U.S. patent application number 14/504581 was filed with the patent office on 2015-03-26 for compositions and methods of selective nucleic acid isolation.
The applicant listed for this patent is APPLIED BIOSYSTEMS, LLC. Invention is credited to Douglas BOST, Lawrence GREENFIELD.
Application Number | 20150086988 14/504581 |
Document ID | / |
Family ID | 23305266 |
Filed Date | 2015-03-26 |
United States Patent
Application |
20150086988 |
Kind Code |
A1 |
BOST; Douglas ; et
al. |
March 26, 2015 |
COMPOSITIONS AND METHODS OF SELECTIVE NUCLEIC ACID ISOLATION
Abstract
The invention relates to methods for isolating and/or
identifying nucleic acids. The invention also provides kits for
isolating and/or identifying nucleic acids.
Inventors: |
BOST; Douglas; (Foster City,
CA) ; GREENFIELD; Lawrence; (Santa Clara,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
APPLIED BIOSYSTEMS, LLC |
Carlsbad |
CA |
US |
|
|
Family ID: |
23305266 |
Appl. No.: |
14/504581 |
Filed: |
October 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13959725 |
Aug 5, 2013 |
8865405 |
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14504581 |
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12471292 |
May 22, 2009 |
8507198 |
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13959725 |
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11789352 |
Apr 23, 2007 |
7537898 |
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12471292 |
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10306347 |
Nov 27, 2002 |
7208271 |
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11789352 |
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60334029 |
Nov 28, 2001 |
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Current U.S.
Class: |
435/6.11 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12N 15/101 20130101; C12Q 1/6806 20130101 |
Class at
Publication: |
435/6.11 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
GRANT INFORMATION
[0002] The present inventions may have been made with support from
the U.S. Government under NIST Grant No. 70NANB8H4002. The U.S.
Government may have certain rights in the inventions recited
herein.
Claims
1. A method for isolating DNA and RNA from a biological sample
comprising: selectively binding DNA to a first solid phase by
contacting the biological sample with the first solid phase under
conditions which selectively bind DNA; separating the first solid
phase with the bound DNA from a first unbound portion of the
biological sample; isolating the DNA from the first solid phase;
and isolating RNA from the first unbound portion of the biological
sample, wherein the isolating of the RNA from the first unbound
portion of the biological sample comprises: exposing the first
unbound portion of the biological sample to a second solid phase
under conditions which bind RNA to the second solid phase, wherein
the conditions which bind RNA to the second solid phase comprise a
neutral or acidic pH; separating the second solid phase with bound
RNA from a second unbound portion of the biological sample; and
isolating the RNA from the second solid phase.
2. The method of claim 1, wherein the isolating the DNA from the
first solid phase comprises eluting the DNA.
3. The method of claim 1, wherein the isolating of the RNA from the
second solid phase comprises eluting the RNA.
4-6. (canceled)
7. The method of claim 1, wherein the conditions which selectively
bind DNA comprise using a binding buffer comprising: an alkaline
pH; and a large anion, wherein the large anion is at least as large
as a bromide ion.
8. The method of claim 7, wherein the large anion is selected from
at least one of picrate, tannate, tungstate, molybdate,
perchlorate, and sulfosalicylate.
9. The method of claim 7, wherein the large anion is selected from
at least one of trichloroacetate, tribromoacetate, thiocyanate, and
nitrate.
10. The method of claim 7, wherein the large anion is selected from
at least one of iodide and bromide.
11. The method of claim 7, wherein the alkaline pH is equal to, or
above 8.0.
12. The method of claim 7, wherein the alkaline pH is equal to, or
above 9.0.
13. The method of claim 7, wherein the alkaline pH is equal to, or
above 10.0.
14. A method of isolating nucleic acid from a biological sample
comprising: binding nucleic acid to a first solid phase by
contacting the biological sample with the first solid phase under
conditions which bind both RNA and DNA; separating the first solid
phase with bound nucleic acid from a first unbound portion of the
biological sample; eluting RNA from the first solid phase with
bound nucleic acid under conditions which selectively bind DNA;
removing the first solid phase with bound DNA from a first eluate;
and isolating the DNA from the first solid phase.
15. The method of claim 14, wherein the isolating the DNA from the
first solid phase comprises eluting the DNA from the first solid
phase.
16-18. (canceled)
19. The method of claim 14, wherein the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion.
20. The method of claim 19, wherein the large anion is selected
from at least one of picrate, tannate, tungstate, molybdate,
perchlorate, and sulfosalicylate.
21. The method of claim 19, wherein the large anion is selected
from at least one of trichloroacetate, tribromoacetate,
thiocyanate, and nitrate.
22. The method of claim 19, wherein the large anion is selected
from at least one of iodide and bromide.
23. The method of claim 19, wherein the alkaline pH is equal to, or
above 8.0.
24. The method of claim 19, wherein the alkaline pH is equal to, or
above 9.0.
25. The method of claim 19, wherein the alkaline pH is equal to, or
above 10.0.
26. The method of claim 14, further comprising: exposing the first
eluate to a second solid phase under conditions which bind RNA to
the second solid phase; separating the second solid phase with the
bound RNA from an unbound portion of the first eluate; and
isolating the RNA from the second solid phase.
27. The method of claim 26, wherein the isolating the RNA from the
second solid phase comprises eluting the RNA.
28-30. (canceled)
31. The method of claim 26, wherein the conditions which bind RNA
to the second solid phase comprise a neutral or acidic pH.
32. The method of claim 26, wherein the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion.
33. The method of claim 32, wherein the conditions which bind RNA
to the second solid phase comprise a neutral or acidic pH.
34-35. (canceled)
36. A method of identifying nucleic acid in a biological sample
comprising: binding nucleic acid to a first solid phase by
contacting the biological sample with the first solid phase under
conditions which bind both DNA and RNA; separating the first solid
phase with bound nucleic acid from a first unbound portion of the
biological sample; eluting RNA from the first solid phase with
bound nucleic acid under conditions which selectively bind DNA;
removing the first solid phase with bound DNA from a first eluate;
and identifying the DNA bound to the first solid phase.
37. The method claim 36, wherein the identifying the DNA bound to
the first solid phase comprises amplifying the DNA bound to the
first solid phase.
Description
PRIORITY DATA
[0001] This application is a divisional of U.S. patent application
Ser. No. 13/959,725 filed Aug. 5, 2013, which is a continuation of
U.S. patent application Ser. No. 12/471,292 filed Jun. 22, 2009
(now U.S. Pat. No. 8,507,198 issued Aug. 13, 2013), which is a
divisional of U.S. patent application Ser. No. 11/789,352 filed
Apr. 23, 2007 (now U.S. Pat. No. 7,537,898 issued May 26, 2009),
which is a divisional of U.S. patent application Ser. No.
10/306,347 filed Nov. 27, 2002 (now U.S. Pat. No. 7,208,271 issued
Apr. 24, 2007), which claims the benefit of U.S. Provisional
Application No. 60/334,029 filed Nov. 28, 2001, which applications
are incorporated by reference herein in their entirety.
FIELD OF THE INVENTION
[0003] The invention relates to methods for isolating and/or
identifying nucleic acids. The invention also provides kits for
isolating and/or identifying nucleic acids.
BACKGROUND OF THE INVENTION
[0004] It may be desirable to isolate nucleic acids from a
biological sample. In certain instances, It would be useful to
selectively isolate DNA from such a biological sample. In certain
instances it would be useful to selectively isolate DNA and to
selectively isolate RNA from a biological sample. Typical protocols
for isolating either RNA or DNA have used selective enzymatic
degradation to remove the undesired nucleic acid.
SUMMARY OF THE INVENTION
[0005] According to certain embodiments, methods of isolating DNA
from a biological sample are provided. In certain embodiments,
methods of isolating DNA from a biological sample comprise:
selectively binding DNA to a solid phase by contacting the
biological sample with the solid phase under conditions which
selectively bind DNA; separating the solid phase with the bound DNA
from an unbound portion of the biological sample; and isolating the
DNA from the solid phase.
[0006] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion.
[0007] According to certain embodiments, methods of isolating DNA
and RNA from a biological sample are provided, comprising:
selectively binding DNA to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
selectively bind DNA; separating the first solid phase with the
bound DNA from a first unbound portion of the biological sample;
isolating the DNA from the first solid phase; and isolating RNA
from the first unbound portion of the biological sample.
[0008] According to certain embodiments, the isolating of the RNA
from the first unbound portion of the biological sample comprises:
exposing the first unbound portion of the biological sample to a
second solid phase under conditions which bind RNA to the second
solid phase; separating the second solid phase with bound RNA from
the second portion of the biological sample; and isolating the RNA
from the second solid phase.
[0009] According to certain embodiments, methods of isolating
nucleic acid from a biological sample are provided, comprising:
binding nucleic acid to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
bind both DNA and RNA; separating the first solid phase with bound
nucleic acid from a first unbound portion of the biological sample;
eluting RNA from the first solid phase with bound nucleic acid
under conditions which selectively bind DNA; removing the first
solid phase with bound DNA from a first eluate; and isolating the
DNA from the first solid phase.
[0010] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion.
[0011] According to certain embodiments, the method of isolating
nucleic acid from a biological sample further comprises: exposing
the first eluate to a second solid phase under conditions which
bind RNA to the second solid phase; separating the second solid
phase with the bound RNA from a second eluate; and isolating the
RNA from the second solid phase.
[0012] According to certain embodiments, isolating nucleic acid
from a solid phase comprises eluting the nucleic acid from the
solid phase.
[0013] According to certain embodiments, methods of identifying DNA
in a biological sample are provided. In certain embodiments,
methods of identifying DNA in a biological sample comprise:
selectively binding DNA to a solid phase by contacting the
biological sample with the solid phase under conditions which
selectively bind DNA; separating the solid phase with the bound DNA
from an unbound portion of the biological sample; and identifying
the DNA bound to the solid phase. According to certain embodiments,
the identifying the nucleic acid on a solid phase comprises
amplifying the nucleic acid bound to the solid phase.
[0014] According to certain embodiments, a kit is provided,
comprising: a buffer with an alkaline pH; a large anion, wherein
the large anion is at least as large as a bromide ion; and a solid
phase.
