U.S. patent application number 10/651445 was filed with the patent office on 2004-02-26 for systems and methods for the rapid isolation of nucleic acids.
This patent application is currently assigned to BIO 101. Invention is credited to Brolaski, Mark, Gautsch, James.
Application Number | 20040039188 10/651445 |
Document ID | / |
Family ID | 27500911 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040039188 |
Kind Code |
A1 |
Gautsch, James ; et
al. |
February 26, 2004 |
Systems and methods for the rapid isolation of nucleic acids
Abstract
The present invention contemplates a system for rapidly
isolating nucleic acids. The system comprises an insoluble silica
matrix and a buffered aqueous salt solution containing salt at a
concentration of at least 3 molar and a buffering agent at a
concentration sufficient to provide a buffering capacity
corresponding to that which either tris(hydroxymethyl)aminomethane
or phosphate ion at a concentration of 0.1 to 1 molar would provide
in the solution. Methods of using the system are also
contemplated.
Inventors: |
Gautsch, James; (Solana
Beach, CA) ; Brolaski, Mark; (Encinitas, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
BIO 101
|
Family ID: |
27500911 |
Appl. No.: |
10/651445 |
Filed: |
August 28, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10651445 |
Aug 28, 2003 |
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09510563 |
Feb 22, 2000 |
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6613895 |
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09510563 |
Feb 22, 2000 |
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08591038 |
Jan 25, 1996 |
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6027750 |
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08591038 |
Jan 25, 1996 |
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08309926 |
Sep 21, 1994 |
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08309926 |
Sep 21, 1994 |
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07962418 |
Oct 16, 1992 |
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07962418 |
Oct 16, 1992 |
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07267530 |
Nov 4, 1988 |
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07267530 |
Nov 4, 1988 |
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06903481 |
Sep 4, 1986 |
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Current U.S.
Class: |
536/24.3 ;
435/320.1; 435/5 |
Current CPC
Class: |
C12N 1/04 20130101; C12N
15/1006 20130101 |
Class at
Publication: |
536/24.3 ; 435/6;
435/320.1 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 015/00 |
Claims
What is claimed is:
1. A system, in kit form, for isolating plasmid DNA from an aqueous
sample, which system comprises, in separate containers, particulate
glass and a buffered aqueous salt solution having a pH value in the
range of 7 to 8, said solution containing; a) a salt at a
concentration of at least 3 molar, and b) a buffering agent at a
concentration sufficient to provide a buffering capacity
corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion
would provide in said solution.
2. The system of claim 1 wherein said buffered salt solution is
substantially free of cyclohexanediamine tetraacetate.
3. The system of claim 1 further containing a sieve at least about
1 inch in diameter and having a mesh size in the range of 90 to
350.
4. The system of claim 1 further containing, in a separate package,
a sample of control plasmid DNA.
5. The system of claim 4 wherein said control plasmid DNA is
present in a viable host cell capable of supporting replication of
said control plasmid DNA.
6. The system of claim 1 further containing, in a separate package,
a unit dose of a dry-concentrate of a culture medium capable of
supporting growth of cells containing said plasmid DNA.
7. The system of claim 6 wherein said medium is in tablet or
capsular form.
8. The system of claim 1 wherein said buffered aqueous salt
solution has a pH value in the range of 7.2 to 7.8.
9. The system of claim 1 wherein said salt is selected from the
group consisting of NaI, NaBr, NaCl, KI, KBr, CsCl, GNHCl and
GNSCN.
10. The system of claim 1 wherein said salt concentration is in the
range of 4 to 6 molar.
11. The system of claim 1 wherein said particulate glass has a
sedimentation rate through still water at unit gravity in the range
of about 0.001 to about 1.0 cm/min.
12. A system, in kit form, for isolating plasmid DNA from a sample
containing RNA and said DNA, which system comprises, in separate
containers; a) particulate glass; and b) a buffered aqueous salt
solution having a pH value in the range of 7.2-7.8, said solution
consisting essentially of: i) 2 M NaI, ii) 2.6 M KBr, and iii) 0.66
M tris(hydroxymethyl)aminomethane.
13. The system of claim 12 further containing, in unit dose form, a
dry-concentrate of a culture medium capable of supporting growth of
cells containing said plasmid DNA.
14. A system, in kit form, for isolating nucleic acid molecules,
which system comprises a composition comprising particulate glass
having a sedimentation rate through still water at unit gravity in
the range of about 0.001 to about 1.0 cm/min.
15. A system, in kit form, for isolating DNA from an aqueous
sample, which system comprises, in separate containers, particulate
glass and a dry buffered salt admixture which upon dissolution in a
predetermined amount of distilled water provides a solution having
a pH value in the range of 7 to 8, said buffered salt admixture
containing: a) a salt in an amount sufficient to provide a
concentration of at least 3 molar upon said dissolution, and b) a
buffering agent at a concentration sufficient to provide a
buffering capacity corresponding to that provided by 0.1 to 1 molar
aqueous tris(hydroxymethyl)aminomethane or 0.1 to 1 molar aqueous
phosphate ion.
16. A method for isolating plasmid DNA from a sample containing RNA
and said DNA, which method comprises: a) forming a binding reaction
admixture by admixing said sample with an insoluble silica matrix
and a buffered aqueous salt solution having a pH value in the range
of 7 to 8, said solution containing i) a salt at a concentration of
at least 3 molar, and ii) a buffering agent at a concentration
sufficient to provide a buffering capacity corresponding to that
which 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1
molar phosphate ion would provide in said solution; b) maintaining
said binding reaction admixture for a time period sufficient for
said DNA to bind to said matrix to form an insoluble DNA-matrix
complex and a remaining admixture; c) separating said remaining
admixture and said complex to form an isolated complex; and d)
recovering said DNA from said isolated complex to form isolated
plasmid DNA.
17. A method for isolating DNA from an agarose gel sample
containing said DNA, which method comprises: a) forming a
gel-dissolving reaction admixture by admixing said sample with a
buffered aqueous chaotropic salt solution having a pH value in the
range of 7 to 8, said solution containing i) a chaotropic salt at a
concentration of at least 3 molar, and ii) a buffering agent at a
concentration sufficient to provide a buffering capacity
corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion
would provide in said solution; b) maintaining said gel-dissolving
reaction admixture at a temperature of about 45 to about 65 degrees
C. for a time period sufficient for said gel sample to dissolve to
form a dissolved sample; c) admixing said dissolved sample with an
insoluble silica matrix to form a binding reaction admixture; d)
maintaining said binding reaction admixture for a time period
sufficient for said DNA present in said sample to bind to said
matrix to form a solution containing dissolved agarose and an
insoluble DNA-matrix complex; e) separating said complex from said
dissolved agarose to form an isolated complex; and f) recovering
said DNA from said isolated complex to form isolated DNA.
18. A dry-concentrate culture medium composition in unit dose
comprising an amount of cell culture medium in dry-concentrate form
sufficient to prepare a preselected amount of culture medium, said
dry medium packaged in unit dose form.
19. The composition of claim 18 wherein said unit dose packaging is
in the form of a capsule containing said dry culture medium.
