U.S. patent application number 11/319146 was filed with the patent office on 2006-10-26 for cell concentration and lysate clearance using paramagnetic particles.
This patent application is currently assigned to Promega Corporation. Invention is credited to Rex Bitner, Braeden L. Butler, Jacqui Sankbeil, Craig E. Smith, Douglas H. White.
Application Number | 20060240448 11/319146 |
Document ID | / |
Family ID | 32929931 |
Filed Date | 2006-10-26 |
United States Patent
Application |
20060240448 |
Kind Code |
A1 |
Bitner; Rex ; et
al. |
October 26, 2006 |
Cell concentration and lysate clearance using paramagnetic
particles
Abstract
Methods are disclosed for using paramagnetic particles to
concentrate or harvest cells. Methods are also disclosed for
clearing a solution of disrupted biological material, such as a
lysate of cells or a homogenate of mammalian tissue. Methods are
also disclosed for using paramagnetic particles to isolate target
nucleic acids, such as RNA or DNA, from a solution cleared of
disrupted biological material using the same type or a different
type of paramagnetic particle. Kits are also disclosed for use with
the various methods of the present invention. Nucleic acids
isolated according to the present methods and using the present
kits are suitable for immediate use in downstream processing,
without further purification.
Inventors: |
Bitner; Rex; (Cedarburg,
WI) ; Smith; Craig E.; (Oregon, WI) ; White;
Douglas H.; (Madison, WI) ; Butler; Braeden L.;
(Madison, WI) ; Sankbeil; Jacqui; (Edgerton,
WI) |
Correspondence
Address: |
MICHAEL BEST & FRIEDRICH, LLP
ONE SOUTH PINCKNEY STREET
P O BOX 1806
MADISON
WI
53701
US
|
Assignee: |
Promega Corporation
Madison
WI
|
Family ID: |
32929931 |
Appl. No.: |
11/319146 |
Filed: |
December 27, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
09475958 |
Dec 30, 1999 |
7078224 |
|
|
11319146 |
Dec 27, 2005 |
|
|
|
60134156 |
May 14, 1999 |
|
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|
Current U.S.
Class: |
435/6.12 ;
435/270; 435/6.1; 435/6.16; 536/25.4 |
Current CPC
Class: |
C12N 1/02 20130101; C12N
15/1013 20130101; C12N 15/1006 20130101; C12Q 1/6806 20130101; Y10T
436/143333 20150115 |
Class at
Publication: |
435/006 ;
435/270; 536/025.4 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 1/08 20060101 C12N001/08; C07H 21/04 20060101
C07H021/04 |
Claims
1. A method for preparing a cleared lysate solution comprising: (a)
contacting a material comprising cells with first magnetic
particles under conditions wherein the cells selectively adsorb to
the first magnetic particles; (b) isolating the first magnetic
particles by applying magnetic force; (c) disrupting the cells to
obtain a preparation comprising disrupted biological material and
target nucleic acids; (d) combining the preparation of step (c)
with second magnetic particles under conditions wherein the
disrupted biological material other than the target nucleic acids
selectively adsorbs to the second magnetic particles; and (e)
separating the second magnetic particles of step (d) by applying
magnetic force to obtain a cleared lysate solution.
2. The method of claim 1, wherein the first magnetic particles and
the second magnetic particles have a particle size of about 1 to
about 15 .mu.m.
3. The method of claim 1, wherein the first magnetic particles are
pH dependent ion exchange magnetic particles.
4. The method of claim 3, wherein the pH dependent ion exchange
magnetic particles are selected from the group consisting of
glycidyl-histidine modified silica magnetic particles and
glycidyl-alanine modified silica magnetic particles.
5. The method of claim 1, wherein the first magnetic particles, the
second magnetic particles, or both the first magnetic particles and
the second magnetic particles, are silica magnetic particles.
6. The method of claim 5, wherein the silica magnetic particles
consist essentially of a magnetic core coated with a siliceous
oxide having a hydrous siliceous oxide adsorptive surface.
7. The method of claim 1, wherein the target nucleic acids are
selected from the group consisting of plasmid DNA, total RNA, mRNA,
and genomic DNA.
8. The method of claim 1, wherein the disrupted biological material
is a bacterial cell lysate.
9. The method of claim 1, wherein the disrupted biological material
is a lysate of blood.
10. The method of claim 1, wherein the disrupted biological
material is a homogenate of mammalian tissue.
11. A method of isolating a target nucleic acid from a disrupted
biological material comprising the target nucleic acid, a first
non-target material, and a second non-target material, comprising
the steps of: (a) contacting the disrupted biological material with
first magnetic particles under conditions wherein the first
non-target material adsorbs to the first magnetic particles; (b)
separating the first magnetic particles by applying magnetic force,
forming a preparation comprising the target nucleic acid and the
second non-target material; (c) combining the preparation of step
(b) with second magnetic particles under conditions wherein the
target nucleic acid adsorbs to the second magnetic particles; and
(d) isolating the second magnetic particles from the preparation of
step (c).
12. The method of claim 11, further comprising combining the second
magnetic particles of step (d) with an elution solution under
conditions wherein the target nucleic acid is desorbed from the
second magnetic particles.
13. The method of claim 12, further comprising washing the second
magnetic particles of step (d) with a wash solution and separating
the second magnetic particles from the wash solution by applying
magnetic force prior to combining the second magnetic particles
with the elution solution.
14. The method of claim 11, wherein the first magnetic particles
are selected from the group consisting of silica magnetic particles
and pH dependent ion exchange magnetic particles.
15. The method of claim 11, wherein the first non-target material
comprises cell debris or homogenized tissue and a precipitate,
wherein the precipitate is comprised of material selected from the
group consisting of proteins, non-target nucleic acids, and
lipids.
16. The method of claim 11, wherein the second non-target material
remains in the preparation of step (b) when the target nucleic acid
is adsorbed to the second magnetic particles in step (c).
17. The method of claim 11, wherein the target nucleic acid is
selected from the group consisting of plasmid DNA, total RNA, mRNA,
and genomic DNA.
18. A kit for isolating a target nucleic acid from a disrupted
biological material comprising a first non-target material and the
target nucleic acid, comprising at least one type of magnetic
particle having the capacity to selectively adsorb (1) the first
non-target material under conditions that promote the adsorption of
the first non-target material to the magnetic particles, and (2)
the target nucleic acid under conditions that promote the
adsorption of the target nucleic acid to the magnetic
particles.
19. The kit of claim 18, wherein the at least one type of magnetic
particle is a silica magnetic particle.
20. The kit of claim 18, wherein the at least one type of magnetic
particle is a pH dependent ion exchange magnetic particle.
21. The kit of claim 18, further comprising a wash solution
configured to wash the magnetic particles prior to desorption of
the target nucleic acid from the magnetic particles.
22. The kit of claim 18, further comprising an elution solution
configured to promote desorption of the target nucleic acid from
the magnetic particles.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 09/475,958, filed Dec. 30, 1999, which claims the benefit of
U.S. Provisional Application No. 60/134,156, filed May 14, 1999,
and is a continuation-in-part of U.S. application Ser. No.
09/064,449, filed Apr. 22, 1998, now U.S. Pat. No. 6,194,562. This
application claims priority to each of these applications and
hereby fully incorporates the subject matter of each of these
applications.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] Not applicable.
TECHNICAL FIELD
[0003] This invention relates generally to the use of magnetically
responsive particles, such as magnetically responsive silica gel
particles or magnetically responsive ion exchange particles, to
harvest or to concentrate cells or biological tissue. This
invention also relates to the use of such particles to clear
lysates or homogenates of such cells or tissue. This invention
relates, furthermore, to the use of such particles to isolate
target nucleic acids, such as plasmid DNA, chromosomal DNA, DNA
fragments, total RNA, mRNA, or RNA/DNA hybrids from non-target
material in a cell lysate.
BACKGROUND OF THE INVENTION
[0004] Cells in a liquid culture must be concentrated or harvested
before they can be preserved for later use, stained for direct
analysis, or processed to isolate target specific materials
therefrom. Most cell harvesting and concentration techniques
involve centrifugation, filtration, or a combination of
centrifugation and filtration. (See, e.g., Molecular Cloning,
(1989) ed. by Sambrook et al., pp. 222 and filtration system
reference). Unfortunately, neither filtration nor centrifugation is
amenable to automation. Specifically, neither can be performed at
basic pipettor-diluter robotics stations, such as the Biomec.RTM..
When it becomes necessary to isolate or analyze certain types of
material in the interior of a cell, such as a target nucleic acid
or a protein, the cell membrane must be disrupted and the contents
of the cell released into the solution surrounding the cell. Such
disruption can be accomplished by mechanical means (e.g., by
sonication or by blending in a mixer), by enzymatic digestion (e.g.
by digestion with proteases), or by chemical means (e.g., by
alkaline lysis followed by addition of a neutralization solution).
Whatever means is used to disrupt a cell, the end product, referred
to herein as a lysate solution, consists of the target material and
many contaminants, including cell debris. The lysate solution must
be cleared of as many of the large contaminants as possible before
the target material can be further isolated therefrom. Either or
both of the same two means described above, i.e. centrifugation and
filtration, have been used to clear lysate solutions prior to
further processing. However, for reasons given above, neither means
of clearing a lysate solution is amenable to automation.
[0005] Many different systems of materials and methods have been
developed for use in the isolation of nucleic acids from cleared
lysate solutions. Many such systems are silica based, such as those
which employ controlled pore glass, filters embedded with silica
particles, silica gel particles, resins comprising silica in the
form of diatomaceous earth, glass fibers or mixtures of the above.
Each such silica-based solid phase separation system is configured
to reversibly bind nucleic acid materials when placed in contact
with a medium containing such materials in the presence of
chaotropic agents. The silica-based solid phases are designed to
remain bound to the nucleic acid material while the solid phase is
exposed to an external force such as centrifugation or vacuum
filtration to separate the matrix and nucleic acid material bound
thereto from the remaining media components. The nucleic acid
material is then eluted from the solid phase by exposing the solid
phase to an elution solution, such as water or an elution buffer.