[0015] According to certain embodiments, a kit is provided
comprising: a solid phase; a nucleic acid binding buffer, wherein
both DNA and RNA bind the solid phase under conditions generated by
the nucleic acid binding buffer; and a selective DNA binding
buffer, wherein the conditions generated by the selective DNA
binding buffer allow selective binding of DNA to the solid
phase.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 shows the effects of various salts on the binding of
DNA to various solid phases at pH 8.
[0017] FIG. 2 compares the binding of DNA to various solid phases
at pH 8 in the presence of either chloride or thiocyanate.
[0018] FIG. 3 shows the effects of various salts on the binding of
RNA to various solid phases at pH 8.
[0019] FIG. 4 shows the effects of various salts on the selectivity
of DNA binding to various solid phases, expressed as a ratio of DNA
to RNA recovery.
[0020] FIG. 5(a) shows the effects of various salts and pH on the
binding of genomic DNA to Organon Teknika Silica.
[0021] FIG. 5(b) shows the effects of various salts and pH on the
binding of genomic DNA to Sigma Diatomaceous Earth.
[0022] FIG. 5(c) shows the effects of various salts and pH on the
binding of genomic DNA to Bio101 Glassmilk.
[0023] FIG. 5(d) shows the effects of pH on the binding of genomic
DNA to various solid phases in the presence of NaCl.
[0024] FIG. 5(e) shows the effects of pH on the binding of genomic
DNA to various solid phases in the presence of GuSCN.
[0025] FIG. 5(f) shows the effects of pH on the binding of genomic
DNA to Organon Teknika Silica in the presence of NaBr.
[0026] FIG. 5(g) shows the effects of pH on the binding of genomic
DNA to various solid phases in the presence of NaI.
[0027] FIG. 5(h) shows the effects of pH on the binding of genomic
DNA to various solid phases in the presence of NaCl (results are
shown as relative efficiencies at different pH levels normalized to
the recovery at pH 6).
[0028] FIG. 5(i) shows the effects of pH on the binding of genomic
DNA to various solid phases in the presence of GuSCN (results are
shown as relative efficiencies at different pH levels normalized to
the recovery at pH 6).
[0029] FIG. 5(j) shows the effects of pH on the binding of genomic
DNA to Organon Teknika Silica in the presence of NaBr (results are
shown as relative efficiencies at different pH levels normalized to
the recovery at pH 6).
[0030] FIG. 5(k) shows the effects of pH on the binding of genomic
DNA to various substrates in the presence of NaI, results are shown
as relative efficiencies at different pH levels normalized to the
recovery at pH 6.
[0031] FIG. 6 shows the effects of pH and salt on the binding of
RNA to either Organon Teknika Silica or to Glassmilk.
[0032] FIG. 7 shows the effects of salt and pH on the selectivity
of nucleic acid binding to Glassmilk in the presence of either NaI
or NaCl.
[0033] FIG. 8(a) shows the effects of pH on the selectivity of
binding of nucleic acid (both RNA and DNA) to Sigma Silica. Binding
was performed in 4.8 M NaI using each of the following buffers: 50
mM MES, pH 6.0; 50 mM HEPES, pH 7.0; 50 mM Tris, pH 8.0; 50 mM
Tris, pH 9.0; or 50 mM AMP, pH 10.0.
[0034] FIG. 8(b) shows the effects of pH on the selectivity of
binding of nucleic acid (both RNA and DNA) to Bangs 2.28 .mu.m
particles. Binding was performed in 4.8 M NaI using each the
following buffers: 50 mM MES, pH 6.0; 50 mM HEPES, pH 7.0; 50 mM
Tris, pH 8.0; 50 mM Tris, pH 9.0; or 50 mM AMP, pH 10.0.
[0035] FIG. 8(c) shows the effects of pH on the selectivity of
binding of nucleic acid (both RNA and DNA) to Davisil Silica Gel.
Binding was performed in 4.8 M NaI using each of the following
buffers: 50 mM MES, pH 6.0; 50 mM HEPES, pH 7.0; 50 mM Tris, pH
8.0; 50 mM Tris, pH 9.0; or 50 mM AMP, pH 10.0.
[0036] FIG. 9 shows the selectivity of DNA versus RNA binding of
various solid phases in the presence of NaI at either pH 8 or pH
10.
[0037] FIG. 10 shows the effect of DNA-selective binding conditions
on binding DNA and RNA to Sigma Silica. Increasing concentrations
of either DNA or RNA were bound to Sigma Silica using 50 mM Tris,
pH 8.0, and 4.8 M NaI.
[0038] FIG. 11 shows the binding of protein (bovine serum albumin)
to silica at various pH levels.
[0039] FIG. 12(a) shows the effects of various anions and cations
on binding of DNA to Sigma Silica at pH 10 (grouped by cation).
[0040] FIG. 12(b) shows the effects of various anions and cations
on binding of RNA to Sigma Silica at pH 10 (grouped by cation).
[0041] FIG. 12(c) shows the effects of various anions and cations
on binding of DNA to Sigma Silica at pH 10 (grouped by anion).
[0042] FIG. 12(d) shows the effects of various anions and cations
on binding of RNA to Sigma Silica at pH 10 (grouped by anion).
[0043] FIG. 12(e) shows the effects of various anions and cations
on binding selectivity to Sigma Silica at pH 10 (grouped by
cation).
[0044] FIG. 12(f) shows the effects of various anions and cations
on binding selectivity to Sigma Silica at pH 10 (grouped by
anion).
[0045] FIG. 13 shows the results of selective binding experiments
with various concentrations of DNA at pH 6 or pH 10.
[0046] FIG. 14 shows the results of DNA and RNA recovery under
selective and nonselective conditions with different ratios of RNA
to DNA.
[0047] FIG. 15 shows the results of extraction of DNA from whole
blood using Sigma Silica at pH 6 and pH 10.
[0048] FIG. 16 shows the recovery of RNA after various incubation
times in order to test for RNA degradation.
[0049] FIG. 17 shows a schematic diagram of certain methods of
isolating nucleic acids by selective binding and elution.
[0050] FIG. 18 shows a stained agarose gel showing RNA and DNA
isolated by an exemplary sequential selective binding method.
[0051] FIG. 19 shows a stained agarose gel showing RNA and DNA
isolated by an exemplary sequential selective elution method.
DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
[0052] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention, as
claimed. In this application, the use of the singular includes the
plural unless specifically stated otherwise. In this application,
the use of "or" means "and/or" unless stated otherwise.
Furthermore, the use of the term "including", as well as other
forms, such as "includes" and "included", is not limiting.
[0053] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described. All documents, or portions of documents, cited in
this application, including but not limited to patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
Definitions
[0054] The term "biological sample" is used in a broad sense and is
intended to include a variety of biological sources that contain
nucleic acids. Such sources include, without limitation, whole
tissues, including biopsy materials and aspirates; in vitro
cultured cells, including primary and secondary cells, transformed
cell lines, and tissue and cellular explants; whole blood, red
blood cells, white blood cells, and lymph; body fluids such as
urine, sputum, semen, secretions, eye washes and aspirates, lung
washes, cerebrospinal fluid, abscess fluid, and aspirates. Included
in this definition of "biological sample" are samples processed
from biological sources, including but not limited to cell lysates
and nucleic acid-containing extracts. Any organism containing
nucleic acid may be a source of a biological sample, including, but
not limited to, any eukaryotes, eubacteria, archaebacteria, or
virus. Fungal and plant tissues, such as leaves, roots, stems, and
caps, are also within the scope of the present invention.
Microorganisms and viruses that may be present on or in a
biological sample are within the scope of the invention.
[0055] The term "buffer," as used herein, refers to aqueous
solutions or compositions that resist changes in pH when acids or
bases are added to the solution. This resistance to pH change is
due to the solution's buffering action. Solutions exhibiting
buffering activity are referred to as buffers or buffer solutions.
Buffers typically do not have an unlimited ability to maintain the
pH of a solution or composition. Rather, typically they are able to
maintain the pH within certain ranges, for example between pH 5 and
pH 7. See, generally, C. Mohan, Buffers, A guide for the
preparation and use of buffers in biological systems, Calbiochem,
1999. Exemplary buffers include, but are not limited to, MES
([2-(N-Morphilino)ethanesulfonic acid]), ADA
(N-2-Acetamido-2-iminodiacetic acid), and Tris
([tris(Hydroxymethyl)aminomethane]; also known as Trizma);
Bis-Tris; ACES; PIPES; and MOPS.
[0056] Buffers that maintain the pH within a certain pH range, for
example, between pH 5 and pH 7, and similar terms as used herein,
are intended to encompass any buffer that exhibits buffering action
at some point within the stated pH range. Thus, that term
encompasses buffers that do not exhibit buffering capacity within
the entire stated range, and buffers with buffering capacity that
extend beyond the stated range. For example, solution A may exhibit
buffering capacity between pH 5.2 and 6.7, solution B may exhibit
buffering capacity between 6.0 and 8.0. For purposes of this
invention, both of those solutions would be considered buffers that
maintain the pH within the range of pH 5.0 to pH 7.0. The skilled
artisan will be able to identify an appropriate buffer for
maintaining the pH between a specified range using a buffer table.
Buffer tables can be found in, among other places, the Calbiochem
2000-2001 General Catalog at pages 81-82, and the Sigma 2000-2001
Biochemicals and Reagents for Life Science Research Catalog at page
1873, both of which are expressly incorporated by reference.
[0057] The term "isolating" nucleic acid refers to the recovery of
nucleic acid molecules from a source. While it is not always
optimal, the process of recovering nucleic acid may also include
recovering some impurities such as protein. It includes, but is not
limited to, the physical enrichment of nucleic acid molecules from
a source. The term "isolating" may also refer to the duplication or
amplification of nucleic acid molecules, without necessarily
removing the nucleic acid molecules from the source.
[0058] The term "salt" as used herein, refers to a compound
produced by the interaction of an acid and a base. Exemplary salts
include, but are not limited to, sodium chloride (table salt),
sodium iodide, sodium bromide, lithium bromide, lithium iodide,
potassium phosphate, sodium bicarbonate, and the like. In water and
other aqueous solutions, salts typically dissociate into an "anion"
or negatively charged subcomponent, and a "cation" or positively
charge subcomponent. For example, when sodium chloride (NaCl) is
dissolved in water, it dissociates into a sodium cation (Na.sup.+)
and a chloride anion (Cl.sup.-). Exemplary salts are discussed,
e.g., in Waser, Jurg, Quantitative Chemistry, A Laboratory Text, W.