20. The composition of claim 19 wherein said dry culture medium is
LB-broth.
21. The composition of claim 18 wherein said unit dose packaging is
dissolvable.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This is a continuation-in-part application of application
Serial Number 06/903,481, filed Sep. 4, 1986, now abandoned, which
is a continuation-in-part application of copending application Ser.
No. 07/267,530, filed Nov. 4, 1988, the disclosures of which are
hereby incorporated herein by reference.
TECHNICAL FIELD
[0002] The present invention relates to methods and systems for
rapidly isolating nucleic acids. More particularly, this invention
contemplates systems and methods for isolating plasmid DNA from
cell lysates and DNA from agarose gels.
BACKGROUND
[0003] The isolation of preparative amounts of biologically active
nucleic acid molecules has been a vexing problem in molecular
biology. This is especially the case with regard to isolation of
DNA for use in recombinant methodologies where it is required to be
in sufficiently pure form to be digestible by restriction
endonucleases, to be a good substrate for polymerases and
topoisomerases, and to be suitable for use as a transfection or
transformation agent.
[0004] Over the years, many methods have been developed to isolate
nucleic acid molecules. However, those methods are typically
tedious, require a high level of skill to perform, take extended
periods of time to accomplish, require the processing of relatively
large volumes of materials and often give variable results. In
addition, most of the isolation techniques reported are costly in
terms of equipment and materials. See, Gamper et al., DNA,
4:157-164 (1985); Yang et al., Meth. Enzol., 68:176-182 (1979); and
Vogelstein et al., Proc. Natl. Acad. Sci. USA, 7:615-619
(1979).
[0005] To date, the art has applied several different technologies
to the problem of preparing nucleic acids in quantities and
purities sufficient for use in recombinant methodologies.
Classically, the final step in the isolation of plasmid DNA is a
cesium chloride-ethidium dye isopycnic gradient ultracentrifugation
through a neutral or alkaline density gradient, gel
electrophoresis, high-pressure liquid chromatography through RPC-5,
alkaline extraction and column chromatography on methylated albumin
kieselguhr, hydroxyapatite, or benzoylated naphthoylated
DEAE-cellulose. See Gamper et al., DNA, 4:157-164 (1985); Yang et
al., Meth. Enzol. 68:176-182 1979); Birnboim. Meth. Enzol., 100:
243-255 (1983); Mizutani. J. Chrom., 262:441-445 (1983); and the
references cited therein.
[0006] Of particular interest to the present invention are methods
wherein the nucleic acid to be isolated is adsorbed onto an
insoluble silica matrix, e.g., particulate glass. While there are
several reports of using the binding of nucleic acids to
particulate glass as an isolation means, the physio-chemical
mechanism(s) responsible for the binding phenomenon and the
conditions under which it occurs are poorly characterized. Advances
in the art have therefore proceeded on an empirical basis.
[0007] The use of adsorption onto glass as a means for isolating
nucleic acids is based on the observation that both DNA and RNA
bind to glass in highly concentrated aqueous salt solutions, i.e.,
salt concentrations of at least about 3 molar, and can be eluted
therefrom by lowering the salt (ionic) concentration. While the pH
value of the salt solution appears to have some effect on the
adsorption process, that effect has not been characterized.
[0008] There have been several reports on the use of the
glass-adsorption technique to isolate DNA from agarose gels. In
each case, the salt solutions used to mediate the binding of the
nucleic acids to the glass contained the buffering agent tris
(hydroxymethyl) aminomethane at a concentration of less than 50
millimolar. Those solutions therefore had a low buffering capacity.
See, Mizutani, J. Col. Inter. Sci., 93:270-273 (1983); Mizutani, J.
Chrom., 262:441-445 (1983); Marko et al., Anal. Biochem.,
121:382-387 (1982); Chen et al., Anal. Biochem., 101:339-341
(1980); Vogelstein et al., Proc. Natl. Acad. Sci. USA, 76:615-619
(1979); and Yang et al., Meth. Enzol., 68:176-182 (1979).
[0009] None of the previously reported methods of isolating nucleic
acids by glass-adsorption has gained widespread acceptance by those
skilled in the art of recombinant DNA technology. This is probably
due to the inability of those methods to consistently separate DNA
from sample contaminants such as RNA, protein and agarose. For
instance, Marko et al., supra, reported that the buffered salt
solution used to mediate DNA binding to glass was required to
contain the chelating agent cyclohexanediamine tetraacetate (CDTA)
in order to prevent binding of tRNA to glass and co-purification of
the tRNA with the plasmid DNA.
[0010] From the foregoing it can be seen that there has been a long
felt need by those practicing recombinant DNA technology for a
reliable, rapid method for isolating nucleic acid molecules.
BRIEF SUMMARY OF THE INVENTION
[0011] The present invention contemplates systems and methods for
isolating nucleic acids. The systems and methods take advantage of
solutions to the problems, discovered by the inventor, of
insufficient buffering capacity and excessive glass particle
heterogeneity.
[0012] In addition, the novel systems approach described herein
permits a significant reduction in the level of skill and time
required to produce isolated DNA.
[0013] In one embodiment, the present invention contemplates a
system, in kit form, for isolating plasmid DNA from an aqueous
sample. The system comprises, in separate containers, particulate
glass and a buffered aqueous salt solution having a pH value in the
range of 7 to 8. The solution contains:
[0014] a) a salt at a concentration of at least 3 molar, and
[0015] b) a buffering agent at a concentration sufficient to
provide a buffering capacity corresponding to that 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate ion
would provide in solution.
[0016] Another aspect of the present invention is a system, in kit
form, for isolating plasmid DNA from a sample. The system
comprises, in separate containers:
[0017] a) particulate glass; and
[0018] b) a buffered aqueous salt solution having a pH value in the
range of 7.2-7.8. The solution consists essentially of:
[0019] i) 2 M NaI,
[0020] ii) 2.6 M KBr, and
[0021] iii) 0.66 M tris(hydroxymethyl)aminomethane.
[0022] Also contemplated is a system, in kit form, for isolating
nucleic acid molecules. The system comprises a composition
comprising particulate glass having a sedimentation rate through
still water at unit gravity in the range of about 0.001 to about
1.0 cm/min.
[0023] A further aspect of this invention is a system, in kit form,
for isolating DNA from an aqueous sample. The system comprises, in
separate containers, particulate glass and a buffered salt
admixture which upon dissolution in a predetermined amount of
distilled water provides a solution having a pH value in the range
of 7 to 8. The buffered salt admixture contains:
[0024] a) a salt in an amount sufficient to provide a concentration
of at least 3 molar upon said dissolution, and
[0025] b) a buffering agent at a concentration sufficient to
provide a buffering capacity corresponding to that provided by 0.1
to 1 molar aqueous tris(hydroxymethyl)aminomethane or 0.1 to 1
molar aqueous phosphate ion.