Numerous commercial sources offer silica-based resins designed for
use in centrifugation and/or filtration isolation systems, e.g.
Wizard.RTM. DNA purification systems products from Promega
Corporation (Madison, Wis., U.S.A.), or the QiaPrep.RTM. DNA
isolation systems from Qiagen Corp. (Chatsworth, Calif., U.S.A.).
Unfortunately, the type of silica-based solid phases described
above all require one use centrifugation or filtration to perform
the various isolation steps in each method, limiting the utility of
such solid phases in automated systems.
[0006] Magnetically responsive solid phases, such as paramagnetic
or superparamagnetic particles, offer an advantage not offered by
any of the silica-based solid phases described above. Such
particles could be separated from a solution by turning on and off
a magnetic force field, or by moving a container on to and off of a
magnetic separator. Such activities would be readily adaptable to
automation.
[0007] Magnetically responsive particles have been developed for
use in the isolation of nucleic acids. Such particles generally
fall into either of two categories, those designed to reversibly
bind nucleic acid materials directly, and those designed to
reversibly bind nucleic acid materials through an intermediary. For
an example of particles of the first type, see silica based porous
particles designed to reversibly bind directly to DNA, such as
MagneSil.TM. particles from Promega, or BioMag.RTM. magnetic
particles from PerSeptive Biosystems. For examples of particles and
systems of the second type designed to reversibly bind one
particular type of nucleic acid (mRNA), see the PolyATract.RTM.
Series 9600.TM. mRNA Isolation System from Promega Corporation
(Madison, Wis., U.S.A.); or the streptavidin coated microsphere
particles from Bangs Laboratories (Carmel, Ind., U.S.A.). Both of
these systems employ magnetically responsive particles with
streptavidin subunits covalently attached thereto, and biotin with
an oligo(dT) moiety covalently attached thereto. The
biotin-oligo(dT) molecules act as intermediaries, hybridizing to
the poly(A) tail of mRNA molecules when placed into contact
therewith, then binding to the streptavidin on the particles. The
mRNA molecules are then released in water.
[0008] Indirect binding magnetic separation systems for nucleic
acid isolation or separation require at least three components,
i.e. magnetic particles, an intermediary, and a medium containing
the nucleic acid material of interest. The intermediary/nucleic
acid hybridization reaction and intermediary/particle binding
reaction often require different solution and/or temperature
reaction conditions from one another. Each additional component or
solution used in the nucleic acid isolation procedure adds to the
risk of contamination of the isolated end product by nucleases,
metals, and other deleterious substances.
[0009] Various types of magnetically responsive silica based
particles have been developed for use as solid phases in direct or
indirect nucleic acid binding isolation methods. One such particle
type is a magnetically responsive glass bead, preferably of a
controlled pore size. See, e.g. Magnetic Porous Glass (MPG)
particles from CPG, Inc. (Lincoln Park, N.J., U.S.A.); or porous
magnetic glass particles described in U.S. Pat. Nos. 4,395,271;
4,233,169; or 4,297,337. Nucleic acid material tends to bind very
tightly to glass, however, so that it can be difficult to remove
once bound thereto. Therefore, elution efficiencies from magnetic
glass particles tend to be low compared to elution efficiencies
from particles containing lower amounts of a nucleic acid binding
material such as silica.
[0010] Another type of magnetically responsive particle designed
for use as a solid phase in direct binding and isolation of nucleic
acids, particularly DNA, is a particle comprised of agarose
embedded with smaller ferromagnetic particles and coated with
glass, e.g. U.S. Pat. No. 5,395,498. Yet another type of
magnetically responsive particle designed for direct binding and
isolation of nucleic acids is produced by incorporating magnetic
materials into the matrix of polymeric silicon dioxide compounds,
e.g. German Patent Application No. DE 43 07 262. The latter two
types of magnetic particles, the agarose particle and the polymeric
silicon dioxide matrix, tend to leach iron into a medium under the
conditions required to bind nucleic acid materials directly to each
such magnetic particle. It is also difficult to produce such
particles with a sufficiently uniform and concentrated magnetic
capacity to ensure rapid and efficient isolation of nucleic acid
materials bound thereto.
[0011] Magnetically responsive beads designed for use in the
isolation of target polymers, such as nucleic acids, and methods
for their use therein are described in U.S. Pat. No. 5,681,946 and
in International Publication No. WO 91/12079. These last beads are
designed to become nonspecifically associated with the target
polymer, only after the target polymer is precipitated out of a
solution comprising the target polymer and the beads. Magnetic
force is used to isolate the beads and polymer associated therewith
from the solution. The magnetically responsive beads recommended
for use in this last system are "finely divided magnetizable
material encapsulated in organic polymer." ('946 Patent, col. 2,
line 53).
[0012] A variety of solid phases have also been developed with ion
exchange ligands capable of exchanging with nucleic acids. However,
such systems are generally designed for use as a solid phase of a
liquid chromatography system, for use in a filtration system, or
for use with centrifugation to separate the solid phase from
various solutions. Such systems range in complexity from a single
species of ligand covalently attached to the surface of a filter,
as in DEAE modified filters (e.g., CONCERT.RTM. isolation system,
Life Technology Inc., Gaithersburg, Md., U.S.A.), to a column
containing two different solid phases separated by a porous divider
(e.g., U.S. Pat. No. 5,660,984), to a chromatography resin with pH
dependent ionizable ligands covalently attached thereto (e.g., U.S.
Pat. No. 5,652,348).
[0013] Materials and methods are needed which enable one to
automate as many steps as possible to quickly and efficiently
isolate target nucleic acids from cells or mammalian tissue.
Specifically, methods and materials are needed for the
concentration or harvesting of cells, for the clearing of solutions
of disrupted cells or tissue, and for the isolation of target
nucleic acids from such cleared solutions, wherein labor-intensive
steps such as filtration or centrifugation are not required. The
present invention addresses each of these needs. Nucleic acids
isolated according to the present method can be used in a variety
of applications, including restriction digestion and
sequencing.
BRIEF SUMMARY OF THE INVENTION
[0014] In the methods of the present invention, magnetic particles
are used to process biological material. In one embodiment, the
present invention is a method of concentrating or harvesting cells
comprising the steps of: (a) combining a solution with cells
contained therein, such as an overnight culture of bacteria in a
growth medium or white cells in whole blood with magnetic particles
under conditions wherein the cells form a complex with the magnetic
particles; and (b) isolating the magnetic particle/cell complex
from the solution by application of magnetic force, e.g., by means
of a magnet.
[0015] In another embodiment, the present invention is a method of
clearing disrupted biological material, such as a cell lysate or a
homogenate of mammalian tissue, comprising the steps of: (a)
providing a solution comprising a disrupted biological material,
such as a cell lysate or homogenized tissue; (b) combining the
solution with magnetic particles under conditions wherein the
disrupted biological material forms a complex with the magnetic
particles; and (c) isolating the complex from the solution by
application of magnetic force.
[0016] In yet another embodiment, the present invention is a method
of isolating a target nucleic acid from a solution of disrupted
biological material, comprising the target nucleic acid, a first
non-target material, and a second non-target material, comprising
the steps of: (a) combining a solution of the disrupted biological
material with first magnetic particles under conditions wherein the
first non-target material forms a first complex with the first
magnetic particles; (b) separating the first complex from the
solution of disrupted biological material by application of
magnetic force, forming a cleared solution comprising the target
nucleic acid and the second non-target material; (c) combining the
cleared solution with second magnetic particles under conditions
wherein the target nucleic acid adsorbs to the second magnetic
particles, forming a second complex; (d) isolating the second
complex from the cleared solution; (e) washing the second complex
by combining the second complex with a wash solution and separating
the second complex from the wash solution by magnetic force; and
(f) combining the washed second complex with an elution solution,
under conditions wherein the target material is desorbed from the
second magnetic particles.
[0017] In yet another embodiment, the present invention also
consists of kits with at least one type of magnetic particle and at
least one solution needed to practice one or more of the methods of
the invention, described above. In one such embodiment, the present
invention is a kit comprising: (a) a first container of first
magnetic particles with the capacity to form a first complex with
first non-target material in a first solution of disrupted
biological material comprising the first non-target material and
the target nucleic acid; and (b) a second container of second
magnetic particles with the capacity to form a second complex with
the target nucleic acid, under solution conditions designed to
promote the specific adsorption of the target nucleic acid to the
second magnetic particles.
[0018] The methods and materials of the present invention can be
used to isolate target nucleic acids including, but not limited to
plasmid DNA, total RNA, mRNA, RNA/DNA hybrids, amplified nucleic
acids, and genomic DNA from a variety of contaminants, including
but not limited to agarose and components of a bacteria, animal
tissue, blood cells, and non-target nucleic acids. Applications of
the methods and compositions of the present invention to isolate
nucleic acids from a variety of different media will become
apparent from the detailed description of the invention below.
Those skilled in the art of this invention will appreciate that the
detailed description of the invention is meant to be exemplary only
and should not be viewed as limiting the scope of the
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 is a photograph of samples of plasmid DNA isolated
with MagneSil.TM. particles (Promega) or varying amounts of
Mag-IE-glycidyl-histidine particles, fractionated by gel
electrophoresis and visualized by staining with ethidium bromide,
as described in Example 6.
[0020] FIG. 2 is a photograph of samples of plasmid DNA isolated
from varying amounts of a culture of transformants of E.coli
DH5.alpha. cells using centrifugation ("Spin") on MagneSil.TM.
particles (Promega Corp.) ("Mag"), followed by fractionation by gel
electrophoresis on a short run gel, and visualization by staining
with ethidium bromide, as described in Example 7.
[0021] FIG. 3 is a photograph of the same gel shown in FIG. 2, shot
after electrophoresis was continued for a longer period of
time.
[0022] FIG. 4 is a photograph of samples of DNA and RNA isolated
from a mouse liver homogenate, using MagIE-glycidyl-histidine
particles, as described in Example 9, after fractionation by gel
electrophoresis and visualization by staining with ethidium
bromide.