A. Benjamin, Inc., New York, page 160, (1966).
[0059] The term "nucleic acid," as used herein, refers to a polymer
of ribonucleosides or deoxyribonucleosides typically comprising
phosphodiester linkages between subunits. Other linkages between
subunits include, but are not limited to, methylphosphonate,
phosphorothioate, and peptide linkages. Such nucleic acids include,
but are not limited to, genomic DNA, cDNA, hnRNA, mRNA, rRNA, tRNA,
fragmented nucleic acid, nucleic acid obtained from subcellular
organelles such as mitochondria or chloroplasts, and nucleic acid
obtained from microorganisms or DNA or RNA viruses that may be
present on or in a biological sample.
[0060] Solid phase components (also called solid phases) that are
capable of binding to nucleic acids released from a biological
sample include a variety of materials that are capable of binding
nucleic acids under suitable conditions. Exemplary solid phase
components include, but are not limited to, silica particles,
silicon dioxide, diatomaceous earth, glass, alkylsilica, aluminum
silicate, borosilicate, nitrocellulose, diazotized paper,
hydroxyapatite, nylon, metal oxides, zirconia, alumina,
diethylaminoethyl- and triethylaminoethyl-derivatized supports
(Chromegabond SAX, LiChrosorb-AN, Nucleosil SB, Partisil SAX, RSL
Anion, Vydac TP Anion, Zorbax SAX, Nucleosil NMe.sub.2, Aminex
A-series, Chromex, and Hamilton HA Ionex SB, DEAE sepharose, QAE
sepharose), hydrophobic chromatography resins (such phenyl- or
octyl-sepharose), and the like.
[0061] The term "selective binding" with regard to nucleic acid
refers to binding of a type or species of nucleic acid (e.g., DNA)
to a solid phase under conditions in which other types or species
of nucleic acid (e.g., RNA) bind less efficiently. For example,
conditions for binding may be said to be selective for DNA when the
amount of DNA bound to a solid phase is greater than the amount of
RNA, when the DNA and RNA are in equimolar ratios in solution.
Further, conditions for the binding of DNA to a solid phase may be
said to be selective when the efficiency of binding of DNA to a
solid phase is unaffected by the amount of RNA in solution with the
DNA.
EXEMPLARY EMBODIMENTS
[0062] A. According to certain embodiments, methods of isolating
DNA from a biological sample are provided, which comprise:
selectively binding DNA to a solid phase by contacting the
biological sample with the solid phase under conditions which
selectively bind DNA; separating the solid phase with the bound DNA
from an unbound portion of the biological sample; and isolating the
DNA from the solid phase.
[0063] According to certain embodiments, methods of identifying DNA
in a biological sample are provided. In certain embodiments,
methods of identifying DNA in a biological sample comprise:
selectively binding DNA to a solid phase by contacting the
biological sample with the solid phase under conditions which
selectively bind DNA; separating the solid phase with the bound DNA
from an unbound portion of the biological sample; and identifying
the DNA bound to the solid phase. According to certain embodiments,
the identifying the DNA bound to the solid phase comprises
amplifying the DNA bound to the solid phase.
[0064] According to certain embodiments, the solid phase is a
siliceous material. In certain embodiments, the siliceous material
is selected from a group comprising silica, silica dioxide,
diatomaceous earth, glass, Celite, and silica gel. In certain
embodiments, the solid phase is in a form selected from a group
comprising a particle, a bead, a membrane, a frit, and a side of a
container. Exemplary, but nonlimiting, examples of solid phases are
discussed in U.S. Pat. Nos. 4,648,975; 4,923,978; 5,075,430;
5,175,271; 5,234,809; 5,438,129; 5,658,548; 5,804,684; and
5,808,041; European Application Nos. EP 0 391 608 and EP 0 757 106;
and PCT Publication Nos. WO 87/06621; WO 91/00924; WO 92/18514; WO
97/30062; WO 99/51734; and WO 99/40098.
[0065] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion. In certain embodiments, the large anion
is selected from at least one of a group comprising picrate,
tannate, tungstate, molybdate, perchlorate, and sulfosalicylate. In
certain embodiments, the large anion is selected from at least one
of a group comprising trichloroacetate, tribromoacetate,
thiocyanate, and nitrate. In certain embodiments, the large anion
is selected from at least one of a group comprising iodide and
bromide. In certain embodiments, the alkaline pH is equal to, or
above 8.0. In certain embodiments, the alkaline pH is equal to, or
above 9.0. In certain embodiments, the alkaline pH is equal to, or
above 10.0. In certain embodiments, the alkaline pH is any pH range
or point between 8.0 and 12.0.
[0066] In certain embodiments, the isolating the DNA from the solid
phase comprises eluting the DNA.
[0067] B. According to certain embodiments, methods of isolating
DNA and RNA from a biological sample are provided, which comprise:
selectively binding DNA to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
selectively bind DNA; separating the first solid phase with the
bound DNA from a first unbound portion of the biological sample;
isolating the DNA from the first solid phase; and isolating RNA
from the first unbound portion of the biological sample. In certain
embodiments, the isolating the DNA from the first solid phase
comprises eluting the DNA.
[0068] According to certain embodiments, methods of identifying DNA
and RNA in a biological sample are provided, which comprise:
selectively binding DNA to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
selectively bind DNA; separating the first solid phase with the
bound DNA from a first unbound portion of the biological sample;
identifying the DNA bound to the first solid phase; and identifying
the RNA from the first unbound portion of the biological sample. In
certain embodiments, the isolating the DNA from the first solid
phase comprises eluting the DNA. In certain embodiments, the
identifying the DNA bound to the first solid phase comprises
amplifying the DNA bound to the first solid phase.
[0069] One of ordinary skill will appreciate that there are many
methods of identifying nucleic acid (both DNA and RNA) bound to a
solid phase, according to certain embodiments. Such methods
include, but are not limited to, hybridization to labeled probes,
reverse transcription, mass spectrometry, and detection by a
reaction of the bound DNA with a label, e.g., detection of
fluorescence following the addition of a DNA-binding
fluorophore.
[0070] According to certain embodiments, the isolating of the RNA
from the first unbound portion of the biological sample comprises:
exposing the first unbound portion of the biological sample to a
second solid phase under conditions which bind RNA to the second
solid phase; separating the second solid phase with bound RNA from
the second portion of the biological sample; and isolating the RNA
from the second solid phase by eluting the RNA.
[0071] According to certain embodiments, the conditions which bind
RNA to the second solid phase comprise a neutral or acidic pH. In
certain embodiments, the conditions which bind RNA to the second
solid phase comprise reducing the pH to 8.0 or below. In certain
embodiments, the conditions which bind RNA to the second solid
phase comprise use of a salt with an anion smaller than
bromide.
[0072] According to certain embodiments, the second solid phase is
selected from any of the materials discussed above to the first
solid phase in A above. The second solid phase may be the same
material as the first solid phase or it may be different
material.
[0073] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion. Conditions which selectively bind DNA
for these methods may comprise the conditions discussed above for
selectively binding DNA in section A.
[0074] As a non-limiting example, one may add a buffer or salt to a
cell lysate, making the cell lysate very alkaline. A solid phase,
such a silica bead, would then be exposed to the alkaline lysate.
DNA selectively binds to the bead, which is then removed. Another
buffer or salt is added to the alkaline lysate to make the lysate
neutral in pH. A second solid phase is added to the neutral lysate,
and the RNA in the lysate binds to the second solid phase. The DNA
bound to the first solid phase is then eluted with a neutral or
alkaline, low salt buffer. The second solid phase is removed from
the neutral lysate, and the RNA is eluted from the second solid
phase with a neutral or alkaline, low salt buffer.
[0075] C. According to certain embodiments, methods of isolating
nucleic acid from a biological sample are provided, which comprise:
binding nucleic acid to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
bind both RNA and DNA; separating the first solid phase with bound
nucleic acid from a first unbound portion of the biological sample;
eluting RNA from the first solid phase with bound nucleic acid
under conditions which selectively bind DNA; removing the first
solid phase with bound DNA from a first eluate; and isolating the
DNA from the first solid phase.
[0076] According to certain embodiments, the isolating the DNA from
the first solid phase comprises eluting the DNA from the first
solid phase.
[0077] According to certain embodiments, methods of identifying
nucleic acid in a biological sample are provided, which comprise:
binding nucleic acid to a first solid phase by contacting the
biological sample with the first solid phase under conditions which
bind both RNA and DNA; separating the first solid phase with bound
nucleic acid from a first unbound portion of the biological sample;
eluting RNA from the first solid phase with bound nucleic acid
under conditions which selectively bind DNA; removing the first
solid phase with bound DNA from a first eluate of the biological
sample; and identifying the DNA bound to the first solid phase.
According to certain embodiments, the identifying the DNA bound to
the first solid phase comprises amplifying the DNA bound to the
first solid phase.
[0078] One of ordinary skill will appreciate that there are many
methods of identifying nucleic acid (both DNA and RNA) bound to a
solid phase, according to certain embodiments. Such methods
include, but are not limited to, hybridization to labeled probes,
reverse transcription, mass spectrometry, and detection by a
reaction of the bound DNA with a label, such as detection of
fluorescence following the addition of a DNA-binding
fluorophore.
[0079] According to certain embodiments, the first solid phase is
selected from any of the materials discussed above to the first
solid phase in A above.
[0080] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: a
buffer with an alkaline pH; and a large anion, wherein the large
anion is at least as large as a bromide ion. Conditions which
selectively bind DNA for these methods may comprise the conditions
discussed above for selectively binding DNA in section A.
[0081] According to certain embodiments, the method of isolating
nucleic acid from a biological sample further comprises: exposing
the first eluate to a second solid phase under conditions which
bind RNA to the second solid phase; separating the second solid
phase with the bound RNA from a second eluate of the biological
sample; and isolating the RNA from the second solid phase.
[0082] According to certain embodiments, the isolating the RNA from
the second solid phase comprises eluting the RNA. According to
certain embodiments, the isolating the RNA from the second solid
phase comprises amplifying the RNA bound to the second solid
phase.
[0083] According to certain embodiments, the second solid phase is
selected from any of the materials discussed above to the first
solid phase in A above. The second solid phase may be the same
material as the first solid phase or it may be different
material.