[0026] A further embodiment of this invention is a method for
isolating plasmid DNA from an aqueous sample. The method comprises
the steps of:
[0027] a) forming a binding reaction admixture by admixing said
sample with an insoluble silica matrix and a buffered aqueous salt
solution having a pH value in the range of 7 to 8, said solution
containing i) a salt at a concentration of at least 3 molar, and
ii) a buffering agent at a concentration sufficient to provide a
buffering capacity corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar sodium phosphate
would provide in said solution;
[0028] b) maintaining said binding reaction admixture for a time
period sufficient for said DNA to bind to said matrix to form an
insoluble DNA-matrix complex and a remaining admixture;
[0029] c) separating said remaining admixture and said complex to
form an isolated complex; and
[0030] d) recovering said DNA from said isolated complex to form
isolated plasmid DNA.
[0031] The present invention also contemplates a method for
isolating DNA from an agarose gel sample. The method comprises the
steps of:
[0032] a) forming a gel-dissolving reaction admixture by admixing
said sample with a buffered aqueous chaotropic salt solution having
a pH value in the range of 7 to 8, said solution containing i) a
chaotropic salt at a concentration of at least 3 molar, and ii) a
buffering agent at a concentration sufficient to provide a
buffering capacity corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate would
provide in said solution;
[0033] b) maintaining said gel-dissolving reaction admixture at a
temperature of about 45 to about 65 degrees C. for a time period
sufficient for said gel sample to dissolve to form a dissolved
sample;
[0034] c) admixing said dissolved sample with an insoluble silica
matrix to form a binding reaction admixture;
[0035] d) maintaining said binding reaction admixture for a time
period sufficient for said DNA present in said sample to bind to
said matrix to form a solution containing dissolved agarose and an
insoluble DNA-matrix complex;
[0036] e) separating said complex from said dissolved agarose to
form an isolated complex; and
[0037] f) recovering said DNA from said isolated complex to form
isolated DNA.
[0038] In another embodiment, the invention describes a
dry-concentrate culture medium composition packaged in unit dose
form comprising an amount of cell culture medium in dry-concentrate
form sufficient to prepare a preselected amount of culture medium.
Preferably, the unit dose packaging is in the form of a capsule
containing the dry culture medium. In a related embodiment, the
packaging is comprised of a dissolvable material.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] In the drawings, forming a portion of this disclosure:
[0040] FIGS. 1-4 illustrate electron micrographs of the particulate
glass fractions prepared in Example 1. Each sample is shown in two
magnifications in an upper and lower panel. A white bar line in the
lower right corner of each panel indicates the relative size of the
glass particles in each sample.
[0041] FIG. 1 depicts fraction 1 at magnifications of .times.250
and .times.500, and includes a bar line of 100 .mu.m and 100 .mu.m,
in the upper and lower panels, respectively.
[0042] FIG. 2 depicts fraction 4 at magnifications of .times.1,000
and .times.2,000, and includes a bar line of 10 .mu.m and 10 .mu.m,
in the upper and lower panels, respectively.
[0043] FIG. 3 depicts fraction 5 at magnifications of .times.10,000
and .times.20,000, and includes a bar line of 1 .mu.m and 1.mu., in
the upper and lower panels, respectively.
[0044] FIG. 4 depicts fraction 6 at magnifications of .times.10,000
and .times.35,000, and includes a bar line of 1 .mu.m and 1 .mu.m,
in the upper and lower panels, respectively.
[0045] FIG. 5 illustrates the relative binding capacity of
particulate glass fractions used to isolate DNA from agarose gels
by the procedure described in Example 2. Plasmid DNA was isolated
from an agarose gel using particulate glass fraction 4 (Lanes 1 and
4), fraction 5 (Lanes 3 and 6) or fraction 6 (Lanes 2 and 5) and
analyzed by agarose gel electrophoresis. DNA isolated from the
particulate glass after a first elution (Lanes 4-6) and after a
second elution (Lanes 1-3) are shown. A sample of DNA was also
analyzed (Lane 7) as a control that corresponds to the starting
plasmid DNA in an amount equal to the amount present in each
agarose sample before isolation.
DETAILED DESCRIPTION OF THE INVENTION
[0046] A. Systems for Isolating Nucleic Acids
[0047] The present invention contemplates a system, in kit form,
for isolating nucleic acid molecules. The system includes, as a
separately packaged component, an insoluble silica matrix,
preferably particulate glass, in an amount sufficient to bind at
least 2-3 micrograms (.mu.g) of nucleic acid. In preferred
embodiments, the particle size of the glass is in the range of
about 0.001 to about 1.0 centimeters/minute (cm/min), more
preferably about 0.1 to 0.01 cm/mm determined by sedimentation rate
through water at unit gravity.
[0048] The size of the glass particles can also be described in
terms of an average longest-diameter and/or range thereof. Thus, in
preferred embodiments the average glass particle size is about 2 to
8 microns in a composition having a glass particle size range of
about 0.8 to 30 microns. More preferably, the average glass
particle size is about 8 microns in a composition having a glass
particle size range of about 1.5 to 30 microns.
[0049] In addition to an insoluble silica matrix, a preferred
system of the present invention includes a buffered aqueous salt
solution. The salt concentration, determined using entire salt
formula as opposed to a formula representing an ionized form of the
salt, of the solution is at least about 3 molar, and is preferably
in the range of about 3 to about 7 molar, and more preferably is in
the range of about 4 to about 6 molar.
[0050] Preferred salts are those that upon dissolution in
CO.sub.2-free distilled water provide a pH value of about 6.5 to
about 7.8, i.e., neutral salts such as NaCl, LiCl, KCl and the
like. In another preferred embodiment, the salt is chaotropic and
has an anion such as perchlorate, iodide, thiocyanate, acetate,
trichloroacetate, hexafluorosilicate, tetrafluoroborate and the
like. Preferred cations for a chaotropic salt are provided by the
elements lithium, sodium, potassium, cesium, rubidium, guanidine
and the like. More than one salt can be present in the buffered
aqueous salt solution, a preferred combination of salts being
NaI/KBr, preferably with the ratio of NaI to KBr such that the salt
solution does not promote dissolution of agarose gels.
[0051] As previously discussed, it has now been discovered that one
of the problems associated with previously known methods for
isolating nucleic acids by adsorption to a silica matrix was
failure to maintain a pH value that mediates binding, i.e., a pH
value in the range of 7 to 8, preferably 7.2-7.8, for DNA and 4 to
6 for RNA. The solution provided by the present invention is in
providing of an aqueous salt solution that has sufficient buffering
capacity so that when it is admixed with a nucleic acid-containing
sample the pH value of the admixture formed favors binding of
either RNA or DNA, but not a significant amount of both.
[0052] Thus, an aqueous buffered salt solution of the present
invention further contains at least one buffering agent and has a
buffering agent concentration sufficient to provide a buffering
capacity corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethane or 0.1 to 1 molar sodium phosphate
would provide in the solution, i.e., in a solution having the same
salt at the same salt concentration.