[0023] FIG. 5 is a photograph of samples of DNA and RNA isolated
from mouse spleen (lanes 2-5) and kidney (lanes 7-9), using
MagIE-glycidyl-histidine particles, as described in Example 9,
after the samples were fractionated by gel electrophoresis and
visualized by staining with ethidium bromide, as described in
Example 9.
[0024] FIG. 6 is a photograph of mouse liver RNA and DNA, after
digestion with DNase, fractionation by gel electrophoresis, and
visualization by staining with ethidium bromide.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention will now be described in detail, in
part, by reference to the following definitions.
[0026] The term "solid phase" is used herein in a standard
chromatographic sense, to refer to an insoluble, usually rigid,
matrix or stationary phase which interacts with a solute, in this
case a tissue or cell or target nucleic acid, in a solute mixture.
In the methods and kits of the present invention magnetic particles
function as a solid phase when added to various solute
mixtures.
[0027] The term "surface", as used herein, refers to the portion of
the support material of a solid phase which comes into direct
contact with a solution when the solid phase is combined
therewith.
[0028] The term "silica gel" as used herein refers to
chromatography grade silica gel, a substance which is commercially
available from a number of different sources. Silica gel is most
commonly prepared by acidifying a solution containing silicate,
e.g. by acidifying sodium silicate to a pH of less than 11, and
then allowing the acidified solution to gel. See, e.g. silica
preparation discussion in Kurt-Othmer Encyclopedia of Chemical
Technology, Vol. 21, 4th ed., Mary Howe-Grant, ed., John Wiley
& Sons, pub., 1997, p. 1021.
[0029] As used herein, the term "silica magnetic particles" refers
to silica based solid phases which are further comprised of
materials which have no magnetic field but which form a magnetic
dipole when exposed to a magnetic field, i.e., materials capable of
being magnetized in the presence of a magnetic field but which are
not themselves magnetic in the absence of such a field.
[0030] The term "magnetic", as used herein refers to temporarily
magnetic materials, such as ferrimagnetic or ferromagnetic
materials. The term encompasses paramagnetic and superparamagnetic
materials.
[0031] The term "magnetic particle" refers to a matrix comprising a
core of paramagnetic or superparamagnetc materials and a solid
phase capable of forming a complex with a solute of interest.
[0032] The term "silica magnetic particles", as used herein refers
to paramagnetic particles comprising a superparamagnetic core
coated with siliceous oxide, having a hydrous siliceous oxide
adsorptive surface (i.e. a surface characterized by the presence of
silanol groups).
[0033] The term "magnetic ion exchange particles", as used herein,
refers to paramagnetic particles with ion exchange ligands
covalently attached thereto.
[0034] The term "pH dependent ion exchange magnetic particles", as
used herein, refers to magnetic particles with a plurality of ion
exchange ligands covalently attached thereto, which can act as
cation exchangers at one pH and as anion exchangers at another pH.
Such magnetic particles are particularly well suited for use in the
methods and kits of the present invention, as their binding
capacity to different substrates can be adjusted merely by varying
the pH or salt conditions in a solution.
[0035] The term "pH dependent ion exchange silica magnetic
particles", as used herein, refers to silica magnetic particles
with a plurality of ion exchange ligands covalently attached
thereto, which can act as cation exchangers at one pH and as anion
exchangers at another pH. Such magnetic particles are particularly
well suited for use in the methods and kits of the present
invention, because substrates can selectively adsorb to the hydrous
siliceous oxide adsorptive surface of the particle through
hydrophobic interactions, to the ion exchange ligands through ion
exchange, or to both the surface and ion exchange ligands,
depending upon solution conditions.
[0036] The term "nucleic acid" as used herein refers to any DNA or
RNA molecule or a DNA/RNA hybrid molecule. The term includes
plasmid DNA, amplified DNA or RNA fragments, total RNA, mRNA,
genomic DNA, and chromosomal DNA.
[0037] The term "target nucleic acid" as used herein refers to any
particular species of nucleic acid to be isolated using magnetic
particles according to a method of the present invention. The
target nucleic acid is preferably at least 20 nucleotides long,
more preferably at least 100 nucleotides long, and most preferably
at least 1,000 nucleotides long.
[0038] The methods and kits of the present invention can be used to
harvest or concentrate cells, to clear a solution of disrupted
biological material, and/or to isolate a target nucleic acid from a
solution, preferably from a solution of cleared disrupted
biological material. In at least one step of each such method, a
complex is formed in a solution between a solute and magnetic
particles. The resulting complex is then isolated from or removed
from the solution by the application of magnetic force. Magnetic
particles suitable for use in any given step of the methods and
kits of the present invention have the capacity to form a complex
with the solute of interest in that particular step of the
method.
[0039] The solute is the type of material to be isolated from or
removed from a solution, using magnetic particles, according to a
method of the present invention. Cells to be concentrated or
harvested are the solute in the harvesting method of the present
invention. Disrupted biological material is the solute in the
lysate or homogenate clearing method of the invention. A target
nucleic acid is the solute when magnetic particles are used to
isolate the target nucleic acid from any solution comprising the
target nucleic acid and other material, such as a cleared lysate or
homogenate solution.
[0040] In one aspect of the methods of the present invention, cells
are harvested or concentrated using magnetic particles which can
form a complex with the cells, under solution conditions designed
to promote the formation of the complex. Silica magnetic particles
and pH dependent ion exchange magnetic particles are both suitable
for use in harvesting or concentrating cells according to the
method of the present invention. However, one of ordinary skill in
the art could readily select other suitable magnetic particles for
use in this particular embodiment of the invention.
[0041] Conditions which promote the formation of a magnetic
particle/solute complex vary, depending upon the nature of the
solute and on the characteristics of the solid phase component of
the magnetic particle. For example, when the magnetic particles are
ion exchange magnetic particles or pH dependent ion exchange
particles, the complex is preferably formed as a result of ion
exchange between the solute and ion exchange ligands at the surface
of the particles. In order to promote such ion exchange
interaction, there must be at least some salt present in the
solution to promote ion exchange with the solute, and the pH of the
solution must be within the range wherein the ion exchange ligand
has a charge appropriate to exchange with the solute. When the
magnetic particles are silica magnetic particles, the complex is
preferably formed as a result of hydrophobic interactions between
the solute and particles. When the magnetic particles are pH
dependent ion exchange silica magnetic particles, the complex can
be formed as a result of hydrophobic interactions between the
solute and the siliceous oxide surface of the particles, as a
result of ion exchange between the solute and the ion exchange
ligands, or as a result of a combination of the two types of
interactions. Preferred salt, pH, and other solution conditions to
be used to promote formation of a complex with any given preferred
substrate isolated according to the present methods or using the
present kits are described below.
[0042] When the solute is intact cells, the complex is preferably
formed in the presence of a low molecular weight alcohol, such as
ethanol or isopropanol.
[0043] When the solute is disrupted biological material, such as
one finds in a cell lysate or tissue homogenate, and the magnetic
particles are silica-based particles, the magnetic particle/solute
complex is preferably formed in a solution which does not contain
any more than trace amounts of alcohol or of chaotropic salts. Both
alcohol and chaotropic salts, such as guanidine thiocyanate or
guanidine isothiocyanate, promote adsorption of nucleic acid
materials to such particles. It is contemplated, however, that one
could practice the present method of cell lysate clearance in the
presence of alcohol or chaotropic salts if the concentration of
magnetic particles in a homogenate or lysate solution were low
enough to clear the solution, but not high enough to adhere to a
significant amount of the target nucleic acid in the solution.
[0044] When the solute is a target nucleic acid, formation of the
complex is preferably done in the presence of at least one agent
known to promote reversible adsorption of the target nucleic acid
to the magnetic particles. The reversible adsorption reaction is
preferably done through specific adsorption between the target
nucleic acid and magnetic particles, leaving non-target material in
solution. For example, when the target nucleic acid is plasmid DNA
being isolated from a cleared lysate solution, the plasmid DNA is
combined with magnetic particles under conditions wherein the
plasmid DNA forms a complex therewith while non-target materials,
such as proteins, lipids, and chromosomal DNA remain in solution.
When the magnetic particle is an ion exchange magnetic particle,
the complex is formed in the presence of a counterion and in a
solution with a pH at which the ion exchange ligands have the
capacity to exchange with the target nucleic acid. When the
magnetic particles are silica magnetic particles, formation of the
complex is preferably done in the presence of an agent selected
from the group consisting of a low molecular weight alcohol, a high
concentration of a non-chaotropic salt, and a chaotropic salt, or a
combination of any of the above. For methods of adsorption and
desorption of target nucleic acids to silica magnetic particles,
which are suitable for use in the present invention, see
international patent application number PCT/US98/01149 for METHODS
OF ISOLATING BIOLOGICAL TARGET MATERIALS USING SILICA MAGNETIC
PARTICLES, published as WO 98/31840, incorporated by reference
herein.
[0045] The solid phase of the magnetic particles used in the
present methods can be made of any common support material,
including soft gel supports such as agarose, polyacrylamide, or
cellulose, or hard support material such as polystyrene, latex,
methacrylate, or silica.
[0046] When the solid phase support material is silica, it is
preferably in the form of silica gel, siliceous oxide, solid silica
such as glass or diatomaceous earth, or a mixture of two or more of
the above. Silica based solid phases suitable for use in the pH
dependent ion exchange matrixes of the present invention include
the mixture of silica gel and glass described in U.S. Pat No.
5,658,548, the silica magnetic particles described in PCT
Publication Number WO 98/31840, and solid phases sold by Promega
Corporation for use in plasmid DNA isolation, i.e. Wizard.RTM.
Minipreps DNA Purification Resin. Silica gel particles are
particularly preferred for use as the solid phase in the pH
dependent ion exchange matrix and methods of the present invention.
Silica gel particles are stable at much higher pressures than solid
phases made from soft gel support material, making the silica gel
solid phases suitable for HPLC as well as LC and batch separation
applications.