[0084] According to certain embodiments, the conditions which
selectively bind DNA comprise using a binding buffer comprising: an
alkaline pH; and a large anion, wherein the large anion is at least
as large as a bromide ion. Conditions which selectively bind DNA
for these methods may comprise the conditions discussed above for
selectively binding DNA in section A. In certain embodiments, the
conditions which bind RNA to the second solid phase comprise a
neutral or acidic pH.
[0085] In certain embodiments, the conditions which bind RNA to the
second solid phase comprise reducing the pH to 8.0 or below. In
certain embodiments, the conditions which bind RNA to the second
solid phase comprise use of a salt with an anion smaller than
bromide.
[0086] As a non-limiting example, a solid phase, such a silica
bead, would be exposed to a cell lysate. Nucleic acid, both DNA and
RNA, then binds to the solid phase, which is then removed. The
solid phase is then placed in a high pH buffer, which allows the
RNA to elute from the solid phase, but keeps the DNA bound to the
solid phase. The solid phase is then removed, and placed in a low
salt buffer, which elutes the DNA.
[0087] D. According to certain embodiments, a kit is provided,
which comprises: a buffer with an alkaline pH; a large anion,
wherein the large anion is at least as large as a bromide ion; and
a solid phase. In certain embodiments, the large anion is selected
from any of the large anions discussed in section A. In certain
embodiments, the alkaline pH is equal to, or above 8.0. In certain
embodiments, the alkaline pH is equal to, or above 9.0. In certain
embodiments, the alkaline pH is equal to, or above 10.0. According
to certain embodiments, the solid phase is selected from any of the
materials discussed above to the solid phase in A above.
[0088] E. According to certain embodiments, a kit is provided,
which comprises: a solid phase; a nucleic acid binding buffer,
wherein both DNA and RNA bind the solid phase under conditions
generated by the nucleic acid binding buffer; and a selective DNA
binding buffer, wherein the conditions generated by the selective
DNA binding buffer allow selective binding of DNA to the solid
phase. The conditions which selectively bind DNA are those
discussed in section A, above. In certain embodiments, the kit
further comprises an RNA binding buffer, wherein the conditions
generated by the RNA binding buffer allow RNA to bind to a solid
phase. Conditions which allow the binding of RNA are those
discussed in section B, above. In certain embodiments, the
selective binding buffer has a pH equal to, or above 8.0. In
certain embodiments, the selective binding buffer has a pH equal
to, or above 9.0. In certain embodiments, the selective binding
buffer has a pH equal to, or above 10.0. According to certain
embodiments, the solid phase is selected from any of the materials
discussed above to the solid phase in A above. In certain
embodiments, the nucleic acid binding buffer has a pH equal to, or
below 8.0.
EXAMPLES
[0089] The following examples illustrate certain embodiments of the
invention, and do not limit the scope of the invention in any
way.
[0090] The following terms, abbreviations, and sources apply to the
materials discussed throughout Examples 1 to 4.
[0091] These substrates were obtained from the following sources:
Silica (Organon Teknika, Product Number 82951, Lot 00030302),
Diatomaceous Earth (Sigma, Product Number D-3877, Lot 128H3702),
Empore Filter Aid 400 (3M, Product Number 56221-746, Lot 990020),
Silica Gel (JT Baker, Product Number 3405-01, Lot N36338), Silicon
dioxide (Sigma, Product Number S-5631, Lot 58H0154), Binding Matrix
(BIO-101, Product Number 6540-408, Lot Number 6540-408-0B13),
Glassmilk Spin Buffer #4 (BIO 101, Product Number 2072-204, Lot
Number 2072-204-8A17), Davisil Grade 643 Silica Gel (Spectrum,
Product Number Sil 66, Lot NE 0387), and Uniform Silica
Microspheres (Bangs Laboratories, Inc. Catalog Code SS05N, Inv #
L0002188).
[0092] Abbreviations or names of the following reagents and sources
for them are as follows: guanidine hydrochloride (Sigma, Lot
38H5432), guanidine thiocyanate (Sigma, Product Number G-9277),
sodium iodide (Aldrich Chemical Company, Product Number 38,311-2,
Lot Number 07004TS), sodium perchlorate (Aldrich Chemical Company,
Product Number 41,024-1, Lot KU 06910HU), sodium bromide (Aldrich,
Product Number 31050-6, Lot 11805KR), sodium chloride (Aldrich
Chemical Company, Product Number 33,251-4, Lot Number 16524CS),
Tris (Trizma base, Tris[Hydroxymethyl]aminomethane, Sigma, Product
Number T-6791, Lot Number 1261-15738)--pH 8, MES
(2-[N-Morpholino]ethanesulfonic acid, Sigma, product number M-5287,
lot number 58H5411)--pH 6.0, AMP (2-amino-2-methyl-1-propanol,
Sigma, Product Number 221)--pH 10, Hepes
(n-[2hydroxyethyl]piperazine-N'-[2-ethane sulfonic acid], Sigma,
product number H-4034, lot number 19H54101), ethanol (Ethyl
alcohol, absolute, Aldrich, catalog number E702-3), HCl (Sigma,
Product Number H-7020, Lot Number 97H3562), sodium hydroxide
(Sigma, Product Number S-8045, Lot Number 127H0531 and 69H1264),
ammonium bifluoride (ammonium hydrogen fluoride, Aldrich Product
Number 22,482-0), nitric acid (Aldrich, Product number 22571-1, lot
number 00261 A1), and ammonium hydroxide (Aldrich product number
22,122-8, lot number 02308KR).
[0093] Nucleic acids and tissue samples and sources for them are as
follows: calf thymus genomic DNA (deoxyribonucleic acid, type 1,
highly polymerized from calf thymus, Sigma, Product Number D-1501,
Lot 87H7840); rat liver total RNA (Biochain Institute, lot numbers
A304057, A305062, or A306073); and whole blood (Blood Centers of
the Pacific).
[0094] The spectrophotometry was performed with a Hewlett-Packard
Model 8453 Spectrophotometer.
[0095] Gel electrophoresis of nucleic acid samples in Examples 12
to 14 was carried out using SeaKem.RTM.) agarose (Teknova);
1.times.TBE (89 mM Tris, 89 mM Boric Acid, 2 mM EDTA, Teknova,
catalog number 0278-1L, lot number 17F801); and 0.5 .mu.g/ml
ethidium bromide buffer (BIO-RAD). Molecular weight markers used in
electrophoresis were an AmpliSize DNA molecular weight standard
(BIO-RAD), a High Molecular Weight DNA Marker (Gibco BRL), and an
RNA ladder (GIBCO BRL).
Example 1
[0096] Silica and glass matrices from a variety of suppliers were
evaluated for their ability to bind genomic DNA using different
salts at pH 8.
[0097] Silica (Organon Teknika), Diatomaceous Earth, Empore Filter
Aid 400, J. T. Baker Silica Gel, Silicon dioxide, Binding Matrix,
and Glassmilk Spin Buffer #4 were used for the following example.
Prior to use, all particles (except the Silica from Organon
Teknika) were prepared as follows: the particles were washed once
with 4-8 volumes of 1 N HCl, twice with 4-8 volumes of water, once
with 4-8 volumes of 1 N NaOH, twice with 4-8 volumes of water, once
with 4-8 volumes of ethanol, and four times with 4-8 volumes of
water. As used herein, one "volume" of water or ethanol refers to
an amount of water or ethanol equal in mass to the mass of the
particles being washed. The Binding Matrix and Glassmilk from
BIO-101 were washed 4 times with at least 4 volumes of water before
being treated with HCl, NaOH, and ethanol (as described above). The
Silica particles were used as supplied. Diatomaceous Earth, Empore
Filter Aid 400, Silica Gel, and Silicon dioxide were stored as a
200 mg/ml (20%) slurry in water. The Binding Matrix particles were
stored as a 580 mg/ml (58%) slurry in water and the Glassmilk
particles were stored as a 373 mg/ml (37%) slurry in water.
[0098] Calf thymus DNA was used as the source of the genomic DNA.
Sheared genomic DNA used in the following examples was prepared as
follows. The DNA was resuspended at approximately 10 mg/ml in
water. The DNA was then sheared by passing the material four times
through a 20 G 11/2 needle, three times through a 21 G 11/2 gauge
needle, 22 G 11/2 gauge needle, and once through a 26 G 11/2 gauge
needle.
[0099] Each solid phase and buffer combination shown in FIG. 1 was
assayed once. Sheared calf thymus DNA (25 .mu.g of DNA, 50 .mu.l of
a 0.5 mg/ml concentration) was added to 1.5 ml microcentrifuge
tubes containing 0.45 ml of one of the following buffers: (1) 50 mM
Tris-HCl, pH 8, 4.75 M guanidine thiocyanate; (2) 50 mM Tris HCl,
pH 8; 4.75 M guanidine hydrochloride; (3) 50 mM Tris HCl, pH 8;
4.75 M Sodium chloride; (4) 50 mM Tris-HCl, pH 8, 4.75 M sodium
bromide; (5) 50 mM Tris-HCl, pH 8, 4.75M sodium iodide; or (6) 50
mM Tris-HCl, pH 8, 4.75 M sodium perchlorate. The nucleic acid was
incubated for up to 10 minutes at ambient temperature in the
buffered solution, with occasional mixing. Those mixtures were
incubated for 10 minutes at ambient temperature with occasional
mixing. Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed twice with 0.5 ml
of the binding buffer that had been used for the binding
incubation. Subsequently, the particles were washed three to four
times with 0.5 ml of 70% ethanol.
[0100] Following the last ethanol wash, the particles were allowed
to air dry at ambient temperature or at 56.degree. C. for 5-10
minutes. The bound nucleic acid was first eluted with 0.25 ml of 10
mM Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing
and the eluted nucleic acid was collected. Any residual nucleic
acid bound to the particles was subsequently eluted with 0.25 ml of
0.1 N NaOH at 56.degree. C. for 5 minutes with constant mixing and
the eluted nucleic acid was collected. The amount of nucleic acid
was quantified by spectrophotometry. The results are shown in FIG.
1.