Tris(hydroxymethyl)aminomethane and sodium phosphate are preferred
buffering agents, that when present at a concentration of about 0.1
to about 1 molar in the salt solution provide sufficient buffering
capacity, in the pH value range of 4 to 6 for RNA and 7 to 8 for
DNA, to maintain the desired pH values upon admixture with the
nucleic acid-containing sample at a ratio of at least 2 volumes of
buffered aqueous salt solution to 1 volume of nucleic
acid-containing sample.
[0053] Buffering agents other than tris(hydroxymethyl)aminomethane
can be used, but they must be present in the solution at a
concentration that has the capacity to resist a change in pH value
upon the addition of either H+ or OH- in a manner that is similar
to that of 0.1 to 1 molar tris(hydroxymethyl)aminomethane or 0.1 to
1 molar phosphate ion. A combination of buffering agents can be
used, so long as the solution has the required buffering capacity.
Methods for determining the buffering capacity of a solution are
well known in the art.
[0054] Of course, the comparison of buffering capacity is carried
out in the presence of the salt to be used, at the salt
concentration to be used, and with the solutions being compared at
about the same temperature, preferably at a temperature within the
range of about 15.degree. C. to about 25.degree. C. Other exemplary
buffering agents include sodium acetate, and the like.
[0055] Preferred buffered aqueous salt solutions include the
following:
[0056] Formula I: salt at a concentration in the range of 3 to 6
molar, 0.1 molar tris(hydroxymethyl) aminomethane, pH value range
from about 7.1 to about 7.8;
[0057] Formula II: salt at a concentration in the range of 3 to 6
molar, about 0.6 to about 0.7 molar
tris(hydroxymethyl)aminomethane, pH value range from about 7.1 to
about 7.5; and
[0058] Formula III: salt at a concentration of at least about 3
molar, 0.1 to about 1 molar tris(hydroxymethyl)aminomethane or 0.1
to about 1 molar sodium phosphate, pH value range from about 4 to
about 6.
[0059] Preferably, the buffered aqueous salt solution is
substantially free of cyclohexanediamine tetraacetate (CDTA), i.e.,
contains less than about 1 mM CDTA.
[0060] In one embodiment, the invention contemplates a system for
media preparation that includes, in a package, a unit dose of a
dry-concentrate of a culture medium capable of supporting growth of
cells containing plasmid DNA. A dry concentrate of medium comprises
the dry reagent components that make up a conventional growth
medium, such as LB-broth and the like bacterial culture media.
Exemplary media are described herein.
[0061] The term "unit dose" as it pertains to the media of the
present invention refers to physically discrete units each suitable
for providing, upon dissolution in a predetermined amount of water,
such as about 50 to about 500 ml, preferably 50 ml, 100 ml, 150 ml,
200 ml and the like, a complete culture medium capable of
supporting the growth of cells containing plasmid DNA. The
specifications for the novel unit dose of a medium of this
invention are dictated by and are directly dependent on (a) the
unique characteristics of the nutrients and the particular nutrient
requirements of the organism to be grown, and (b) the limitations
inherent in the art of compounding such nutrients for use in
culture media, as are disclosed in detail herein, these being
features of the present invention.
[0062] The unit dose of dry-concentrate medium can be supplied in a
variety of formats, including packaged in containers, pressed into
tablets, and the like. Particularly preferred are gelatin capsule
containers for ease of manipulation and ease of uniform dissolution
of the concentrated medium.
[0063] The packaging for a unit dose form can vary widely. In one
embodiment, the packaging is comprised of a dissolvable material,
which can be either inert, inactive or nutritional, from the
perspective of the nutrient medium the packaging contains.
Preferably, the material is dissolvable in water or other aqueous
solutions. The art of dissolvable packaging is well known and will
not be recited here, but exemplary dissolvable materials include
gelatin, polysaccharides, sugar, corn starch, short water-soluble
inert polymers, binders, and the like, and combinations
thereof.
[0064] A preferred system for isolating nucleic acids includes a
unit dose form of the above dry-concentrate medium in a separate
package to be used for the preparation of culture media for
culturing cells or microorganisms for the preparation of a nucleic
acid to be isolated according to the methods of the invention.
Preferred are bacterial media such as L-broth and the like.
Exemplary media are described herein.
[0065] Thus, particularly preferred systems further include a one
or more unit dose capsules, containing an amount sufficient to
prepare culture medium for growing at least one sample of
microorganisms for the preparation of nucleic acids to be isolated
by the present invention.
[0066] In another preferred embodiment, a system of this invention
includes a sieve for separating insoluble globular protein from the
supernatant formed upon centrifugation of a cell lysate.
Preferably, the sieve has a mesh size in the range of 90 to 350,
preferably 100 to 325, and more preferably 200, in U.S. Mesh Size
American AMSI/ASTM Series units. It should be noted that the
AMSI/ASTM Series mesh sizes 90, 200 and 325 correspond to aperture
sizes of 160, 75 and 45 microns, respectively. The sieve has a
diameter or long-axis of at least 1 inch and is preferably a square
having sides of about 2 inches. Useful sieves can be prepared from
commercially available materials such as screen made of
monofilamentous nylon, polyfilamentous polyester and the like. An
exemplary sieve was used herein to form the filtered solution as
described in Example 2.
[0067] Still another preferred system further includes a filter for
use in the filter-based separation step described in Examples 5 and
7 to accommodate an isolation method of this invention having a
simplified wash and elution step. In that method, the filter is
used to separate the glass particles from the various buffers
during binding, wash and/or elution steps. A separation filter can
be provided in a variety of formats depending upon the particular
separation means being utilized so long as the filter has the
capacity to retain the glass particle while allowing aqueous
solutions to pass. For example, the separation means may be
facilitated by pressurized liquid such as from a syringe as
described in Example 5, or by gravity from centrifugation as
described in Example 7.
[0068] The separation filter, in these embodiments has a pore size
selected to retain the particulate glass yet allow the various
buffers to conveniently pass. The pore size can be varied, and
depends on the minimum diameter of the particulate glass to be
filtered. Typical and preferred filters have a pore size of about
0.1 to 1.0 micrometers (".mu." or microns), and preferably about
0.45.mu..
[0069] Thus, in one embodiment, a separation filter for use in the
present invention can be adapted for use in a pressurizable chamber
adapted for attachment to a pressurized liquid supply, thereby
allowing the delivery of the particulate glass suspension to the
filter, together with any of the various buffers. The chamber
contains an outlet after the filter for collecting the aqueous
solution that passes the filter. An exemplary separation filter
chamber is the syringe-mounted filter and system described in
Example 5. Particularly preferred is a separation filter according
to the design of the Gelman Acrodisc, and the like chamber-based
filter units.
[0070] In another embodiment, a separation filter for use in the
present invention can be adapted for use in a centrifugation step.
Such filters are conveniently in the form of a centrifugation tube
adapted to contain a separation filter with an upper chamber above
the filter and a lower chamber below the filter. Typically, the
tube's lower chamber is detachable from the filter to allow the
removal of liquids from the lower chamber collected during
centrifugation. Exemplary centrifugable separation filters are
described in Example 7, and are available from a variety of
commercial vendors, such as the SPIN-X centrifuge filter unit
(COSTAR, Cambridge, Mass.) or the MC Filter Units from Millipore
(Bedford, Mass.).