[0047] Silica magnetic particles can be used to concentrate cells,
clear lysates, or isolate target nucleic acids according to the
methods the present invention. When silica magnetic particles are
employed, the silica-based surface material of the particle
specifically interacts with the various solutes isolated or removed
therewith.
[0048] When the silica magnetic particles have ion exchange ligands
covalently attached thereto, the silica-based surface material acts
primarily as a solid support for the ion exchange ligands, which
enable the particles to form complexes with the various solutes to
be isolated or removed from any given solution. When used to
isolate a target nucleic acid, the ion exchange ligands are
preferably capable of forming a complex with the target nucleic
acid by exchanging therewith at one pH, and of releasing the target
nucleic acid at another pH. The most preferred ion exchange ligands
are ones which complex with the target nucleic acid at a pH which
is lower than a neutral pH, and which release the target nucleic
acid at about a neutral pH and in low salt conditions, so the
target nucleic acid released therein can used immediately, without
concentration or further isolation. Such preferred ion exchange
ligands and pH dependent ion exchange matricies which incorporate
such ligands are described in U.S. patent application Ser. No.
09/312,172, now U.S. Pat. No. 6,310,199, for an invention titled pH
DEPENDENT ION EXCHANGE MATRIX AND METHOD OF USE IN THE ISOLATION OF
NUCLEIC ACIDS, incorporated by reference herein, an application
filed concurrently with the provisional patent application on which
the present non-provisional patent application is based.
[0049] When the solid support component of the pH dependent ion
exchange matrix is a silica magnetic particle, the size of the
particle is preferably selected as follows. Smaller silica magnetic
particles provide more surface area (on a per weight unit basis)
for covalent attachment to the plurality of ion exchange ligands,
but smaller particles are limited in the amount of magnetic
material which can be incorporated into such particles compared to
larger particles. The median particle size of the silica magnetic
particles used in a particularly preferred embodiment of the
present invention is about 1 to 15 .mu.m, more preferably about 3
to 10 .mu.m, and most preferably about 4 to 7 .mu.m. The particle
size distribution may also be varied. However, a relatively narrow
monodal particle size distribution is preferred. The monodal
particle size distribution is preferably such that about 80% by
weight of the particles are within a 10 .mu.m range of the median
particle size, more preferably within an 8 .mu.m range, and most
preferably within a 6 .mu.m range.
[0050] The magnetic particles of the present invention can be
porous or non-porous. When the magnetic particles are porous, the
pores are preferably of a controlled size range sufficiently large
to admit the target nucleic acid material into the interior of the
solid phase particle, and to bind to functional groups or silica on
the interior surface of the pores. When the magnetic particles are
porous silica magnetic particles, the total pore volume of each
silica magnetic particle, as measured by nitrogen BET method, is
preferably at least about 0.2 ml/g of particle mass. The total pore
volume of porous silica magnetic particles particularly preferred
for use as components of the pH dependent ion exchange matrix of
the present invention, as measured by nitrogen BET, is preferably
at least about 50% of the pore volume is contained in pores having
a diameter of 600 .ANG. or greater.
[0051] Silica magnetic particles may contain substances, such as
transition metals or volatile organics, which could adversely
affect the utility of target nucleic acids substantially
contaminated with such substances. Specifically, such contaminants
could affect downstream processing, analysis, and/or use of the
such materials, for example, by inhibiting enzyme activity or
nicking or degrading the target nucleic acids isolated therewith.
Any such substances present in the silica magnetic particles used
in the present invention are preferably present in a form which
does not readily leach out of the particle and into the isolated
biological target material produced according to the methods of the
present invention. Iron is one such undesirable at least one
contaminant, particularly when the biological target material is a
target nucleic acid.
[0052] Iron, in the form of magnetite, is present at the core of
particularly preferred forms of silica magnetic particles used as
the solid phase component of the pH dependent ion exchange matrixes
of the present invention. Iron has a broad absorption peak between
260 and 270 nanometers (nm). Target nucleic acids have a peak
absorption at about 260 nm, so iron contamination in a target
nucleic acid sample can adversely affect the accuracy of the
results of quantitative spectrophotometric analysis of such
samples. Any iron containing silica magnetic particles used to
isolate target nucleic acids using the present invention preferably
do not produce isolated target nucleic acid material sufficiently
contaminated with iron for the iron to interfere with
spectrophotometric analysis of the material at or around 260
nm.
[0053] The most preferred silica magnetic particles used in the
matrixes and methods of the present invention, siliceous oxide
coated silica magnetic particles, leach no more than 50 ppm, more
preferably no more than 10 ppm, and most preferably no more than 5
ppm of transition metals when assayed as follows. Specifically, the
particles are assayed as follows: 0.33 g of the particles (oven
dried at 110.degree. C.) are combined with 20 ml. of 1N HCl aqueous
solution (using deionized water). The resulting mixture is then
agitated only to disperse the particles. After about 15 minutes
total contact time, a portion of the liquid from the mixture is
then analyzed for metals content. Any conventional elemental
analysis technique may be employed to quantify the amount of
transition metal in the resulting liquid, but inductively coupled
plasma spectroscopy (ICP) is preferred.
[0054] At least two commercial silica magnetic particles are
particularly preferred for use in the present invention,
BioMag.RTM. Magnetic Particles from PerSeptive Biosystems, and the
MagneSil.TM. Particles available from Promega Corporation (Madison,
Wis.). Any source of magnetic force sufficiently strong to separate
the silica magnetic particles from a solution would be suitable for
use in the nucleic acid isolation methods of the present invention.
However, the magnetic force is preferably provided in the form of a
magnetic separation stand, such as one of the MagneSphere.RTM.
Technology Magnetic Separation Stands (cat. no.'s Z5331 to 3, or
Z5341 to 3) from Promega Corporation.
[0055] When magnetic particles are used to both clear a solution of
disrupted biological material and to isolate a target nucleic acid
therefrom, one can use the same type of particles or a different
type of particles for clearing and isolation. For purposes of this
disclosure, and to emphasize the flexibility in the invention, the
particles used to clear the solution of disrupted biological
material are referred to as first magnetic particles, while the
particles used to isolate the target nucleic acid are referred to
as second magnetic particles.
[0056] When the target nucleic acid is plasmid DNA, the second
magnetic particles can be added directly to cleared lysate of
bacteria transformed with the plasmid DNA, wherein the lysate is
formed by alkaline lysis followed by clearance using first magnetic
particles as described above. Alkaline lysis procedures suitable
for use in the present invention can be found in Sambrook et al,
Molecular Cloning, Vol. 1, 2.sup.nd ed. (pub. 1989 by Cold Spring
Harbor Laboratory Press), pp. 1.25-1.28, and in Technical Bulletin
No's 202, 225, and 259 (Promega Corp.). When the second silica
magnetic particle is a pH dependent ion exchange particle, plasmid
DNA from a lysate solution prepared as described above will form a
complex with the pH dependent ion exchange particles upon
combination therewith, provided the overall charge of the matrix is
positive, and provided the charge density is sufficiently high to
enable to plasmid DNA to participate in anion exchange with the ion
exchange ligands of the matrix at a first pH. Once adsorbed to the
matrix to form a complex, the complex can be washed in a wash
solution with buffer and salt solution conditions designed to
ensure the plasmid DNA remains adsorbed to the matrix throughout
any such washing steps, while removing at least one contaminant.
Finally, the plasmid DNA is eluted from the complex by combining
the complex with an elution buffer having a second pH above that of
the lysate and wash solutions, wherein the second pH is
sufficiently high to promote desorption of the plasmid DNA from the
matrix.
[0057] The materials and methods of the present invention can be
used to isolate genomic DNA from living tissue, including but not
limited to blood, semen, vaginal cells, hair, buccal tissue,
saliva, tissue culture cells, plant cells, placental cells, or
fetal cells present in amniotic fluid and mixtures of body fluids.
When the target nucleic acid is genomic DNA, it is necessary to
disrupt the tissue to release the target genomic DNA from
association with other material in the tissue, so the target
genomic DNA can adhere to the pH dependent ion exchange matrix in
the presence of a solution at the first pH. The resulting complex
of matrix and genomic DNA is separated from the disrupted tissue,
and washed to remove additional contaminants (if necessary). The
genomic DNA is then eluted from the complex by combining the
complex with an elution solution having a second pH which is higher
than the first pH.
[0058] The following, non-limiting examples teach various
embodiments of the invention. In the examples, and elsewhere in the
specification and claims, volumes and concentrations are at room
temperature unless specified otherwise. The magnetic silica
particles used in the examples below were all either porous or
nonporous MagneSil.TM. particles having the general preferred
dimensions and siliceous oxide coating described as preferred
above. More specifically, the porous MagneSil.TM. Particles used in
the Examples below were taken from either of two batches of
particles having the following characteristics: (1) a surface area
of 55 m.sup.2/g, pore volume of 0.181 ml/g for particles of <600
.ANG. diameter, pore volume of 0.163 ml/g for particles of >600
.ANG. diameter, median particle size of 5.3 .mu.m, and iron leach
of 2.8 ppm when assayed as described herein above using ICP; or (2)
a surface area of 49 m.sup.2/g, pore volume of 0.160 ml/g (<600
.ANG. diameter), pore volume of 0.163 ml/g (>600 .ANG.
diameter), median particle size of 5.5 .mu.m, and iron leach of 2.0
ppm.
[0059] One skilled in the art of the present invention will be able
to use the teachings of the present disclosure to select and use
magnetic particles other than the silica-based magnetic particles
and ion exchange magnetic particles used to illustrate the methods
and kits of the invention in the Examples, below.
[0060] The Examples should not be construed as limiting the scope
of the present invention. Other magnetic silica particles and their
use in the present method to concentrate cells, to clear solutions
of disrupted biological material, and to isolate target nucleic
acids from disrupted biological material will be apparent to those
skilled in the art of chromatographic separations and molecular
biology.