[0101] At pH 8, the effect of salt composition on DNA binding
appeared to be a function of the source of the solid phase. For a
particular matrix, the recovery of DNA varied up to 43-fold,
depending on the choice of salt. The preferred anions for most of
the matrices in this work were the bulky anions thiocyanate,
bromide, iodide, and perchlorate. In general, recovery of DNA was
poor with salts containing the less bulky chloride anion. A
comparison of the effect of GuHCl and GuSCN on DNA binding
demonstrated greater DNA binding in the presence of the larger
thiocyanate anion.
Example 2
[0102] The solid phases used in the present example were prepared
as described in Example 1. Sheared genomic DNA from calf thymus was
prepared as described in Example 1.
[0103] Each solid phase and buffer combination was assayed once.
Sheared calf thymus DNA (25 .mu.g of DNA, 50 .mu.l of a 0.5 mg/ml
concentration) was added to a 1.5 ml microcentrifuge tube
containing 0.45 ml of either (1) 50 mM Tris-HCl, pH 8 and 4.75 M
guanidine thiocyanate; or (2) 50 mM Tris-HCl, pH 8 and 4.75 M
guanidine hydrochloride. Nucleic acid was incubated up to 10
minutes at ambient temperature in the buffered solution, with
occasional mixing. Each of the seven solid phases (10-187 mg) was
added separately to each of the two buffered nucleic acid solutions
so that there were 14 containers with each combination of the
individual solid phases and buffers. Those mixtures were incubated
for 10 minutes at ambient temperature with occasional mixing.
Following the binding incubation, the particles were centrifuged
(15,800.times.g, 1 minute) and washed twice with 0.5 ml of the
binding buffer that had been used for the binding incubation.
Subsequently, the particles were washed three to four times with
0.5 ml of 70% ethanol.
[0104] Following the last ethanol wash, the particles were allowed
to air dry at ambient temperature or at 56.degree. C. for 5-10
minutes. The bound nucleic acid was first eluted with 0.25 ml of 10
mM Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing
and the eluted nucleic acid was collected. Any residual nucleic
acid bound to the particles was subsequently eluted with 0.25 ml of
0.1 N NaOH at 56.degree. C. for 5 minutes with constant mixing and
the eluted nucleic acid was collected. The amount of nucleic acid
was quantified by spectrophotometry. The results are shown in FIG.
2. In general, recovery in the presence of thiocyanate appeared
superior to recovery in the presence of chloride ions.
Example 3
[0105] The binding characteristics of RNA to silica and glass solid
phases from a variety of suppliers were evaluated using different
salts at pH 8. Silica, Diatomaceous Earth, Binding Matrix, and
Glassmilk Spin Buffer #4 were used for the following studies. The
solid phase particles were prepared as described in Example 1.
[0106] Each solid phase and buffer combination was assayed once.
Rat liver total RNA (15 .mu.g of RNA, 6 .mu.l of a 2.5 mg/ml
concentration in water) was added to a 1.5 ml microcentrifuge tube
containing 0.45 ml of one of the following buffers: (1) 50 mM
Tris-HCl, pH 8, 4.75 M guanidine thiocyanate; (2) 50 mM Tris HCl,
pH 8; 4.75 M guanidine hydrochloride; (3) 50 mM Tris HCl, pH 8;
4.75 M sodium chloride; or (4) 50 mM Tris HCl, pH 8; 4.75 M sodium
iodide. Nucleic acid was incubated up to 5 minutes at ambient
temperature in the buffered solution, with occasional mixing. Those
mixtures were incubated 10 minutes at ambient temperature with
occasional mixing.
[0107] Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed twice with 0.5 ml
of the binding buffer that had been used for the binding
incubation. Subsequently, the particles were washed two or four
times with 0.5 ml of 70% ethanol. The particles which were washed
twice with ethanol were then washed once with 0.5 ml of acetone and
were allowed to dry for 5 minutes at 56.degree. C. The bound
nucleic acid was first eluted with 0.25 or 0.275 ml of 50 mM Tris,
pH 9 for 5 minutes at 56.degree. C. with constant mixing and the
eluted nucleic acid was collected. Any residual nucleic acid was
eluted with 0.25 or 0.275 ml of 0.1 N NaOH at 56.degree. C. for 5
minutes with constant mixing and the eluted nucleic acid was
collected. The amount of nucleic acid was quantified by
spectrophotometry. The results are shown in FIG. 3.
[0108] In contrast to the results found with DNA, recovery of RNA
when bound to the solid phase at pH 8 did not appear to show a
strong dependence on salt composition. The selection of salt
resulted in less than a threefold variation in the recovery of RNA
for a particular matrix.
[0109] When binding at pH 8, many of the matrices showed a
moderately higher binding of DNA as compared to RNA. Selectivity
for DNA binding was increased with sodium iodide (see FIG. 4).
Example 4
[0110] In order to investigate the relationship between DNA binding
to solid-phases, DNA was bound to several solid-phases using
buffers with different salt compositions and pH levels.
[0111] The following solid phases used in this study were prepared
as described in Example 1: Silica, Diatomaceous Earth, and
Glassmilk Spin Buffer #4. For these studies, sheared calf thymus
DNA prepared as described in Example 1 was used as the source of
genomic DNA.
[0112] Each solid phase and buffer combination was assayed once.
Sheared calf thymus DNA (25 .mu.g-50 .mu.l at 0.5 mg/ml) was added
to different 1.5 ml microcentrifuge tubes containing 0.45 ml of one
of the following buffers: (1) 50 mM MES, pH 6, 4.75 M guanidine
thiocyanate; (2) 50 mM MES, pH 6.0, 4.75 M sodium chloride; (3) 50
mM MES, pH 6.0, 4.75 M sodium bromide; (4) 50 mM MES, pH 6.0, 4.75
M sodium iodide; (5) 50 mM Tris-HCl, pH 8, 4.75 M guanidine
thiocyanate; (6) 50 mM Tris-HCl, pH 8, 4.75 M sodium chloride; (7)
50 mM Tris-HCl, pH 8, 4.75M sodium bromide; (8) 50 mM Tris-HCl, pH
8, 4.75 M sodium iodide; (9) 50 mM AMP, pH 10, 4.75M guanidine
thiocyanate; (10) 50 mM AMP, pH 10, 4.75 M sodium chloride; (11) 50
mM AMP, pH 10, 4.75 M sodium bromide; or (12) 50 mM AMP, pH 10,
4.75 M sodium iodide.
[0113] Nucleic acid was incubated for 5 to 10 minutes at ambient
temperature in the buffered solution, sometimes with occasional
mixing. Each of the three solid phases (10 mg) was added separately
to each of the buffered nucleic acid solutions. Those mixtures were
incubated for 10 minutes at ambient temperature with occasional
mixing. following the binding incubation, the particles were
centrifuged (4000.times.g, 1 minute) and washed twice with 0.5 ml
of the binding buffer that had been used for the binding
incubation. The particles were then washed three times with 0.5 ml
of 70% ethanol.
[0114] Following the last ethanol wash, the particles were allowed
to air dry at ambient temperature for 5-10 minutes. The bound
nucleic acid was first eluted with 0.25 ml of 50 mM Tris, pH 9 for
5 minutes at 56.degree. C. with constant mixing, and the eluted
nucleic acid was collected. Any residual nucleic acid bound to the
particles was eluted with 0.25 ml of 0.1 N NaOH at 56.degree. C.
for 5 minutes with constant mixing and the eluted nucleic acid was
collected. The amount of nucleic acid was quantified by
spectrophotometry. Results for each set of experiments are shown in
FIGS. 5(a)-(k).
[0115] Previous investigators showed that DNA binding to silica
decreases as the pH of the buffer is increased above 7 (Melzak,
Kathryn A. et al. (1996), Driving Forces for DNA Adsorption to
Silica in Perchlorate Solutions, Journal of Colloid and Interface
Science 181: 635-644). The results in this example showed that the
salt composition influenced the effect of pH on DNA binding. The
results also demonstrated that source of the solid phase altered
the magnitude of the effect of the salt and the absolute amount of
nucleic acid that was bound. The effect of salt on pH sensitivity
of DNA binding shows the following order of sensitivity to
pH:NaCl>GuSCN>NaBr>NaI
Example 5
[0116] The effect of pH and particular salts on RNA binding was
also evaluated. The solid phases Binding Matrix and Glassmilk
particles were prepared as described in Example 1. Rat liver total
RNA was the source of RNA.
[0117] Each solid phase and buffer combination was assayed once.
Rat liver total RNA (15 .mu.g of RNA, 6 .mu.l of a 2.5 mg/ml
concentration) was added to 1.5 ml microcentrifuge tubes containing
0.45 ml of one of the following buffers: (1) 50 mM Tris-HCl, pH 8,
4.75 M guanidine thiocyanate; (2) 50 mM Tris-HCl, pH 8, 4.75 M
sodium chloride; (3) 50 mM Tris-HCl, pH 8, 4.75 M sodium iodide;
(4) 50 mM MES, pH 6, 4.75 M guanidine thiocyanate; (5) 50 mM MES,
pH 6, 4.75 M sodium iodide; (6) 50 mM MES, pH 6, 4.75 M sodium
chloride; (7) 50 mM AMP, pH 10, 4.75 M guanidine thiocyanate; (8)
50 mM AMP, pH 10, 4.75 M sodium iodide; or (9) 50 mM AMP, pH 10,
4.75 M sodium chloride. Nucleic acid was incubated up to 5 minutes
at ambient temperature in the buffered solution, with occasional
mixing. Each of the two solid phases (10-187 mg) was added
separately to the buffered nucleic acid solutions. Those mixtures
were incubated 10 minutes at ambient temperature with occasional
mixing. Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed twice with 0.5 ml
of the binding buffer that had been used for the binding
incubation. Subsequently, the particles were washed two or four
times with 0.5 ml of 70% ethanol. The particles which were washed
twice with ethanol were then washed once with 0.5 ml of acetone and
were allowed to dry for 5 minutes at 56.degree. C. The bound
nucleic acid was first eluted with 0.25 ml or 0.275 ml of 10 mM
Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing, and
the eluted nucleic acid was collected. Any residual nucleic acid
bound to the particles was subsequently eluted with 0.25 or 0.275
ml of 0.1 N NaOH for 5 minutes with constant mixing and the eluted
nucleic acid was collected. The amount of nucleic acid was
quantified by spectrophotometry.