[0071] A preferred system of this invention further includes, in a
separate container, plasmid DNA as a control. Any plasmid capable
of production in eucaryotic or procaryotic culture is suitable,
such plasmids being well known in the art and commercially
available from many sources. Exemplary plasmids suitable for
inclusion as a control or standard in a system of this invention
include pUC18, pBR322, YEP24, YRP17, M13amp18, bacteriophage X174
and the like. The plasmid can be provided in isolated form or in a
host cell transformed therewith.
[0072] A preferred system of this invention further includes
instruction for use of at least one of the system components.
[0073] "Instructions for use" typically include a tangible
expression describing or identifying a system component or at least
one parameter for using the system such as the relative amounts of
the sample and component(s) to be admixed, maintenance time periods
for component/sample admixtures, temperature and the like.
[0074] The packages and containers discussed herein in relation to
systems are those customarily used in the chemical arts. Such
packages and containers include glass and plastic (e.g.,
polyethylene, polypropylene and polycarbonate) bottles, vials,
tubes, plastic and plastic-foil laminated envelopes and the
like.
[0075] A preferred embodiment of a system of this invention
includes a syringe and a filler adapted for attachment to the
nozzle of the syringe. The pore size of the filter is sufficiently
small to retain the particulate glass that forms a part of the
system. The binding and elution processes described herein can be
performed in the syringe using the filter attached thereto as a
means for separating DNA-matrix complexes from solution and
separating eluted DNA from glass particles.
[0076] In another embodiment, the system of the invention can
further contain a centrifuge tube according to the present
invention having a filter means for retaining particulate glass
that allows the elution of aqueous solutions by centrifugal elution
methods, as described herein.
[0077] B. Methods for Isolating Nucleic Acids
[0078] The present invention contemplates any method of isolating
nucleic acid molecules using a system of this invention.
[0079] In one embodiment, a method of this invention is useful for
isolating plasmid DNA from a sample containing the plasmid and
other host-cell components such as chromosomal DNA, tRNA, mRNA,
protein and the like. Preferably the sample is substantially free
of cell-membranes or fragments thereof. The method includes the
following steps:
[0080] a) A binding reaction admixture is formed by admixing the
sample with an insoluble silica matrix, preferably particulate
glass as described hereinbefore, and a before described buffered
aqueous salt solution having a pH value of about 7 to about 8. The
buffered salt solution includes, i) salt at a concentration of at
least 3 molar, preferably in the range of about 4 to about 6 molar,
and ii) a buffering agent at a concentration sufficient to provide
a buffering capacity corresponding to that which 0.1 to 1 molar
tris(hydroxymethyl)aminomethan- e or 0.1 to 1 molar phosphate ion
would provide in the solution. Preferred buffered aqueous salt
solutions are those corresponding to formulas I and II.
[0081] b) The binding reaction admixture is maintained for a
predetermined time period sufficient for the plasmid DNA to bind to
the silica matrix to form an insoluble DNA-matrix complex and a
remaining admixture. The time period ranges from minute to hours,
such as about 1 min to about 4 hours and is preferably in the range
of 5 to 30 min.
[0082] c) The insoluble DNA-matrix complex is then separated from
the remaining admixture to form isolated complex. Separation can be
performed by any of the well known methods for partitioning an
insoluble material from an aqueous solution. Preferred methods
include centrifugation, filtration, gravimetric sedimentation and
the like. In preferred embodiments, the complex is washed with a
buffer (wash buffer) one to several times to insure removal of any
residual remaining admixture, e.g., RNA, protein and the like.
[0083] d) The plasmid DNA is then recovered from the isolated
DNA-matrix complex to form isolated plasmid DNA. Recovery is
typically performed by elution with a low ionic buffer, e.g., 0 to
1 M salt, preferably about 0 to 50 mM salt. A preferred elution
buffer is distilled water. The elution buffer can also be buffered
such that it has a buffering capacity equivalent to about 1 to 100
mM Tris at a pH of about 6 to 8.
[0084] The concentration of the isolated plasmid DNA thus produced
can be determined by methods well known in the art and adjusted, if
necessary, by dilution or concentration by ethanol
precipitation.
[0085] The above method can be adapted for the isolation of RNA by
substituting a buffered aqueous salt solution according to formula
III for that described in step a).
[0086] In another embodiment, the present invention contemplates a
method for isolating DNA from an agarose gel sample containing the
DNA. The method includes the following steps:
[0087] a) A gel-dissolving reaction admixture is formed by admixing
the gel sample with a before described buffered aqueous chaotropic
salt solution having a pH value of about 7 to about 8. The salt
solution contains a chaotropic salt at a concentration of at least
3 molar, preferably about 4 to about 6 molar. In addition the salt
solution includes a buffering agent at a concentration sufficient
to provide a buffering capacity corresponding to that which 0.1 to
1 molar tris(hydroxymethyl)aminomethane or 0.1 to 1 molar phosphate
ion would provide in the solution. Preferred buffered chaotropic
salt solutions include those corresponding to formulas I and
II.
[0088] b) The gel-dissolving reaction admixture is maintained at
about 45 to about 65, preferably about 55 degrees C. for a
predetermined time period sufficient for the gel sample to dissolve
to form a dissolved sample.
[0089] c) The dissolved sample is admixed with an insoluble silica
matrix, preferably particulate glass as described hereinbefore, to
form a binding reaction admixture.
[0090] d) The binding reaction admixture is maintained for a
predetermined period of time sufficient for the DNA present in the
sample to bind to the matrix to form a solution containing
dissolved agarose and an insoluble DNA-matrix complex. The period
of time is similar to that previously described in step b) of the
plasmid DNA isolation method.
[0091] e) The insoluble DNA-matrix complex is separated from the
dissolved agarose to form an isolated DNA-matrix complex. This is
accomplished in a manner similar to that described in step c) of
the plasmid DNA isolation method.
[0092] f) The DNA is recovered from the isolated complex to form
isolated DNA. This is accomplished in a manner similar to that
described in step d) of the plasmid isolation method. In addition
the DNA can be quantitated, concentrated or diluted as previously
described.
[0093] The above method can be adapted to isolate RNA from an
agarose gel using a buffer according to formula III wherein the
salt is a chaotrope in place of the buffer described in step
a).
EXAMPLES
[0094] The following examples are intended to illustrate, but not
limit, the present invention. Accordingly, variations and
equivalents, now known or later developed, that would be within the
purview of one skilled in this art are to be considered to fall
within the scope of this invention, which is limited only as set
forth by the appended claims.
[0095] 1. Preparation of Particulate Glass
[0096] Silica in the form of grade AH glass beads was obtained from
Cal-Chem (San Diego, Calif.). The glass beads were suspended in 50%
nitric acid and boiled for 1 hour. The beads were allowed to settle
for one week without agitation and the excess liquid was decanted
off of the settled bead layer. Thereafter the beads were washed 6
times by sequential cycles of a resuspension in water followed by a
one week settling period through still water and decantation to
form washed glass beads.