EXAMPLES
[0061] The following examples are given to illustrate various
aspects of the invention, without limiting the scope thereof:
Example 1
Gel Electrophoresis
[0062] Samples of target nucleic acids isolated according to
procedures described in Examples below were analyzed for
contamination with non-target nucleic acids, and for size as
follows. The samples were fractionated on an agarose gel of
appropriate density (e.g., a 1.0% agarose gel was used to analyze
plasmid DNA, while a 1.5% agarose gel was used to analyze RNA). The
fractionated nucleic acid was visualized using a fluorescent label
or by dying the gel with a DNA sensitive stain, such as ethidium
bromide or silver staining. The resulting fractionated, visualized
nucleic acid was either photographed or visualized using a
fluorimager and the resulting image printed out using a laser
printer.
[0063] In some cases, size standards were fractionated on the same
gel as the target nucleic acid, and used to determine the
approximate size of the target nucleic acid. In every case where a
gel assay was done, the photograph or fluorimage of the
fractionated nucleic acid was inspected for contamination by
non-target nucleic acids. For example, images of fractionated
samples of plasmid DNA were inspected for RNA, which runs
considerably faster than DNA on the same gel, and for chromosomal
DNA, which runs considerably slower than plasmid DNA on the same
gel. Images of isolated plasmid DNA were also inspected to
determine whether most of the plasmid DNA shown in the image is
intact, supercoiled plasmid DNA.
Example 2
Absorption Spectrophotometry
[0064] Samples of target nucleic acids isolated from various media,
as described below, were also analyzed using absorption
spectrophotometry. Absorption measurements were taken at
wavelengths of 260, 280, and 230 nanometers (nm).
A.sub.260/A.sub.280 absorption ratios were computed from the
measurements. An A.sub.260/A.sub.280 of greater than or equal to
1.80 was interpreted to indicate the sample analyzed therein was
relatively free of protein contamination. The concentration of
nucleic acid in each sample was determined from the absorption
reading at 260 nm (A.sub.260).
Example 3
Synthesis of Glycidyl-Histidine and Glycidyl-Alanine Silica
Magnetic Ion Exchange Particles
[0065] Various two different pH dependent ion exchange ligands,
glycidyl-histidine and glycidyl-alanine, were attached to porous
silica magnetic particles according to the following procedure. The
silica magnetic pH dependent ion exchange particles synthesized as
described herein were used to concentrate cells, clear lysates, or
isolate target nucleic acids, as described in subsequent Examples,
below.
A. Preparation of Glycidyl Modified Silica Magnetic Particles
[0066] 1. Silica magnetic particles were activated by heating under
vacuum at 110.degree. C. overnight.
[0067] 2. 10 g of the activated particles were suspended in 100 ml
of toluene in a flask, and 3.2 ml of
3-glycidylpropyl-trimethoxysilane was added thereto.
[0068] 3. The flask containing the mixture was fitted with a
condenser and the reaction was refluxed for 5 hr. After cooling to
room temperature, the reaction mixture sat for 48 hr at room
temperature.
[0069] 4. The reaction mixture was then filtered and the retentate,
including glycidyl-modified silica magnetic particles produced in
the reflux reaction, were washed with toluene (2.times.100 ml),
hexanes (2.times.100 ml) and ethyl ether (1.times.150 ml). The
washed product was then left to dry in the air.
[0070] 5. A small portion of the product was further dried in a
110.degree. C. oven and submitted for elemental analysis. The
results (%C 0.75; %H 0.58) are consistent with glycidyl
modification of silica gel particles, as illustrated in Formula
(I), below. The wavy line in this and other formulae depicted
herein and in the remaining Examples below represents the surface
of a solid phase, a porous silica magnetic particle in this
particular Example. ##STR1## wherein, R is --OH, OCH.sub.3, or 13
OCH.sub.2CH.sub.3.
[0071] 6. The glycidyl-modified silica magnetic particles produced
as described above were then further modified by the linkage of an
amino acid, such as histidine, alanine, or cysteine to the
particles, by reaction with the terminal ring of the glycidyl
moiety, as described below.
B. Synthesis of Glycidyl-Histidine Modified Silica Magnetic
Particles
[0072] 1. 2.0 g. of D,L-histidine was dissolved in a mixture of 20
ml of tetrahydrofuran and 20 ml of water by heating the solution to
reflux.
[0073] 2. To this solution, 2 g of glycidyl-modified silica
magnetic particles was added and the resulting suspension was
refluxed overnight (18 hr).
[0074] 3. After cooling to room temperature the reaction mixture
was filtered, and the retentate, which included glycidyl-histidine
modified silica magnetic particles, was washed once with 100 ml of
acetone, three times with 150 ml of water, and once with 150 ml of
ether. The solid was air dried.
[0075] 4. A small portion of the dried solid from step 3 was
further dried at 110.degree. C. and submitted for elemental
analysis. Results: %C 1.35; %H 0.68; %N 0.50. This results are
consistent with glycidyl-histidine linkage, such as is as shown in
Figure (II), below: ##STR2## wherein, R is --OH, OCH.sub.3, or
--OCH.sub.2CH.sub.3.
C. Synthesis of Glycidyl-Alanine Modified Silica Magnetic
Particles
[0076] 1. 3-(3-pyridyl)-D-alanine (1 g) was dissolved in 20 ml of
water.
[0077] 2. To this solution 2 g. of glycidyl-modified silica
magnetic particles were added, and the resulting mixture was
refluxed overnight.
[0078] 3. After cooling, the reaction mixture was filtered and
washed twice with water, and once with ethyl ether.
[0079] 4. Elemental analysis of a sample of the product from step 3
showed: %C 0.98; %H 0.56; %N 0.20. This result is consistent with
glycidyl-alanine modification, as illustrated in Formula (III),
below: ##STR3## wherein, R is --OH, OCH.sub.3, or
--OCH.sub.2CH.sub.3.
Example 4
Preparation of a Lysate of Plasmid DNA
[0080] E. coli bacteria cells, DH5.alpha. strain, were transformed
with pGL3-Control Vector (Promega) plasmid DNA, grown overnight
Luria Broth ("LB") medium at 37.degree. C., then harvested by
centrifugation.
[0081] The following solutions were used to prepare a lysate of the
harvested cells, as described below:
[0082] Cell Resuspension Solution: [0083] 50 mM Tris-HCl, pH 7.5
[0084] 10 mM EDTA [0085] 100 .mu.g/ml DNase-free ribonuclease A
(RNase A)
[0086] Wizard.RTM. Neutralization Buffer (Promega Corp.): [0087]
1.32M KOAc (potassium acetate), pH 4.8
[0088] Cell Lysis Solution: [0089] 0.2M NaOH [0090] 1% SDS (sodium
dodecyl sulfate)
[0091] A lysate of the transformed cells was produced as
follows:
[0092] 1. The cells from 1 to 10 ml of bacteria culture were
harvested by centrifuging the culture for 1-2 minutes at top speed
in a microcentrifuge. The harvested cells were resuspended in 250
.mu.l of Cell Resuspension Solution, and transferred to a
microcentrifuge tube. The resulting solution of resuspended cells
was cloudy.
[0093] 2. 250 .mu.l of Cell Lysis Solution was then added to the
solution of resuspended cells and mixed by inversion until the
solution became relatively clear, indicating the resuspended cells
had lysed.
[0094] 3. 350 .mu.l of Wizard.RTM. Neutralization Buffer was added
to the lysate solution, and mixed by inversion. The lysate became
cloudy after the Neutralization Solution was added.
[0095] Each sample of lysate prepared as described above was
cleared, either by centrifugation (control samples), or by using
silica magnetic particles or silica magnetic ion exchange particles
(test samples), as described in the Examples below.
Example 5
Lysate Clearance by Centrifugation or Silica Magnetic Particles,
Followed by Plasmid DNA Isolation using Glycidyl-Histidine or
Glycidyl-Alanine Silica Magnetic Particles
A. Preparation of Cleared Lysates
[0096] Four samples of lysates of 1 ml cultures of DH5.alpha.
(pGL3) were prepared as described in Example 4, above, except that
24 hour cultures in Circlegrow medium were used instead of
overnight LB. Two of the samples were cleared by centrifugation.
The other two samples were cleared by mixing the lysate with 150
.mu.l of silica magnetic particles (100 mg/ml), vortexing the
resulting mixture until debris in the lysate has adsorbed to the
particles, and separating the silica magnetic particles from the
solution by magnetic force, using a magnetic separator.
B. Isolation of Plasmid DNA from Cleared Lysates
[0097] Plasmid DNA was then isolated from the samples of cleared
lysate, as follows:
[0098] 1. The cleared lysate solutions from both sets of samples
were transferred to clean tubes containing 150 .mu.l of either
glycidyl-histidine silica magnetic ion exchange particles
(hereinafter, "Mag-IE-glycidyl-histidine" particles) or
glycidyl-alanine silica magnetic ion exchange particles
(hereinafter, "Mag-IE-glycidyl-alanine particles"), and mixed by
vortexing. The Mag-IE-glycidyl-alanine and
Mag-IE-glycidyl-histidine particles were produced as described in
Example 3, above.
[0099] 2. After waiting 5 minutes for DNA binding to the particles,
the solutions were placed on a magnetic rack, allowed to sit for 2
minutes, and the solutions removed.
[0100] 3. The particles were then resuspended in 1.0 ml of nanopure
water, the tubes inverted to wash the side-walls and cap, and
placed back into a magnetic separator, which was inverted to wash
the tube cap to removed suspended particles.
[0101] 4. Step 3 (a water wash) was repeated 3 times, for a total
of four washes.
[0102] 5. The solution was removed from the tubes, and the DNA was
eluted using (1) 10 mM Tris HCl pH 8.5 for
Mag-IE-glycidyl-histidine or (2) 20 mM Tris HCl pH 9.5 for
Mag-E-glycidyl-alanine.