[0118] The results are shown in FIG. 6. RNA binding to the two
solid-phases evaluated showed a large dependency on pH. There was a
significant reduction in RNA binding with all salts when the pH of
the buffer was increased.
Example 6
[0119] The selectivity of Glass Milk was evaluated further as a
function of pH and ionic composition during binding. Glassmilk Spin
Buffer #4 was prepared as described in Example 1. Sheared calf
thymus DNA was prepared as described in Example 1. Rat liver total
RNA was the source of RNA.
[0120] Each nucleic acid and buffer combination was assayed once.
Either 25 .mu.g (50 .mu.l of a 0.5 mg/ml solution in water) of
sheared calf thymus DNA or 15 .mu.g (6 .mu.l of a 2.5 mg/ml
solution in water) of rat liver total RNA was added separately to
separate 1.5 ml microcentrifuge tubes containing 0.45 ml of one of
the following buffers: (1) 50 mM MES, pH 6.0, 4.75 M sodium
chloride; (2) 50 mM MES, pH 6.0, 4.75 M sodium iodide; (3) 50 mM
Tris-HCl, pH 8, 4.75 M sodium chloride; (4) 50 mM Tris-HCl, pH 8,
4.75 M sodium iodide; (5) 50 mM AMP, pH 10, 4.75 M sodium iodide;
or (6) 50 mM AMP, pH 10, 4.75 M sodium chloride. Thus, there were
12 separate containers with each combination of the DNA or RNA and
individual buffers. The solid phase (186 mg) was added to the
buffered nucleic acid solutions and the mixtures were incubated 10
minutes at ambient temperature with occasional mixing. Following
the binding incubation, the particles were centrifuged
(15,800.times.g, 1 minute) and washed twice with 0.5 ml of the
binding buffer that had been used for the binding incubation.
Subsequently, the particles were washed four times with 0.5 ml of
70% ethanol. The bound nucleic acid was first eluted with 0.25 ml
of 10 mM Tris, pH 9 for 5 minutes at 56.degree. C. with constant
mixing and the eluted nucleic acid was collected. Any residual
nucleic acid bound to the particles was eluted with 0.25 of 0.1 N
NaOH for 5 minutes with constant mixing and the eluted nucleic acid
was collected. The amount of nucleic acid was quantified by
spectrophotometry.
[0121] The results are shown in FIG. 7. With sodium iodide as the
binding salt, selectivity for DNA binding increased when the pH was
increased. In contrast, binding in the presence of sodium chloride
did not show increased specificity at an alkaline pH.
Example 7
[0122] The effect of pH on DNA and RNA binding in the presence of
sodium iodide was evaluated with several solid phases. Silicon
dioxide (Sigma, Product Number S-5631, Lot 58H0154), Davisil Grade
643 Silica Gel (Spectrum, Product Number Sil 66, Lot NE 0387) and
Uniform Silica Microspheres (Bangs Laboratories, Inc. Catalog Code
SS05N, Inv # L0002188) were used for the following study. Prior to
use, the silicon dioxide and Davisil Grade Silica Gel particles
were prepared as follows. The particles were washed once with
water, once with 500 mM ammonium bifluoride, once with 100 mM
nitric acid, twice with 100 mM ammonium hydroxide, twice with 300
mM ammonium hydroxide, once with ethanol, and nine times with
water. All particles of the three solid phases were prepared and
stored as a 200 mg/ml (20%) slurry in water.
[0123] Total rat liver RNA was the source of RNA. For genomic DNA,
sheared calf thymus DNA was prepared as described in Example 1.
[0124] Each solid phase, nucleic acid, and buffer combination was
performed in triplicate. Either sheared calf thymus DNA (30 .mu.g,
addition of 50 .mu.L of a 590 .mu.g/mL stock) or total rat liver
RNA (30 .mu.g, addition of 50 .mu.L of a 600 .mu.g/mL stock) was
added separately to separate 1.5 ml microcentrifuge tubes
containing 50 .mu.L of silica particles (10 mg) along with 0.45 ml
of one of the following buffers: (1) 50 mM MES, pH 6.0, 4.8 M NaI;
(2) 50 mM Hepes, pH 7.0, 4.8 M NaI; (3) 50 mM Tris, pH 8, 4.8 M
NaI; (4) 50 mM Tris, pH 9, 4.8 M NaI; or (5) 50 mM AMP, pH 10, 4.8
M NaI. For each of the three solid phases, binding of either DNA or
RNA was evaluated with each of the five buffers in triplicate,
resulting in a total of thirty containers per solid phase. Nucleic
acid was incubated 5-30 minutes at ambient temperature in the
buffered solution, with continuous mixing using a Vortex Genie-2
mixer at setting 7 (Scientific Industries).
[0125] Following binding, the particles were centrifuged 14,000 rpm
for 1 minute and the supernatant removed. The particles were
subsequently washed four times with 1 mL of 70% ethanol. Following
addition of 250 .mu.L of 50 mM Tris, pH 9.0, the particles were
incubated for 5-6 minutes at 56.degree. C. with continuous shaking
(1400 rpm) on an Eppendorf Thermomixer R. The particles were
centrifuged at 14,000 rpm for 1 minute and the supernatant
containing eluted nucleic acid was collected. In order to detect
the presence of residual bound nucleic acid, 250 .mu.L of 100 mM
NaOH was added, the particles were incubated for 5-70 minutes at
56.degree. C. with continuous shaking (1400 rpm) on an Eppendorf
Thermomixer R, and the eluted nucleic acid was collected. The
amount of nucleic acid in each fraction was quantified
spectrophotometrically.
[0126] The results are shown in FIGS. 8(a)-(c). Almost all
siliceous solid phases that were examined showed an increased
selectivity for DNA binding at alkaline pH when sodium iodide was
the binding salt. DNA binding to Sigma Silica in the presence of
sodium iodide was relatively insensitive to pH. In contrast,
increasing the pH resulted in a dramatic decrease in binding of RNA
to the same solid phase. Thus, DNA selectivity was seen at an
alkaline pH in the presence of NaI.
Example 8
[0127] The effect of pH on DNA selectivity was examined using
several solid phases when sodium iodide was the binding salt.
Silica (Organon Teknika), Diatomaceous Earth, silicon dioxide
(Sigma Silica), Binding Matrix, and Glassmilk were prepared as
described in Example 1. Binding was carried out in the presence of
sheared calf thymus DNA (as described in Example 1) or rat liver
total RNA. Binding was performed in the following binding buffers:
(1) 50 mM Tris-HCl, pH 8, 4.75 M NaI; or (2) 50 mM AMP, pH 10, 4.75
M NaI.
[0128] Each solid phase, nucleic acid, and buffer combination was
assayed one to three times. Either sheared calf thymus DNA (25
.mu.g) or total rat liver RNA (15-25 .mu.g) was added to separate
1.5 ml microcentrifuge tubes containing 4.5 ml of one of the
following buffers: (1) 50 mM Tris-HCl, pH 8, 4.75 M NaI; or (2) 50
mM AMP, pH 10, 4.75 M NaI.
[0129] Each of the five solid phases (10-186 mg) was added
separately to the combinations of RNA or DNA and the individual
buffers. The data in FIG. 9 reflects for each of the RNA and DNA
experiments: (1) three separate containers with Glassmilk at pH 8
and 2 containers with Glassmilk at pH 10; (2) two separate
containers with Binding Matrix at pH 8 and 1 container with Binding
Matrix at pH 10; (3) one separate container with Sigma Silica at pH
8 and 1 container with Sigma Silica at pH 10; (4) one separate
container with Silica (Organon Teknika) at pH 8; and (5) two
separate containers with Diatomaceous Earth at pH 8 and 1 container
with Diatomaceous Earth at pH 10. Thus, there were 14 different
containers with each of the possible combinations of solid phase
and buffer for the RNA, and there were 14 different containers with
each of the possible combinations of solid phase and buffer for the
DNA. The mixtures were incubated for 10 minutes at ambient
temperature with occasional mixing. Following the binding
incubation, the particles were centrifuged (15,800.times.g, 1
minute) and washed twice with 0.5 ml of the binding buffer that had
been used in the binding incubation. Subsequently, the particles
were washed three to four times in 0.5 ml of 70% ethanol.
[0130] Following the last ethanol wash, particles were allowed to
air dry at ambient temperature or at 56.degree. C. for 5-10
minutes. The bound nucleic acid was first eluted with 0.25 ml of 10
mM Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing
and the eluted nucleic acid was collected. Any residual nucleic
acid bound to the particles was eluted with 0.25 ml of 0.1 N NaOH
at 56.degree. C. for 5 minutes with constant mixing and the eluted
nucleic acid was collected. The amount of nucleic acid was
quantified by spectrophotometry.
[0131] The results are shown in FIG. 9. Each of the solid phases
that were examined showed a greater specificity for DNA binding at
the alkaline pH. There appeared to be a reduced affinity for RNA at
alkaline pH. Binding of DNA at concentrations below saturation of
the solid phase was efficient. Virtually all of the added DNA was
bound and recovered from the siliceous solid phase. In contrast,
binding of RNA under these conditions was inefficient even when the
amount added was high.
Example 9
[0132] To measure the difference between the binding of DNA and RNA
to silicon dioxide, and the level of saturation, the following
experiment was performed. The solid phase examined was silicon
dioxide, which was prepared as described in Example 1. The nucleic
acids studied were sheared calf thymus DNA (prepared as described
in Example 1) and total rat liver RNA.
[0133] Sheared calf thymus DNA (126 .mu.g, 60 .mu.g, 30 .mu.g, 15
.mu.g, or 5 .mu.g) or total rat liver RNA (125 .mu.g, 60 .mu.g, 30
.mu.g, 15 .mu.g, or 5 .mu.g) was added in 50 .mu.L to separate
Eppendorf tubes (1.5 ml) containing 450 .mu.l of binding buffer (50
mM Tris, pH 8, 4.8 M sodium iodide), and 10 mg of Sigma silicon
dioxide particles (Sigma, prepared as described in Example 7). The
work was performed in triplicate, so there were 15 containers for
the five different amounts of DNA and 15 containers for the five
different amounts of RNA. The samples were incubated at ambient
temperature for 5-10 minutes on a Vortex Genie-2 mixer at setting 7
(Scientific Industries).