[0097] Size fractionation of the washed glass beads was
accomplished by first mixing the washed glass beads into a
suspension followed by maintaining the glass beads in a stationary
cylindrical container over time to allow the beads (particles) to
settle at unit gravity over a distance of 100 centimeters (cm). At
any given time particles in suspension are referred to as fines and
the settled particles form a layer at the bottom of the cylinder
below the fines. Separation of the fines from the settled layer
creates a population of glass particles that may be defined by the
rate of sedimentation in centimeters per minute (cm/min) or defined
by the time that the fines were removed after mixing.
[0098] Five minutes after a first mixing, the fines were removed
from above the settled particle layer. The removed fines were then
allowed to settle for an additional 20 minutes in a similar
cylindrical container and the resulting fines were then removed
from the resulting settled layer. The process was repeated and by
this method, the above washed glass suspension was fractionated
into samples of particulate glass that settled at various times as
indicated in Table 1 below.
[0099] The resulting particulate glass fractions were analyzed by
scanning electron microscopy to evaluate the characteristics of
size and shape of the glass particles present in each fraction.
Electron microscopy was conducted by MICRON, INC. (Wilmington; DE),
photographs were prepared and are shown in FIGS. 1 through 4. By
comparison of the particles to the size marker in each sample, the
range of sizes and average size were visually estimated for each
sample and is reported in Table 1.
1TABLE 1 Particulate Glass Fractions Fraction Sediment.sup.a
Sediment.sup.b Size Avg. # Time Rate Range Size 1 0-5 min >20
15-50.mu..sup.c 25.mu. 2 5-20 min 20-5 nd nd 3 20 min-2 hrs 5-.01
nd nd 4 2 hrs-1 wk .01-.008 1.5-20 8.mu. 5 1 wk-6 wks .008-.001
0.8-3.mu. 2.mu. 6 6 wks+ <.001 0.2-1.mu. 0.5.mu. .sup.aSediment
time connotes that the fraction sedimented between the indicated
times. .sup.bSediment rate is a measure in cm/min of the range of
rates of sedimentation for particles in that fraction. .sup.c".mu."
means micrometers.
[0100] 2. Isolation of DNA from Agarose Gels Using Particulate
Glass
[0101] All DNA manipulations were done according to standard
procedures. See Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1982.
[0102] Plasmid pUC18 DNA was prepared by techniques well known in
the art and was then electrophoresed on a 1% agarose gel to deposit
the plasmid into an agarose gel. The gel and its contents were then
stained with ethidium bromide. Next the DNA sample in the gel was
visualized by exposure to long-wave ultraviolet light. The gel was
cut to isolate the agarose containing the band of visualized
plasmid DNA. The agarose sample had a volume of about 0.5 ml and
contained about 3 ug of plasmid DNA and was placed into a 1.5
microcentrifuge tube.
[0103] A solution of sodium iodide having a molarity of about 6
molar (M) was prepared by admixing 0.75 gm sodium bisulfite with 40
ml distilled water (dH2O) and 45 gms of sodium iodide. One ml of
the sodium iodide solution was added to the microcentrifuge tube
containing the agarose sample, and the tube was then maintained at
55.degree. C. for about 5 minutes with a periodic agitation of the
solution about every minute for about 5 seconds per agitation to
allow the agarose to dissolve to form an agarose-plasmid DNA
solution.
[0104] Five microliters (.mu.l) of a suspension of particulate
glass, prepared as in Example 1, obtained from a sample of either
fraction 1, 4, 5, or 6 and each suspension having a concentration
of about 50 percent (v/v) of glass per dH2O, were then admixed with
the agarose DNA solution to form a binding reaction admixture. The
admixture was then maintained for about 5 minutes at about
4.degree. C. in an ice bucket with periodic agitation of the
suspension about every minute for about 5 seconds for each
agitation to allow the DNA present in the solution to form an
insoluble DNA-particulate glass complex. The microcentrifuge tube
was then centrifuged for 15 seconds at about 12,000.times.g and the
resulting supernate was removed to form isolated DNA-matrix complex
in a pellet.
[0105] About 200 .mu.l of wash buffer (100 mM NaCl, 10 mM Tris, 1
mM EDTA, pH 7.5, 50% ethanol) was added to the DNA-glass pellet.
The complex was resuspended by agitation and the suspension was
centrifuged as before. The resulting pellet was recovered by
removing the supernatant and this wash procedure was repeated twice
to form an isolated complex. Five .mu.l of dH2O was then added to
the thrice washed isolated complex and maintained at 55.degree. C.
for about 5 minutes. The microcentrifuge tube containing the added
dH2O and particulate glass was centrifuged at 12,000.times.g for
about 15 seconds and the resulting supernate was recovered to form
a first eluted DNA solution. Five ul of dH20 was again added to the
glass pellet, the above maintenance at 55.degree. C. and
centrifugation steps repeated, and the supernate was recovered to
form a second eluted DNA solution.
[0106] The various first and second eluted DNA solutions, prepared
using the different particulate glass fractions, were then applied
to and electrophoresed on 1% agarose gels to evaluate the relative
binding capacity of each particulate glass fraction prepared in
Example 1. As shown in FIG. 5, plasmid DNA was recovered from
agarose gel slices at efficiencies that depend upon the size
fraction of the particulate glass. Fractions 8 and 10 exhibited the
highest binding capacity. Comparison of the first and second
elution of isolated DNA indicates that most of the isolated DNA was
released by a first elution.
[0107] 3. Preparation of Media in Unit Dose Formulation
[0108] Culture medium capable of supporting growth of the bacterial
cell Escherichia coli (E. coli) containing plasmid DNA was prepared
by first admixing 7 gms yeast extract, 14 gms bacto-tryptone, 1.25
.mu.m sodium chloride (all from DIFCO Laboratories, Detroit,
Mich.), 0.3 gm Tris Base and 1.6 gm Tris-HCl (both Tris reagents
[tris(hydroxymethyl)aminomethane] were obtained from Sigma Chemical
Co., St. Louis, Mo.) to form dry concentrate media powder, i.e.,
having less than 5% by weight, preferably less than 1% by weight,
water.
[0109] A culture medium designated as LB-Medium (Luria-Bertani
Medium) was also separately prepared by admixing 10 gm tryptone, 5
gm yeast extract, 10 gm sodium chloride, and a small amount of
sodium hydroxide sufficient to form a medium having a pH of
approximately 7.0 when reconstituted into a liquid medium. The
powders are mixed, milled in a ball mill to a consistent 300 mesh
size or smaller for admixture into a one liter medium formulation.
The ingredients are available from a variety of sources such as
DIFCO Laboratories, or BBL (Becton-Dickinson).
[0110] The resulting powder admixture was packaged into either a 00
or 000 size gelatin capsule using manual or automated
capsule-filling machinery. A 00 size capsule will hold about 0.7 gm
of LB medium, and a 000 capsule will hold about 1 gm of LB medium.
Due to the hygroscopic nature of the media ingredients, it is
important that the admixing of powdered reagents, and addition to
capsules be conducted in an environment having relative humidity of
less that 10%. To achieve this degree of humidity, it may be
necessary to utilize air conditioners that are outfitted with
dehumidifiers, and allow the dehumidifiers to form a relatively dry
environment.