C. Assay of Results
[0103] A spectrophotometric assay was conducted on each eluent
sample, as described in Example 2. Spectrophotometric results from
the Mag-IE-glycidyl-histidine particle eluent showed a yield of 26
.mu.g of DNA and a high purity, with an A.sub.260/A.sub.280 ratio
of 1.85. Assay results from the Mag-IE-glycidyl-alanine particle
eluent showed a yield of 25 .mu.g of DNA and a A.sub.260/A.sub.280
ratio of 1.90, indicating a comparable purity to the eluent from
the other species of IE particle described above.
[0104] All the eluents produced as described above were also
assayed by gel electrophoresis, as described in Example 1, above.
Intact plasmid DNA was detected in each sample, with no evidence of
degradation or RNA contamination in any of the samples.
Example 6
Lysate Clearance with Silica Magnetic Particles or Varying Amounts
of Mag-IE-Glycidyl-Histidine Particles
[0105] The assay described below was performed to determine whether
small quantities of silica magnetic ion exchange particles could
clear lysate with sufficient efficiency that one could isolate
intact plasmid DNA therefrom, which is substantially free of
contaminants. Lysate cleared with 4 mg of silica magnetic particles
was used as a control. Plasmid DNA was isolated from both the
control and test samples of cleared lysate, using
Mag-IE-glycidyl-histidine, according to the same procedure, set
forth below.
A. Lysate Clearing
[0106] Silica magnetic particles and varying amounts of
Mag-IE-glycidyl-histidine particles were used, as follows, to
prepare a cleared lysate. All the steps below were conducted in 1.5
ml tubes, and at room temperature.
[0107] 1. A pellet of cells harvested, by centrifugation of a 50 ml
overnight culture of DH5.alpha. E. coli bacteria transformed with
pGEM-3Zf.sup.+ plasmid DNA, were resuspended in 2.5 ml of
Wizard.RTM. Resuspension Solution.
[0108] 2. 265 .mu.l of the resuspended cells was added to each of
eight tubes.
[0109] 3. 250 .mu.l Wizard.TM. Lysis solution was added to each
tube of resuspended cells, and mixed gently, to avoid possible
sheering of genomic DNA.
[0110] 4. 350 .mu.l Wizard Neutralization solution was added to
each tube of lysed cells, and mixed gently and thoroughly.
[0111] 5. Mag-IE-histidine particles (100 mg/ml) were added to six
of the samples from step 4, as follows: 10 .mu.l or 20 .mu.l or 40
.mu.l per lysate tube (in duplicate). 40 .mu.l of silica magnetic
particles (100 mg/ml) were added to each of the remaining two
samples. All the samples were mixed thoroughly, by vortexing.
[0112] 6. The resulting particle/cell debris complex was separated
from the lysate within each tube, using a magnetic separator. The
caps of the tubes were washed four times, by inversion of each
tube. The tubes allowed to sit for 1 minute.
B. DNA Isolation
[0113] DNA was isolated from each of the cleared lysate samples,
above, as described below:
[0114] 1. Each cleared lysate solution sample, above, was
transferred to a clean 1.5 ml tube containing 150 .mu.l of
Mag-IE-glycidyl-histidine (100 mg/ml), vortexed, and allowed to sit
5 minutes.
[0115] 2. The resulting Mag-IE-glycidyl-histidine/DNA complex was
then separated from the solution within each tube, using a magnetic
separator. The tube caps were each washed four times, by inversion.
The tubes were allowed to sit for 1 minute.
[0116] 3. The liquid was removed from each tube, and discarded.
[0117] 4. The particles were washed with nanopure water, as
follows. 1.0 ml nanopure water was added to each tube, and the
particles resuspended therein. The Mag-IE-glycidyl-histidine
particles were separated from the solution within each tube, using
a magnetic separator. The tube caps were each washed four times, by
inversion. The tubes were allowed to sit for 1 minute. The liquid
was removed from each tube and cap, and discarded, using the
magnetic separator to retain the particles in each tube while the
wash solution was discarded.
[0118] 5. Step 4 was repeated twice, for a total of 3 washes.
[0119] 6. Added 100 .mu.l 10 mM Tris HCl, pH 8.0, to each tube, and
resuspend the particles contained therein by vortexing
[0120] 7. The plasmid DNA was magnetically separated from the
particles from the resulting eluent solution in each tube, and
transferred to a clean tube.
C. Assay of Results
[0121] Each of the eluent samples produced as described above was
assayed spectrophotometrically, as described in Example 2. The
assay results are summarized in Table 2, below: TABLE-US-00001
TABLE 1 NUCLEIC PARTICLES & AMOUNT A.sub.260/A.sub.280 ACID
YIELD 1 mg of Mag-IE-glycidyl-histidine 1.73 37 .mu.g 1.73 43 .mu.g
2 mg of Mag-IE-glycidyl-histidine 1.75 36 .mu.g 1.76 38 .mu.g 4 mg
of Mag-IE-glycidyl-histidine 1.76 40 .mu.g 1.76 38 .mu.g 4 mg of
Magnesil .TM. 1.80 36 .mu.g 1.80 37 .mu.g
[0122] The samples assayed by spectrophotometric analysis, as
described above, were also analyzed by gel electrophoresis, as
described in example 1. FIG. 1 shows a photograph of samples of
each of the eluents, above, after being fractionated by gel
electrophoresis and stained with ethidium bromide. The samples were
loaded on the gel, from left to right, in the same order shown in
Table 1, above. None of the samples showed any visible RNA, and the
intensity of the plasmid DNA bands is consistent with the yield
data obtained by absorption spectrophotometry (as described in
example 2).
Example 7
Lystate Clearance by Centrifugation VS. Using Silica Magnetic
Particles, Followed by Isolation of Plasmid DNA from Cleared Lysate
using Silica Magnetic Particles
[0123] In the following assay, centrifugation or silica magnetic
particles were used to clear cell lysates of varying volumes of
overnight cultures of the same transformants. Plasmid DNA was then
isolated from each cleared lysate solution, using silica magnetic
particles, and tested as described below.
A. Lysate Clearing
[0124] 1. An overnight culture of DH5.alpha.(pGL3) was centrifuged
to obtain, in six replicates, 1.0 ml, 2.0 ml, and 3 ml cell pellets
in 1.5 ml tubes. To each tube, 250 .mu.l of Resuspension Buffer was
added, and the cells resuspended by vortexing.
[0125] 2. 250 .mu.l of Wizard Lysis solution was added per tube,
and gently mixed to avoid sheering genomic DNA.
[0126] 3. 350 .mu.l Wizard Neutralization solution was added per
tube, mixed gently and thoroughly.
[0127] 4. To one set of triplicate samples, the tubes were
centrifuged for 10 minutes at 12,000.times.g to clear the lysate
debris. The cleared supernatants were transferred to clean 1.5 ml
tubes and processed as described in section B, below.
[0128] 5. To the other set of triplicate samples (3 of 1.0 ml, 3 of
2 ml, 3 of 3 ml), 50 .mu.l of resuspended silica magnetic particles
(100 mg/ml) were added per lysate tube, and vortexed
thoroughly.
[0129] 6. The resulting particles/cell debris complex was separated
from the solution in the tube, in a magnetic separator. Tube caps
were washed by tube inversion (4.times.). Tubes were allowed to sit
for 1 minute. The resulting cleared lysate was transferred from the
each tube and processed as described in section B, below.
B. Isolation of DNA from Cleared Lysates
[0130] 1. The cleared solutions from steps 4 and 6, above, were
each placed in a clean 1.5 ml tube containing 200 .mu.l of 5.0M
guanidine thiocyanate, and vortexed. 150 .mu.l of silica magnetic
particles (15 mg) was added per tube, vortexed, and allowed to sit
10 minutes.
[0131] 2. The resulting silica magnetic particle/ DNA complex was
separated from the solution in the tube, on a magnetic separator.
Tube caps were washed four times, by tube inversion, and allowed to
sit in the separator for 1 minute.
[0132] 3. Liquid was removed from each tube, including caps, and
discarded.
[0133] 4. Each tube was washed with 1 ml of 60 mM KOAc/10 mM
Tris-HCl (pH 7.5 at 25.degree. C.)/60% ethanol, using vortexing to
resuspend the particles.
[0134] 5. The silica magnetic particle/DNA complex was separated
from the wash solution in the tube, using a magnetic separator.
Tube caps were washed four times, by tube inversion, and allowed to
sit in the separator for 1 minute.
[0135] 6. Liquid was removed from tube and caps, and discarded.
[0136] 7. Steps 4-6 were repeated, for a total of 2 washes.
[0137] 8. The tubes were allowed to air dry for 30 minutes to
remove residual ethanol.
[0138] 9. 100 .mu.l of nanopure water was added per tube, and the
particles were resuspended thoroughly by vortexing. After 10
minutes at ambient temperature, the tubes were placed in a magnetic
separator, and the resulting eluent was transferred to clean 1.5 ml
tubes.
C. Analysis of Results
[0139] The eluent from each sample was analyzed with an absorption
spectrophotometer at 230, 260, and 280 nm, as described in Example
3, above. The average value of test results obtained from each set
of three samples of eluent, prepared as described above, is set
forth in Table 2, below: TABLE-US-00002 TABLE 2 VOL. CULTURE, YIELD
(.mu.g CLERANCE MEANS A.sub.230 A.sub.260 A.sub.280
A.sub.260/A.sub.280 DNA) 1 ml & Centrifugation 0.160 0.072
0.039 1.83 7.17 1 ml & Silica Magnetic 0.176 0.094 0.053 1.77
9.36 Particles 2 ml & Centrifugation 0.197 0.121 0.667 1.82
12.0 2 ml & Silica Magnetic 0.189 0.103 0.058 1.79 10.3
Particles 3 ml & Centrifugation 0.495 0.149 0.082 1.82 14.9
[0140] The results shown in Table 2, above, indicate comparable
amounts of plasmid DNA were isolated from the same volumes of
lysate cleared either by centrifugation or by silica magnetic
particles. The A.sub.230 and A.sub.260/A.sub.280 measurements from
samples isolated from the same volumes of cultures lysed and
cleared with each of the two means described above, indicates that
both methods of isolation produced isolated DNA which appears to be
free from contamination with low molecular weight alcohol or
proteins.