[0134] Following binding, the particles were centrifuged 14,000 rpm
for 1 minute and the supernatant was removed. The particles were
subsequently washed four times with 1 ml of 70% ethanol. Following
addition of 250 .mu.L of 50 mM Tris, pH 9.0, the particles were
incubated for 5-10 minutes at 56.degree. C. with continuous shaking
(1400 rpm) on an Eppendorf Thermomixer R. The particles were
centrifuged at 14,000 rpm for 1 minute and the supernatant
containing eluted nucleic acid was collected. In order to detect
the presence of residual bound nucleic acid, 250 .mu.L of 100 mM
NaOH was added, the particles were incubated for 5-10 minutes at
56.degree. C. with continuous shaking (1400 rpm) on an Eppendorf
Thermomixer R, and the eluted nucleic acid was collected. The
amount of nucleic acid in each fraction was quantitated
spectrophotometrically.
[0135] The results are shown in FIG. 10. Under the binding
conditions used in this study, 10 mg of the silicon dioxide
particles had a capacity of .about.25 .mu.g for genomic DNA. There
was virtually complete recovery of added genomic DNA at
concentrations below saturation, indicating that the DNA recovery
is efficient. In contrast, RNA recovery was low over the entire
range of added RNA. The low amount of RNA recovered may, in fact,
have been a result of contaminating RNA in the initial
preparation.
Example 10
[0136] In order to test whether selectivity resulted from RNA
degradation due to the alkalinity of the binding buffer, RNA was
incubated either at pH 6 or pH 10 prior to binding to silica under
conditions compatible with RNA binding as follows. Total rat liver
RNA (25 .mu.g in a total volume of 10 .mu.L) was incubated, in
duplicate, in 100 .mu.L of either 50 mM AMP containing pH 10, 4.8 M
NaI, or 50 mM MES, pH 6 containing 4.8 M NaI at ambient temperature
for 5, 10, 15 30 or 60 minutes. At the end of the indicated time, 1
mL of 50 mM MES, pH 6 containing 4.8 M NaI was added to each tube
in order to render conditions compatible with RNA binding to
silica. The reactions were mixed and 10 mg of Sigma silicon dioxide
(prepared as described in Example 1) in a total volume of 50 .mu.L
was added. The samples were incubated at ambient temperature for 10
minutes on a Vortex Genie-2 mixer at setting 7 (Scientific
Industries).
[0137] Following binding, the particles were centrifuged 14,000 rpm
for 1 minute and the supernatant was removed. The particles were
subsequently washed twice with 1 ml of 50 mM MES, pH 6 containing
4.8 M NaI followed by four times with 1 mL of 70% ethanol.
Following addition of 250 .mu.L of 50 mM Tris, pH 9.0, the
particles were incubated for 5-10 minutes at 56.degree. C. with
continuous shaking (1400 rpm) on an Eppendorf Thermomixer R. The
particles were centrifuged at 14,000 rpm for 1 minute and the
supernatant containing eluted nucleic acid was collected. In order
to detect the presence of residual bound nucleic acid, 250 .mu.L of
100 mM NaOH was added, the particles were incubated for 5-10
minutes at 56.degree. C. with continuous shaking (1400 rpm) on an
Eppendorf Thermomixer R, and the eluted nucleic acid was collected.
The amount of nucleic acid in each fraction was quantified
spectrophotometrically.
[0138] As shown in FIG. 16, the half-life of RNA is 86 minutes and
260 minutes at pH 10 and 6, respectively. Based on these
half-lives, only 7.7% and 2.6% of the added RNA would be expected
to degrade during a 10 minute binding incubation at pH 10 and 6,
respectively.
Example 11
[0139] The effect of pH on protein binding was examined. Each
condition was assayed once. Purified bovine serum albumin (1 mg,
100 .mu.l of a 10 mg/ml solution in water, New England Biolabs, Lot
938) was added to 1.5 ml microcentrifuge tubes containing 1 ml of:
(1) 50 mM MES, pH 6.0, 4.75 M NaI; (2) 50 mM Tris-HCl, pH 8, 4.75 M
NaI; or (3) 50 mM AMP, pH 10, 4.75 M NaI. The solid phase (10 mg)
was added to the separate buffered protein solutions and the
mixtures were incubated at ambient temperature with occasional
mixing.
[0140] Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute), and then washed four times
with 0.5 ml of 70% ethanol. Following the last ethanol wash, the
particles were allowed to air dry at ambient temperature for 10
minutes. The bound protein was first eluted with 0.25 ml of 50 mM
Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing and
the eluted protein was collected. Any residual protein bound to the
particles was eluted with 0.25 ml of 0.1 N NaOH at 56.degree. C.
for 5 minutes with constant mixing and the eluted protein was
collected. The recovery of protein was quantified by
spectrophotometry.
[0141] The results are shown in FIG. 11. Like RNA binding, protein
binding to silica was reduced as the pH of the binding buffer was
increased.
Example 12
[0142] The effect of salt composition on DNA and RNA binding to
silica was examined at alkaline pH. Silicon dioxide (which is also
called Sigma silica) was prepared as described in Example 1.
Sheared calf thymus DNA was prepared according to Example 1. Rat
liver total RNA was the source of RNA.
[0143] Each nucleic acid and buffer combination was assayed once.
Either 25 .mu.g (50 .mu.l of a 0.5 mg/ml solution in water) sheared
calf thymus DNA or 25 .mu.g (10 .mu.l of a 2.5 mg/ml solution in
water) rat liver total RNA was added separately to separate 1.5 ml
microcentrifuge tubes containing 0.5 ml of one of the following
buffers: (1) 50 mM AMP, pH 10, 3.65 M lithium chloride; (2) 50 mM
AMP, pH 10, 3.65 M lithium bromide; (3) 50 mM AMP, pH 10, 3.65 M
lithium iodide; (4) 50 mM AMP, pH 10, 3.65 M sodium chloride; (5)
50 mM AMP, pH 10, 3.65 M sodium bromide; (6) 50 mM AMP, pH 10, 3.65
M sodium iodide; (7) 50 mM AMP, pH 10, 3.65 M potassium chloride;
(8) 50 mM AMP, pH 10, 3.65 M potassium bromide; or (9) 50 mM AMP,
pH 10, 3.65 M potassium iodide.
[0144] The solid phase (10 mg) was added to each of the 18 buffered
nucleic acid solutions and the mixtures were incubated for 10
minutes at ambient temperature with occasional mixing. Following
the binding incubation, the particles were centrifuged
(15,800.times.g, 1 minute) and washed four times with 0.5 ml of 70%
ethanol. The bound nucleic acid was first eluted with 0.25 ml of 10
mM Tris, pH 9, for 5 minutes at 56.degree. C. with constant mixing
and the eluted nucleic acid was collected. Any residual nucleic
acid bound to the particles was eluted with 0.25 ml of 0.1 N NaOH
at 56.degree. C. for 5 minutes with constant mixing and the eluted
nucleic acid was collected. The recovery of nucleic acid was
quantified by spectrophotometry.
[0145] The results are shown in FIGS. 12(a)-(f). To facilitate
analysis, the data is shown grouped by cation or anion.
[0146] At pH 10, binding of DNA was influenced by the composition
of the anion. DNA binding to silicon dioxide (Sigma silica)
increased as the monovalent anion radius was increased. The radius
of the monovalent cation influenced the magnitude of that effect.
In contrast, the binding of RNA to silica showed a tendency to
decrease as the anion radius was increased. Decreasing the size of
the cation increased the capacity of the silicon dioxide for both
nucleic acid species. As a result, selectivity for DNA binding
could be improved with certain selections of salt composition.
These results showed that the larger the anion, the greater the
selectivity. There was no correlation between the cation radius and
selectivity. Sodium demonstrated the highest degree of selectivity
for DNA binding.
Example 13
[0147] To demonstrate discrimination of the selective conditions
for isolating DNA, the ability of high concentrations of RNA to
inhibit binding of genomic DNA was examined. Silicon dioxide and
genomic DNA was prepared as described in Example 1. Rat liver total
RNA was the source of RNA.
[0148] Each condition was assayed once. Five separate 10 .mu.l
mixtures of RNA and DNA at RNA:DNA ratios of 1:1 (15 .mu.g:15
.mu.g), 10:1 (15 .mu.g:1 .mu.g), 30:1 (15 .mu.g:0.5 .mu.g), or
100:1 (20 .mu.g:0.2 .mu.g), were added to separate 1.5 ml
microcentrifuge tubes containing 0.45 ml of one of the following
buffers: (1) 50 mM AMP, pH 10, 4.75 M NaI; or (2) 50 mM MES, pH
6.0, 4.75 M NaI. The solid phase (10 mg) was added to each of the
10 buffered nucleic acid solution combinations and the mixtures
were incubated for 10 minutes at ambient temperature with
occasional mixing.
[0149] Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed four times with
0.5 ml of 70% ethanol. Following the last ethanol wash, the
particles were washed once with 0.5 ml of acetone. Once the acetone
was removed, the pellets were dried for 5 minutes at 56.degree. C.
The bound nucleic acid was eluted with 50 .mu.l of 10 mM Tris, pH
9, for 5 minutes at 56.degree. C. with constant mixing and the
eluted nucleic acid was collected.
[0150] The recovery of nucleic acid was visualized by agarose gel
electrophoresis (see FIG. 13). Electrophoresis was performed on 2
.mu.l of the nucleic acid stock mixes or 5 .mu.l of the isolated
eluate. Samples were electrophoresed through a 1% SeaKem.RTM.
(Teknova), 0.5 .mu.g/ml ethidium bromide gel using 1.times.TBE, 0.5
.mu.g/ml ethidium bromide buffer (BIO-RAD), at 8V/cm for 30 minutes
to 1 hour. Ethidium-stained material was visualized and
photographed under short wave ultra-violet light. Molecular weight
markers consisted of an AmpliSize DNA molecular weight standard
(BIO-RAD) and an RNA ladder (GIBCO BRL).
[0151] At pH 10 in NaI, silica was at least 40-fold more selective
for DNA than RNA. At pH 6, there was nearly complete capture of the
added RNA. At the highest level of added RNA (20 .mu.g), no
detectable RNA was recovered at pH 10. In contrast, when 0.5 .mu.g
of genomic DNA was added, detectable DNA was recovered. Therefore,
at pH 10, in the presence of sodium iodide, silica showed greater
that 40-fold greater selectivity for DNA than RNA.