[0111] The media powder was packaged into unit dose amounts by
placing 1 gm of the media powder into a size 00 clear gelatin
capsule (CAPSUGEL, Warner Lambert, Greenwood, S.C.) to form media
capsules. The media may alternatively contain binders for the
powdered formulations.
[0112] Two media capsules were admixed with 50 milliliters (mls)
distilled water (dH2O) and subjected to a standard autoclave
procedure to sterilize the liquid admixture. After autoclaving and
an additional time of about 15 minutes for the autoclaved admixture
to reduce in temperature, ampicillin was added to a concentration
of 50 micrograms (mg) per ml to form a unitary culture medium.
[0113] 4. Isolation of Plasmid DNA from Bacterial Cells Using
Particulate Glass
[0114] E. Coli bacteria strain DHl containing plasmid pUC18
(obtainable from Bethesda Research Laboratories, Gaitherburg, Md.)
were inoculated into the 50 mls of unitary culture medium prepared
in Example 3 and the inoculated culture was maintained at
37.degree. C. with moderate aeration for about 16 hours to form a
saturated bacterial culture. The saturated culture was centrifuged
at 8000.times.g for 5 minutes to pellet the bacterial cells, the
pellet was recovered and excess medium removed. The approximate
weight of the resulting cell pellet was determined to form a
weighed cell pellet.
[0115] Prelysis buffer, which is included in preferred embodiments
of the present invention, containing 50 mM Tris, 40 mM EDTA
(ethylenediaminetetraacetic acid) and 50 mM glucose at a pH of
about 8.5 was admixed with the weighed cell pellet at a ratio of 1
ml buffer per 0.5 gms of pellet. The pellet was resuspended in the
prelysis buffer by agitation and the suspension was maintained at
room temperature for 5 minutes. Thereafter, 2 mls of alkaline lysis
buffer, which is included in preferred embodiments of this
invention, containing 200 mM NaOH, and 1% SDS (sodium dodecyl
sulfate) was admixed with the suspension, and the admixture was
maintained for 5 minutes at room temperature with a continuous
gentle agitation. Next, 1.5 mls of neutralizing solution which is
included in preferred embodiments of this invention, prepared by
admixing 40 mls of glacial acetic acid (99.5%) with 1 liter of 3 M
potassium acetate in water, was admixed with the lysed bacterial
pellet suspension, and the resulting admixture was placed onto
crushed ice (frozen water) and maintained for 10 minutes to form a
neutralized solution.
[0116] The neutralized solution was centrifuged at 12,000.times.g
for 15 minutes at 4.degree. C. and the resulting supernatant was
recovered. The recovered supernate was then poured through a nylon
monofilament fabric having a mesh of about 200 and an aperture of
about 75 microns between fibers (DA-KAR, San Diego, Calif.), and
the liquid passing through was recovered to form a filtered
solution. The volume of the filtered solution was determined,
isopropanol was then admixed with the filtered solution in an
amount equal to 0.6 volumes of the filtered solution and the
resulting admixture was centrifuged at 8,000.times.g for 10 minutes
at 4.degree. C. The resulting precipitated cell lysate pellet was
recovered by inverting the centrifuge tube, draining off excess
supernate and adding 0.25 mls of TE buffer (10 mM Tris, 1 mM EDTA,
pH 8.0) to form a dissolved pellet.
[0117] Binding buffer was prepared by first adding excess potassium
bromide to a 1 M Tris (pH 7.3) solution to form a saturated KBr
solution at 25.degree. C., and then admixing 1/3 volume of sodium
iodide solution (prepared in Example 2) with 2/3 volume of the
saturated KBr solution.
[0118] One ml of binding buffer was admixed with the dissolved
pellet in a 1.5 ml microfuge tube, and 75 ml of a suspension of
particulate glass, prepared as in Example 1 from fraction 4 and
having a concentration of about 50% (v/v) of glass per dH2O, were
admixed to form a binding reaction admixture. The admixture was
maintained at room temperature for 5 minutes with periodic
agitation of the admixture about every minute for about 5 seconds
per agitation to allow the plasmid DNA present in the admixture to
bind to the particulate glass and form an insoluble DNA-matrix
complex. Thereafter the admixture was centrifuged for 15 seconds at
about 12,000.times.g to form a plasmid DNA-bound glass pellet. The
DNA-bound glass pellet was washed three times with binding buffer
and then washed three times using wash buffer as described in
Example 2, and the bound plasmid DNA was eluted as in Example 2
except that 100 .mu.l of dH2O was used to produce the first eluted
DNA solution.
[0119] Isolated plasmid DNA present in the eluted DNA solution was
analyzed for yield, purity and quality on agarose gels as described
in Example 2. The results of that gel analysis show that the above
procedure represents a method that produces plasmid DNA that is
substantially free of RNA, and chromosomal DNA. However, numerous
variations of salt concentration, choice of salt, pH, choice of
buffer and buffer concentration were evaluated in the binding
buffer (buffered aqueous salt solution) and also found to
facilitate plasmid DNA isolation. Table 2 shows the results of the
preferred binding buffer, in addition to showing the results when
substituting a variant binding buffer that contains the salt and
buffer conditions indicated in Table 2 in place of the binding
buffer described in the text. In all cases, the buffer was prepared
and the pH of the buffer was adjusted at room temperature before
the salt was admixed to produce the final binding buffer. The
concentrations indicated in Table 2 correspond to binding buffer
before admixture with the dissolved pellet and particulate
glass.
2TABLE 2 Effects of Salt Selection, Salt Concentration and pH on
Plasmid DNA Isolation Using Particulate Glass Binding Pattern.sup.b
Salt.sup.a Concentration Buffer pH DNA RNA NaIO.sub.4 Sat..sup.c --
nd.sup.d - - NaBrO.sub.4 Sat. .sup. -- nd.sup. - - Na Br Sat. .sup.