[0141] Each of the samples of plasmid DNA isolated as described
above was also assayed by agarose gel electrophoresis, as described
in Example 1. Initially, the agarose gel with the above samples
loaded thereon was run only for a sufficient period of time for the
plasmid DNA to migrate into the gel and become separated from any
RNA present in each sample. FIG. 2 is a photograph of the gel taken
under UV light at this initial stage, after staining the gel with
ethidium bromide. No sign of RNA contamination was apparent in any
of the lanes of the gel shown in FIG. 2. The same gel was then
electrophoresed for an additional period of time, to enable the
plasmid DNA to become separated from any chromosomal DNA in each
sample loaded thereon. FIG. 3 is a photograph of the same gel,
taken under the same conditions described above, after the gel had
been run for a longer period of time. No sign of contamination with
chromosomal DNA was apparent in any of the lanes of the gel, in
FIG. 3.
Example 8
Concentration of Cells, Lysate Clearing, and DNA Isolation Using
Mag-IE-Glycidyl-Histidine Particles
[0142] Mag-IE-glycidyl-histidine particles were used to concentrate
cells prior to lysis, to clear the lysate once the concentrated
cells were lysed, and to isolate DNA from the resulting cleared
lysate, as follows:
A. Cell Concentration
[0143] 1. 50 .mu.l of Mag-IE-glycidyl-histidine suspension was
aliquoted into each of two 1.5 ml tubes.
[0144] 2. 500 .mu.l of an overnight culture of DH5.alpha./pGem3Zf+
was aliquoted into the two tubes prepared in step 1. These two
samples were processed to harvest the cells as described in steps
4-6, below.
[0145] 3. 500 .mu.l of the same culture used in step 2 was also
aliquoted into each of two empty 1.5 ml centrifuge tubes, and spun
in a centrifuge to harvest the cells. The supernatant was
discarded, and the harvested cells processed as described in
section B, below.
[0146] 4. 300 .mu.l of 5M NaCl was added to each tube of
Mag-IE-glycidyl-histidine and overnight culture, and mixed
thoroughly.
[0147] 5. 800 .mu.l of room temperature isopropanol was added to
each tube, and mixed thoroughly for a final concentration of 94M
NaCl/50% IPA.
[0148] 6. The resulting Mag-IE-glycidyl-histidine/cells complex was
separated from the solution in each tube, in magnetic separator.
The solution was discarded, and the harvested cells processed as
described in section B, below.
B. Lysate Clearing and DNA Isolation
[0149] 1. 250 .mu.l Wizard Resuspension solution was added to both
sets of tubes, the tubes with cells pelleted in a centrifuge and
the tubes with cells complexed with Mag-IE-glycidyl-histidine
particles. In both cases, the solutions were mixed thoroughly until
the cells were resuspended in each solution.
[0150] 2. 250 .mu.l of Wizard Lysis solution was added to each
tube, and gently mixed to avoid sheering genomic DNA
[0151] 3. 350 .mu.l of Wizard neutralization solution was added,
and mixed gently and thoroughly.
[0152] 4. The resulting Mag-IE-glycidyl-histidine/cell debris
complex was separated from the lysate within each tube, using a
magnetic separator.
[0153] 5. Each resulting cleared lysate solution was transferred to
a clean 1.5 ml tube containing 50 .mu.l Mag-E-glycidyl-histidine,
and incubated 2 minutes at room temperature, to enable DNA to
adhere to the particles.
[0154] 6. The resulting Mag-IE-glycidyl-histidine/DNA complex was
separated from the solution in the tube, using a magnetic
separator.
[0155] 7. The liquid in the tube was removed and discarded.
[0156] 8. Each tube was washed with 1.0 ml nanopure water, and the
particles suspended therein. The particles were separated from the
water in each tube, using a magnetic separator. The liquid was
removed and discarded.
[0157] 9. Step 8 was repeated three times, for a total of four
washes.
[0158] 10. 100 .mu.l of 20 mM Tris pH 9.5, an elution buffer, was
then added to each tube. The particles were resuspended in the
elution buffer.
[0159] 11. Magnetic force was used to separated the
Mag-IE-glycidyl-histidine particles from the resulting eluent
solution.
C. Assay Results
[0160] The four samples of DNA isolated from cells which were
concentrated by either centrifugation or using
Mag-IE-glycidyl-histidine particles, as described above, were
assayed spectrophotometrically, as described in Example 1, above.
The results of the spectrophotometric analysis are presented in
Table 3, below: TABLE-US-00003 TABLE 3 SAMPLE A.sub.260/A.sub.280
YIELD Centrifugation used to 1.84 7.8 .mu.g concentrate cells 1.85
8.1 .mu.g Mag-IE glycidyl-histidine 1.78 9.5 .mu.g used to
concentrated cells 1.80 8.2 .mu.g
Example 9
Clearing Mouse Tissue Homogenates Using Mag-IE-Glycidyl-Histidine,
and Isolating DNA and RNA Therefrom Using
Mag-IE-Glycidyl-Histidine
[0161] The following protocol was used to clear homogenates of
frozen mouse liver, kidney, and spleen tissue, and to isolate RNA
and DNA therefrom:
A. Homogenate Clearance
[0162] 1. A sample of each tissue was homogenized in a solution of
4.5M guanidine thiocyanate (GTC)/132 mM KOAc pH 4.8, wherein, for
every 1 mg of tissue, 1 .mu.l of homogenization solution was used.
120 mg of liver, 320 mg of kidney, and 142 mg of spleen were
homogenized.
[0163] 2. The resulting homogenized mixture was diluted 7.times.
with RNase free nanopure water for mouse liver, 6.times. RNase free
nanopure water for kidney, and 12.times. RNase free nanopure water
for spleen. After the addition of nanopure water (liver=840 .mu.l,
spleen=1.7 ml, and kidney=1.9 ml), each sample was vortexed.
[0164] 3. "1/2 X"volume of Mag-IE-glycidyl-histidine (100 mg/ml)
was added to each solution, and vortexed. The resulting mixture was
then magnetically separated for 10 minutes.
B. Isolation of Nucleic Acids from Cleared Homogenate
[0165] 1. An aliquot of each cleared solution separated from the
Mag-E-glycidyl-histidine particles, as described above, was
transferred to a clean tube containing Mag-IE-glycidyl-histidine
particles. For the liver and spleen samples, 100 .mu.l of cleared
solution was added to 100 .mu.l of Magnesil-IE-glycidyl-histidine
(100 mg/ml), the mixture was vortexed, allowed to sit for 2
minutes, then allowed to sit in a magnetic separator for 2 minutes.
For the kidney sample, 400 .mu.l of cleared solution was added to 1
ml of RNase free nanopure water, then 100 .mu.l of
Magnesil-IE-glycidyl-histidine (100 mg/ml) was added, the mixture
was vortexed, allowed to sit for 2 minutes, then allowed to sit in
a magnetic separator for 2 minutes.
[0166] 2. The solution was then removed from each tube, and each
tube was washed with 1.0 ml RNase free nanopure water, vortexed,
and placed back in the magnetic separator. The tube cap was washed
by inversion of the tubes in the magnetic rack. After 2 minutes,
the wash solution was removed. This wash step was repeated two
times, for a total of 3 washes.
[0167] 3. The nucleic acids were eluted in 100 .mu.l of 10 mM Tris
HCl, pH 9.5.
C. Analysis of Results
[0168] The eluted DNA and RNA was visualized by gel electrophoresis
(see example 1) as shown in FIGS. 4, 5, and 6. FIG. 4 shows a
photograph of mouse liver DNA and RNA isolated as described above,
fractionated by gel electrophoresis along with .lamda. Hind III
marker. Both DNA and RNA appear to be present in each eluent.
[0169] FIG. 5 shows DNA and RNA isolated from mouse spleen and
kidney as described above, after fractionation by gel
electrophoresis. Samples were loaded on the gel as follows: [0170]
Lane 1: .lamda. Hind III marker [0171] Lane 2: Spleen, 0 .mu.l
removed [0172] Lane 3: Spleen, 20 .mu.l removed [0173] Lane 4:
Spleen, 40 .mu.l removed [0174] Lane 5: Spleen, all removed [0175]
Lane 6: .lamda. Hind III marker [0176] Lane 7: Kidney, 0 .mu.l
removed [0177] Lane 8: Kidney, 20 .mu.l removed [0178] Lane 9:
Kidney, all removed [0179] Lane 10: .lamda. Hind III marker
[0180] FIG. 6 shows samples of mouse liver RNA and DNA isolated as
described above, after digestion with DNase and fractionation by
gel electrophoresis. Lanes 1 and 4 contain .lamda. Hind III marker,
while lanes 2 and 3 contain mouse liver nucleic acid isolated from
200 .mu.l and 400 .mu.l of homogenate, respectively, according to
the procedure described above.
Example 10
Concentration of White Blood Cells, Lysate Clearing, and DNA
Isolation from Whole Blood Using Mag-IE-Glycidyl-Histidine
Particles, Non-Porous Magnesil-IE-Gly-Histidine Particles, and
Magnesil.TM. Particles Using Human Whole Blood
[0181] Mag-IE-glycidyl-histidine particles, Non-Porous
Mag-IE-glycidyl-histidine particles and Magnesil.TM. particles were
used to either (a) concentrate white blood cells, clear the lysate
once the concentrated cells were lysed, and to isolate DNA from the
resulting cleared lysate, or (b) clear the lysate produced from
centrifugal concentrated white blood cells, clear the lysate, and
to isolate DNA from the resulting cleared lysate.