Example 14
[0152] To evaluate whether high levels of RNA would compete with
DNA for binding to a solid phase, high levels of RNA were mixed
with genomic DNA and bound under both nonselective and selective
conditions. Sigma silicon dioxide and sheared genomic DNA was
prepared as described in Example 1. Rat liver total RNA (Biochain
Institute, lot numbers A304057, A305062, or A306073) was the source
of RNA.
[0153] Each nucleic acid and buffer combination was assayed twice.
Either (a) 5 .mu.g sheared calf thymus DNA (10 .mu.l of 0.5 mg/ml)
and 5 .mu.g (2 .mu.l of 2.5 mg/ml) of rat liver total RNA, or (b) 5
.mu.g sheared calf thymus DNA (10 .mu.l of 0.5 mg/ml) and 50 .mu.g
(20 .mu.l of 2.5 mg/ml) of rat liver total RNA was added separately
and mixed in separate 1.5 ml microcentrifuge tubes containing 0.45
ml of one of the following buffers: (1) 50 mM AMP, pH 10, 3.5 M
NaI; or (2) 50 mM MES, pH 6.0, 3.5 M NaI. The solutions were
incubated at ambient temperature for 5 minutes.
[0154] The solid phase (10 mg) was added to each of the four
buffered nucleic acid solution combinations and the mixtures were
incubated for 10 minutes at ambient temperature with occasional
mixing. Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute), and washed three times with
0.5 ml of 70% ethanol. The bound nucleic acid was eluted with 100
.mu.l of 10 mM Tris, pH 9 for 5 minutes at 56.degree. C. with
constant mixing and the eluted nucleic acid was collected.
[0155] The recovery of nucleic acid was visualized by agarose gel
electrophoresis (see FIG. 14). Of the isolation eluate, 10 .mu.l
was electrophoresed through a 0.8% SeaKem agarose gel as described
in Example 13. Ethidium-stained material was visualized and
photographed under a short wave ultra-violet light.
[0156] Even at excess RNA concentrations, little RNA was isolated
under the DNA selective conditions. The data showed that the amount
of RNA contamination was lower than the limit of detection when
using selective conditions. Under nonselective conditions, at high
concentrations of nucleic acids, the limited number of nucleic acid
binding sites may reduce overall DNA recovery.
Example 15
[0157] Genomic DNA was isolated from whole human blood as follows.
Silicon dioxide was prepared as described in Example 1.
[0158] Each condition was assayed once. Either 25 .mu.l or 100
.mu.l of whole blood (Blood Centers of the Pacific) was added
separately to separate 1.5 ml microcentrifuge tubes containing
either 0.25 ml or 0.9 ml (respectively) of one of the following
buffers: (1) 50 mM MES, pH 6.0, 4.75 M NaI, or (2) 50 mM AMP, pH
10, 4.75 M NaI. Nucleic acid was incubated 10 minutes at ambient
temperature in the buffered solution, with occasional mixing.
[0159] The solid phase (10 mg) was added to the four buffered
nucleic acid solution combinations, and the mixtures were incubated
10 minutes at ambient temperature with occasional mixing. Following
the binding incubation, the particles were centrifuged
(15,800.times.g, 1 minute), and washed twice with 0.5 ml of the
binding buffer that had been used for the binding incubation.
Subsequently, the particles were washed four times with 70%
ethanol. The bound nucleic acid was eluted with 50 .mu.l of 10 mM
Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing and
the eluted nucleic acid was collected.
[0160] The recovery of nucleic acid was visualized by agarose gel
electrophoresis (see FIG. 15). Either 2 .mu.l or 10 .mu.l of the
isolation eluate was electrophoresed through 1.0% SeaKem agarose
gel as described in Example 13. Ethidium-stained material was
visualized and photographed under short wave ultra-violet
light.
[0161] When silica was added to whole blood in NaI-containing
buffer at pH 6, the particles clump, suggesting that protein also
adsorbed to the particles. As a result, DNA recovery was poor. In
contrast, the silica particles remained in suspension when added to
blood at pH 10. In the absence of protein adsorption to the
particles, DNA recovery was high.
Example 16
[0162] In certain embodiments, both DNA and RNA can be isolated
from a sample mixture by sequential selective binding (sequential
selective binding). An exemplary sequential selective binding is
shown in FIG. 17. In sequential selective binding, the sample is
contacted with the solid phase under conditions compatible with
selective DNA binding. The solid phase containing the bound DNA is
separated from the unbound material, and the conditions in the
unbound fraction are adjusted to those compatible with RNA binding
to the solid phase. A second solid phase is added to this second
fraction to adsorb the RNA. DNA and RNA are then isolated from the
respective solid phases.
[0163] To evaluate an embodiment of sequential selective binding of
DNA and RNA, a mixture of the two was processed under sequential
selective binding conditions. First, the DNA was bound to the solid
phase under DNA selective conditions and then the unbound RNA
fraction was removed and bound to the same solid phase using
nonselective binding conditions. Sigma silicon dioxide and sheared
genomic DNA was prepared as described in Example 1. Rat liver total
RNA (Biochain Institute) was the source of RNA.
[0164] Each nucleic acid and buffer combination was assayed twice.
5pg DNA, 5 .mu.g RNA and a mixture of 5 .mu.g DNA and 5 .mu.g RNA
were added to separate 1.5 ml microcentrifuge tubes containing 50
mM AMP, pH 10, 3.5 M NaI; 50 mM MES, pH 6, 3.5 M NaI; and 50 mM
AMP, pH 10, 3.5 M NaI respectively. The buffered solutions were
incubated for 5 minutes at ambient temperature. 10 mg of the solid
phase was added to each of the 6 buffered nucleic acid solution
combinations and the mixtures were incubated for 10 minutes at
ambient temperature with occasional mixing.
[0165] Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed 3 times with 0.5
ml of 70% ethanol. Once the ethanol was removed, the particles were
allowed to dry at ambient temperature at least 10 minutes.
[0166] The supernatants from the binding reactions of the mixture
of DNA and RNA were taken and added to separate 1.5 ml
microcentrifuge tubes containing 1 mL of 50 mM MES, pH 6, 3.5 M
NaI. To these tubes was added 10 mg of the solid phase and the
mixtures were incubated for 10 minutes at ambient temperature with
occasional mixing.
[0167] Following the binding incubation, the particles were
centrifuged (15,800.times.g, 1 minute) and washed 3 times with 0.5
mL of 70% ethanol. Once the ethanol was removed, the particles were
allowed to dry at ambient temperature at least 10 minutes.
[0168] The bound nucleic acid was eluted from all particles with
0.1 mL of 10 mM Tris-HCl, pH 9, for 5 minutes at 56.degree. C. with
constant mixing and the eluted nucleic acid collected.
[0169] The recovery of nucleic acid was visualized by agarose gel
electrophoresis. Electrophoresis was performed on 10 .mu.L of the
isolated eluate. Samples were electrophoresed through a 0.8%
SeaKem.RTM., 0.5 .mu.g/mL ethidium bromide gel using 1.times.TBE,
0.5 .mu.g/mL ethidium bromide buffer (BIO-RAD), at 7V/cm for 30
minutes to 1 hour. Ethidium-stained material was visualized and
photographed under short wave ultra-violet light. Molecular weight
markers consisted of High Molecular Weight DNA Markers (GIBCO BRL).
The results are shown in FIG. 18.
Example 17
[0170] In certain embodiments, both DNA and RNA can be isolated
from a sample mixture by first binding both species to a solid
phase and sequentially releasing each nucleic acid type under the
appropriate conditions (sequential specific elution). An exemplary
sequential selective elution is shown in FIG. 17. In sequential
selective elution, the sample is contacted with the solid phase
under conditions that bind both DNA and RNA. Following washes, the
RNA is released under conditions that bind only DNA. The solid
phase is removed and the DNA and RNA are subsequently purified from
both fractions.
[0171] To evaluate an embodiment of sequential selective elution,
mixtures of DNA and RNA were contacted with the solid phase under
conditions which would bind both. The RNA is eluted under
conditions which bind only DNA, and the DNA is subsequently eluted
using a low ionic strength buffer. Sigma silicon dioxide and
sheared genomic DNA was prepared as described in Example 1. Rat
liver total RNA (Biochain Institute) was the source of RNA.
[0172] Each nucleic acid and buffer combination was assayed twice,
and the DNA and RNA was processed as follows. Either (1) 10 .mu.g
sheared calf thymus DNA, or (2) 10 .mu.g rat liver total RNA, or
(3) 10 .mu.g sheared calf thymus DNA and 10 .mu.g rat liver total
RNA was added to separate 1.5 ml microcentrifuge tubes containing
0.2 ml 50 mM MES, pH 6, 3.5 M sodium iodide, and incubated five
minutes at ambient temperature with occasional mixing. The solid
phase (10 mg) was added to each of the buffered nucleic acid
solutions and the mixtures were incubated for 10 minutes at ambient
temperature with mixing.
[0173] Following the binding incubation, the particles--with the
exception of one replicate set of particles with both DNA and RNA
bound--were centrifuged (15,800.times.g, 1 minute) and the bound
nucleic acid was eluted with 0.2 mL 10 mM Tris, pH 9 for 5 minutes
at 56.degree. C. with constant mixing, and the eluted nucleic acid
was collected.
[0174] One replicate set of particles with both DNA and RNA bound
were washed with 0.2 ml 50 mM AMP, pH 10, 3.5 M NaI, for 5 minutes
at ambient temperature with occasional mixing, and the RNA was
eluted and collected. The particles were then washed with 0.2 ml 10
mM Tris, pH 9 for 5 minutes at 56.degree. C. with constant mixing,
and the bound DNA was eluted and collected.
[0175] The recovery of nucleic acid was visualized by agarose gel
electrophoresis. Of the isolation eluate, 10 .mu.L was
electrophoresed through a 1% SeaKem.RTM., 0.5 .mu.g/mL ethidium
bromide gel using 1.times.TBE, 0.5 .mu.g/mL ethidium bromide buffer
(BIO-RAD), at 7V/cm for 30 minutes to 1 hour. Ethidium-stained
material was visualized and photographed under short wave
ultra-violet light. Molecular weight markers consisted of High
Molecular Weight DNA Markers (GIBCO BRL). The results are shown in
FIG. 19.
* * * * *