-- nd.sup. + - K Br Sat. .sup. -- nd.sup. + - K Br Sat. .sup. --
nd.sup. - - K Br Sat. .sup. -- nd.sup. + + K Br Sat. .sup. --
nd.sup. - - K Br Sat. .sup. 1 M Tris 6.0 - +++ K Br Sat. .sup. 1 M
Tris 7.0 + + K Br Sat. .sup. 1 M Tris 7.2 +++ - K Br Sat. .sup. 1 M
Tris 7.5 ++ - K Br Sat. .sup. 1 M Tris 7.8 + - K Br Sat. .sup. 1 M
Tris 8.0 - - Na Br Sat. .sup. 1 M Tris 6.0 - +++ Na Br Sat. .sup. 1
M Tris 7.0 - +++ Na Br Sat. .sup. 1 M Tris 8.0 - - Na I 6 M 0.1 M
Tris 7.4 +++ - Na I 6 M 0.1 M Tris 7.8 ++ - NaI/KBr.sup.e 2 M/2.6 M
.66 M Tris 7.3 +++ - Na Cl 3 M 0.1 M Tris 7.2 + ++ Cs Cl 3 M 0.1 M
Tris 7.2 ++ K I 3 M 0.1 M Tris 7.2 +++ + Na ClO.sub.4 3 M 0.1 M
Tris 7.2 ++ ++ GN HCl 3 M 0.1 M Tris 7.2 ++ + GN SCN 3 M 0.1 M Tris
7.2 +++ ++ .sup.a"GN HCl" refers to guanidine hydrochloride, and
"GN SCN" refers to guanidine thiocyanate. .sup.bDNA and RNA binding
patterns were determined by analyzing the material isolated as
described in Example 4 by using agarose gel electrophoresis to
visualize the relative amounts of DNA and RNA obtained. "+++" means
the maximum amount observed in comparison to a control buffer, "++"
means about 20-50 percent of control, "+" means about 20 percent or
less visible material compared to control, and "-" means no visible
material present on the gel. For DNA, the control buffer is binding
buffer, and for RNA, the control buffer is 4 M KBr and 1 M Tris at
pH 6.0. .sup.c"Sat." means a saturated salt solution prepared as in
Example 4. .sup.d"nd" means not done. .sup.e"NaI/KBr" indicates the
binding buffer described in the text of Example 4 that contains
both salts, each at the concentration indicated.
[0120] Table 2 indicates that DNA isolation free of RNA can be
conducted by using a binding buffer having a pH value over a
specific pH range of about 7.2 to 7.5 for KBr and at least about
7.4 to 7.8 for NaI. Further, the combination of KBr and NaI, each
at a concentration lower than 3 M, was observed to produce maximal
plasmid isolation free of RNA, whereas KBr alone at 3 M did not
produce satisfactory yields.
[0121] 5. Particulate Glass Isolation of DNA Using a Filter-Based
Separation Step
[0122] The above methods for isolating DNA from agarose in Example
2 or isolating plasmid DNA from bacterial cultures in Example 4 was
modified to accommodate a procedure having simplified wash and
elution steps. In that modified procedure, a filtration step was
used to separate the particulate glass from the various buffers in
place of a centrifugation step. This modified procedure was the
same as in Example 2 or Example 4 except for the substitution of
the centrifugation steps with the following separation steps as
noted.
[0123] The dissolved pellet prepared in Example 4 was first admixed
with the binding buffer to form a pellet binding solution. The
solution was then drawn into a plastic 3 ml syringe that had fitted
on its inlet a 0.45 micron (.mu.) filter (Gelman Acrodisc, Ann
Arbor, Ill.), i.e., a syringe-mounted filter. The syringe also
contained 100 ul of the particulate glass suspension in the barrel
portion of the syringe such that by drawing up the pellet binding
solution there was an admixing of the solution with the particulate
glass suspension to form a binding reaction admixture in the
syringe barrel. The reaction admixture was then maintained as
before, and then the solution in the syringe was expelled to
separate that solution from the retained insoluble DNA-matrix
complex. In a similar manner, washes and elution were performed by
drawing up the respective wash and elution buffers as described in
Example 4. The eluted sample was collected as it was expelled from
the outlet of the syringe-mounted filter. When this eluted sample
was analyzed on agarose gels for plasmid DNA it was found to be
comparable in yield and purity to the isolated DNA described in
Example 4.
[0124] 6. Particulate Glass Isolation of DNA Using a Sedimentation
Based Separation Step
[0125] The above methods for isolating DNA may be modified to
accommodate wash and elution steps based on sedimentation at unit
gravity as a means for separation in place of centrifugation. This
modified procedure is conducted in the same manner as described in
Example 2 or Example 4 except that the described centrifugation
steps are substituted with the following sedimentation-based
separation steps as noted.
[0126] The dissolved pellet prepared in Example 4 is added to a
well of a microtiter plate and admixed therein with the binding
buffer and the suspension of particulate glass as in Example 4 to
form a binding reaction admixture.
[0127] The fraction of particulate glass used for a
sedimentation-based separation is a fraction prepared as in Example
1 except that is it fractionated such that more than 98% of the
glass particles in the fraction sediment under unit gravity over a
1 cm vertical distance in a time period between 15 seconds and 2
hours. For the purpose of saving time between washes, it is
preferred if the sedimentation time occurs between 2 and 6
minutes.
[0128] The reaction admixture in the microtiter wells is maintained
as in Example 4. Thereafter, in place of centrifugation the plates
are held stationary and the particulate glass is allowed to settle.
The liquid layer present above the settled glass is decanted
(separated) and wash buffer is then admixed with the settled glass
present in the microtiter well. Settling, separating and further
admixing steps are then performed in this sedimentation-based mode
to subject the glass particles to washes and elution essentially as
described in Example 4 thus carried out to conduct the to yield
isolated DNA in the first separated elutant solution.
[0129] 7. Particulate Glass Isolation of DNA Using a Centrifugation
Based Separation Step
[0130] The above methods for isolating nucleic acids was modified
to accommodate a procedure having simplified binding, wash and
elution steps. In the present modified procedure, a centrifugal
filtration step was used to separate the particulate glass from the
various buffers in place of a centrifugation/decanting step, or the
simple pressure filtration step described in Example 5. This
modified procedure was generally the same as in Example 2 or
Example 4 with the following exceptions to the steps for separating
the particulate glass from any of the various buffers as noted.
[0131] As before, a solution containing nucleic acid is admixed
with binding buffer and the particulate glass to form a binding
reaction admixture, and the admixture was maintained as before to
allow the formation of an insoluble DNA-matrix complex. Thereafter,
the admixture was placed into the upper chamber of a SPIN-X
centrifugal filtration unit (COSTAR, Cambridge, Mass.) and
centrifuged on a microfuge apparatus for 15 seconds at about
12,000.times.g to force the liquid phase of the admixture through
the filter into the lower chamber of the unit. The particulate
glass bound nucleic acid molecules remain in the upper chamber of
the filter unit. Thereafter, the particulate glass is washed three
times with binding buffer and three times with wash buffer as
described before, except at each wash, the liquid is removed by a
centrifugation step to pass the liquid through the filter. The
lower chamber was emptied as needed to allow for the repeated
washes. Finally, the bound nucleic acids were eluted by the
addition of elution buffer to the particulate glass as described
before, except that the elution buffer was added to the upper
chamber, and the unit was centrifuged to pass the elution buffer
and eluted nucleic acids into the lower chamber where it was
collected, thereby separating the nucleic acid from the particulate
glass.
[0132] The use of the centrifugal filtration procedure yielded
nucleic acid of similar purity as described before by the present
methods. However, the resulting nucleic acids, when high molecular
weight DNA was isolated by the present method, provides material
that is less sheared (broken) than DNA isolated by multiple
binding, washing and eluting steps that require resuspension and
pipetting manipulations. Thus, the centrifugal filtration method
yields superior high molecular weight DNA.
[0133] The foregoing specification, including the specific
embodiments and examples, is intended to be illustrative of the
present invention and is not to be taken as limiting. Numerous
other variations and modifications can be effected without
departing from the true spirit and scope of the present
invention.
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