. It was found that using
chaotropic salt based purification techniques that the Solder Tape
interfered with purification. However the phase separation reagent
technology allowed for purification to be achieved that was usable
in PCR.
[0192] E. coli transformed with pGEM.RTM. and grown for 16 hours
was used for proof of principle. 1 ml of culture was centrifuged
and the supernatant was discarded. The cells were resuspended with
200 .mu.l water that was dissolved in water. 0.5 inches of tape was
dissolved by allowing it to incubate for 15 minutes at 55.degree.
C. The control sample was resuspended using 200 .mu.l water. 200
.mu.l of the mixture was transferred to BeadBashing Lysis Tube (0.5
mm). 550 .mu.l of 0.91 M NaCl in pH 8.0 Tris/EDTA. The sample was
mechanically lysed using MP Bio Fastprep-24 to bead bash the cells.
Other methods of lysis such as direct chemical lysis are also
possible methods of achieving the same result. The BeadBashing
Lysis Tubes were centrifuged for 1 min at 10,000.times.g for 1 min.
300 .mu.l of lysate was transferred to clean Eppendorf tubes and
100 .mu.l 1% Domiphen Bromide 200 mM Potassium Acetate was added to
the sample and was mixed. 400 .mu.l of mixture was transferred to a
Zymo Spin HIP silica spin column and centrifuged at maximum speed
for 1 min. The sample was subsequently washed with 700 .mu.l 0.6 M
LiCl and centrifuged at .gtoreq.16,000.times.g, for 1 minute more,
and then twice washed with 700 .mu.l 95% ethanol and centrifuged at
.gtoreq.16,000.times.g, for 1 minute. As a comparative sample a
commercially available system from Zymo Research Corporation that
uses chaotropic salts, Quick-gDNA, was used to process the sample
resuspended in the presence of the dissolved tape according to the
standard protocol for processing liquid samples containing cells.
Three volumes of Genomic Lysis Buffer were added to the lysate. The
sample was loaded onto a Zymo-Spin II column centrifuged at
16,000.times.g and subsequently washed with 200 .mu.l DNA Pre-Wash
Buffer and 500 .mu.l g-DNA Wash Buffer.
TABLE-US-00011 TABLE 11 Spectrophotometric results comparing the
Phase Separation Reagent DB to Genomic Lysis Buffer for the
preparation of DNA from a solution containing water dissolvable
tape Sample 260/ 260/ Con- ID Date ng/ul A260 A280 280 230 stant
Control 1 11/12/2014 -0.54 -0.011 -0.018 0.58 0.06 50 Control 2
11/12/2014 -0.41 -0.008 -0.022 0.37 0.05 50 Control 3 11/12/2014
-0.67 -0.013 -0.01 1.38 0.07 50 Experi- 11/12/2014 24.63 0.493
0.232 2.13 2.14 50 mental 1 Experi- 11/12/2014 15.62 0.312 0.128
2.44 -4.05 50 mental 2 Experi- 11/12/2014 18.35 0.367 0.166 2.21
-5.63 50 mental 3
TABLE-US-00012 TABLE 12 qPCR results comparing the Phase Separation
Reagents DB to Genomic Lysis Buffer for the preparation of DNA from
a solution containing water dissolvable tape Phase Separation
Solution Genomic Lysis Buffer Experi- Experi- Control Control
Control Experi- mental mental Neg 1 2 3 mental 1 2 3 C 24.17 23.94
33.47 15.38 16.08 15.43 33.30 25.30 24.08 24.20 15.61 15.96 15.47
33.93
[0193] DNA isolated using the DB based phase separation enabled
direct purification of DNA from cells in a solution of dissolved
brand No. 5414 Water Soluble Wave Solder Tape due to is high
specificity for DNA. This exemplifies the broad utility of these
phase separation reagents in new applications for purification of
nucleic acids. Furthermore, due to the application of this
purification technology the use of water soluble tape could be
utilized for the purification of cells, including human for
applications in forensics.
Example 18--Treatment Solution Boosts Yields but does not Remove
Degraded RNA
[0194] A treatment solution was evaluated with increasing
concentrations of sodium chloride to determine whether the salt
treatment could be used to remove degraded RNA as well as provide
the boost in recovery previously shown.
[0195] For this, JM109 cells transformed with pGEM were grown
overnight for 16 hours. The cells contained within 10 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 a 5 ml
P3 buffer that contained 1.0 M potassium acetate at about pH 4.9
and 200 .mu.g/ml RNAse A. The precipitated cellular debris was
cleared by using a glass fiber filter. 5 ml of the phase separation
solution containing 1% w/v DB and 0.25 M lithium chloride was added
prior to loading the solution onto a glass fiber matrix. The matrix
was washed with 700 .mu.l of 0, 0.1, 0.2, 0.5, and 0.7 M NaCl of
the treatment solution and was subsequently washed with 700 .mu.l
95% ethanol. Finally, the captured nucleic acid was eluted. Each
reaction was visualized by agarose gel electrophoresis at 5 min as
to check for the presence of RNA and again at 45 min to evaluate
the full-length run (FIGS. 20A-B)
[0196] With increasing concentration of sodium chloride in the
treatment solution yields were boosted as previously shown, however
small nucleic acids such as the degraded RNA in this example was
not removed. This is contrary to the popular cationic surfactant
CTAB which uses a salt wash for removal of degraded RNA (U.S. Pat.
App. 2008/0113348). The DB-NA complex not only receives a boost in
recovery when treated with the salt solution it also retains the
RNA complex indicating the uniqueness of these phase separation
reagents and their unpredictability based on current literature
available. Furthermore, DB possesses the ability to remove RNA
during the binding step when the conditions were optimized (See
other examples).
[0197] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
REFERENCES
[0198] The following references, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated herein by reference.
[0199] U.S. Pat. No. 7,754,873 [0200] U.S. Pat. No. 7,867,751
[0201] U.S. Pat. No. 8,679,744 [0202] U.S. Patent App. 2008/0113348
[0203] Bimboim, Meth. in Enzym., 100:243-255, 1983. [0204] Bimboim
and Dolly, Nucl. Acids Res., 7:1513-1523, 1979. [0205] Clewell and
Helinski, Biochemistry, 9:4428-4440, 1970. [0206] Holmes and
Quigley, Anal. Biochem., 114:193-197, 1981. [0207] Lis and Schleif,
Nucl. Acids Res., 2:757, 1975. [0208] Marko et al., Analyt.
Biochem., 121:382-387, 1981. [0209] Vogelstein et al., Proc. Nat.
Acad. Sci., 76:615-619, 1979.
* * * * *