A. Use of Mag-IE-glycidyl-histidine Particles with Ion Exchange
Wash
[0182] Magnetic clearing of blood lysate and purification of
genomic DNA using solutions from Promega's Wizard Genomic DNA
Purification kit (see, Promega's Technical Manual #.TM. 50), and
Mag-IE-glycidyl-histidine particles: All steps were at room
temperature. Mag-IE-glycidyl-histidine particles were used with an
ion exchange wash to concentrate white blood cells, to clear a
lysate of the cells, and to isolate genomic DNA therefrom, as
follows:
[0183] 1. 1.0 ml of blood was placed in a 15 ml tube containing 3.0
ml of Wizard Genomic Cell Lysis solution, mixed, and incubated for
10 minutes.
[0184] 2. 1.0 ml of 5.0 M NaCl was added, and mixed.
[0185] 3. 50 .mu.l of Mag-IE-glycidyl-histidine particles in a 100
mg/ml solution was added to the tube, and mixed.
[0186] 4. 5.0 ml of isopropanol was added and mixed, and incubated
for 2 minutes, then placed on a magnetic rack for 5 minutes.
[0187] 5. The solution was removed and discarded.
[0188] 6. The tubes were removed from the magnetic rack and
vortexed for 5 seconds.
[0189] 7. 1.0 ml of Nuclei Lysis solution was added, the tube was
vortexed for 5 seconds, and incubated for 5 minutes.
[0190] 8. 330 .mu.l of Wizard Genomic Protein Precipitation
solution was added, the tube was vortexed for 5 seconds, and the
tube was placed on a magnetic rack for 5 minutes.
[0191] 9. The cleared lysate solution was removed from the first
tube and placed into a second tube containing 200 .mu.l of
Mag-IE-glycidyl-histidine particles (100 mg/ml), and mixed.
[0192] 10. 0.5 ml of 0.5 M sodium citrate, pH 5.0 (pH adjusted to
5.0 with citric acid) was added, and the solution mixed. 8.0 ml of
nanopure water was added, the solution mixed, the tube was
incubated for 1 minute, and placed on a magnetic rack for 2
minutes.
[0193] 11. The solution was removed and discarded.
[0194] 12. 5.0 ml of 66 mM potassium acetate, pH 4.8 (pH adjusted
with acetic acid) was added, the tube vortexed for 5 seconds, and
the tube placed on a magnetic rack for 2 minutes.
[0195] 13. The solution was removed and discarded, and 2.0 ml of 66
mM potassium acetate/600 mM NaCl, pH 4.8 was added, the tube mixed,
and placed on a magnetic rack for 2 minutes.
[0196] 14. The solution was removed and discarded.
[0197] 15. 2.0 ml of 66 mM potassium acetate, pH 4.8, 450 mM NaCl
was added, the tube was vortexed for 5 seconds, and the tube placed
on a magnetic rack for 2 minutes.
[0198] 16. The solution was removed and discarded.
[0199] 17. 10 ml of nanopure water was added, mixed, and the tube
placed onto a magnetic rack for 2 minutes, after which time the
solution was discarded.
[0200] 18. Step 17 was repeated twice, for a total of 3.times.10 ml
nanopure water washes.
[0201] 19. After removal from the magnetic rack, DNA was eluted in
400 .mu.l of 90 mM Tris HCl, pH 9.5 for 5 minutes. The tube was
then placed on a magnetic rack for 5 minutes.
[0202] Mag-IE-glycidyl-histidine particles were also used to clear
a lysate of white blood cells isolated by centrifugation, before
isolating genomic DNA therefrom using the same particles. The same
procedure described above was used, except that Steps 2-4 were
replaced by centrifugation for 10 minutes at 800.times.g, followed
by removal of the lysed red blood cell debris, and vortexing the
cell pellet to resuspend the white blood cells. Also, in step 8, 50
.mu.l of Mag-IE-glycidyl-histidine particles were added after the
vortexing step, and followed by five seconds of vortexing, prior to
placement of the tube into the magnetic rack.
B. MagneSil.TM. Particles and Guanidine Thiocyanate
[0203] Magnetic clearing of blood lysate and purification of
genomic DNA using solutions from Promega's Wizard.RTM. Genomic DNA
Purification kit, and MagneSil.TM. particles: guanidine thiocyanate
protocol, as described below. All steps were at room
temperature
[0204] 1. 1.0 ml of blood was placed in a 15 ml tube containing 3.0
ml of Wizard Genomic Cell Lysis solution, mixed, and incubated for
10 minutes.
[0205] 2. 1.0 ml of 5.0 M NaCl was added, and mixed.
[0206] 3. 50 .mu.l of MagneSil.TM. Particles, (100 mg/ml) was added
to the tube, and mixed.
[0207] 4. 5.0 ml of isopropanol was added and mixed, and incubated
for 2 minutes, then placed on a magnetic rack for 5 minutes.
[0208] 5. The solution was removed and discarded.
[0209] 6. The tubes were removed from the magnetic rack and
vortexed for 5 seconds.
[0210] 7. 1.0 ml of Nuclei Lysis solution was added, the tube was
vortexed for 5 seconds, and incubated for 5 minutes.
[0211] 8. 330 .mu.l of Wizard Genomic Protein Precipitation
solution was added, the tube was vortexed for 5 seconds, and the
tube was placed on a magnetic rack for 5 minutes.
[0212] 9. 200 .mu.l of MagneSil.TM. Particles (100 mg/ml) was added
to a clean tube, placed on a magnetic rack for 1 minute, and the
solution removed. To this tube, the cleared lysate solution was
added from the tube in step 8, and mixed.
[0213] 10. 2.0 ml of 5 M guanidine thiocyanate (GTC) was added, the
tube mixed, incubated 2 minutes, and placed on a magnetic rack for
5 minutes.
[0214] 11. The solution was removed and discarded.
[0215] 12. 5.0 ml of SV Total RNA Column Wash was added, the tube
was vortexed for 5 seconds, and the tube placed on a magnetic rack
for 2 minutes.
[0216] 13. The solution was removed and discarded
[0217] 14. Steps 12-13 were repeated, for a total of 2 washes.
[0218] 15. 5.0 ml of 80% ethanol was added, and the tube vortexed
for 5 seconds, and the tube placed on a magnetic rack for 2
minutes.
[0219] 16. The solution was removed and discarded.
[0220] 17. Steps 15-16 were repeated 2 times, for a total of 3
washes.
[0221] 18. The tubes were air-dried for 60 minutes in the magnetic
rack.
[0222] 19. After removal from the magnetic rack, DNA was eluted in
400 .mu.l of Wizard Genomic Renaturation Solution for 5 minutes.
The tube was then placed on a magnetic rack for 5 minutes.
[0223] 20. The DNA containing solution was removed to a clean
tube.
[0224] For the isolation of white blood cells by centrifugation,
followed by clearing of the lysate and isolation of DNA with
MagneSil.TM. Particles: Steps 2-4 were replaced by centrifugation
for 10 minutes at 800.times.g, followed by removal of the lysed red
blood cell debris, and vortexing the cell pellet to resuspend the
white blood cells. Additionally, 50 .mu.l of MagneSil.TM. particles
were added in step 8 after the vortexing step, and followed by five
seconds of vortexing, prior to placement of the tube into the
magnetic rack.
C. Non-Porous-Mag-IE-glycidyl-histidine Particles and
Isopropanol
[0225] Magnetic clearing of blood lysate and purification of
genomic DNA using solutions from Promega's Wizard Genomic DNA
Purification kit, and Non-porous MagneSil-IE-glycidyl-histidine
particles, as follows. All steps were at room temperature
[0226] 1. 1.0 ml of blood was placed in a 15 ml tube containing 3.0
ml of Wizard Genomic Cell Lysis solution, mixed, and incubated for
10 minutes.
[0227] 2. 1.0 ml of 5.0 M NaCl was added, and mixed.
[0228] 3. 100 .mu.l of Non-Porous-Mag-IE-glycidyl-histidine in a
solution of 100 mg/ml was added to the tube, and mixed.
[0229] 4. 5.0 ml of isopropanol was added and mixed, and incubated
for 2 minutes, then placed on a magnetic rack for 5 minutes.
[0230] 5. The solution was removed and discarded.
[0231] 6. The tubes were removed from the magnetic rack and
vortexed for 5 seconds.
[0232] 7. 1.0 ml of Nuclei Lysis solution was added, the tube was
vortexed for 5 seconds, and incubated for 5 minutes.
[0233] 8. 330 .mu.l of Wizard Genomic Protein Precipitation
solution was added, the tube was vortexed for 5 seconds, and the
tube was placed on a magnetic rack for 5 minutes.
[0234] 9. The cleared lysate solution was removed from the first
tube and placed into a second tube containing 20 mg of Non-Porous
Mag-IE-glycidyl-histidine (200 .mu.l of 100 mg/ml, placed on a
magnetic rack and the solution removed), and mixed.
[0235] 10. 1.0 ml of isopropanol was added, the solution mixed,
incubated 2 minutes, then placed in a magnetic rack for 2
minutes.
[0236] 11. The solution was removed and discarded.
[0237] 12. 2.0 ml of 66 mM potassium acetate, pH 4.8 (pH adjusted
with acetic acid) was added, and the tube vortexed 5 seconds,
incubated 1 minute, and the tube placed on a magnetic rack for 2
minutes.
[0238] 13. The solution was removed and discarded.
[0239] 14. 2.0 ml of nanopure water was added, mixed, and the tube
placed onto a magnetic rack for 2 minutes, after which time the
solution was discarded.
[0240] 15. Step 18 was repeated twice, for a total of 3.times.2 ml
nanopure water washes.
[0241] 16. After removal from the magnetic rack, DNA was eluted in
400 .mu.l of 90 mM Tris HCl, pH 9.5 for 5 minutes. The tube was
then placed on a magnetic rack for 5 minutes.
[0242] 17. The DNA containing solution was removed to a clean
tube.
[0243] For the isolation of white blood cells by centrifugation,
followed by clearing of the lysate and isolation of DNA with Non
Porous-Mag-IE-glycidyl-histidine: Steps 2-4 were replaced by
centrifugation for 10 minutes at 800.times.g, followed by removal
of the lysed red blood cell debris, and vortexing the cell pellet
to resuspend the white blood cells. Additionally, 1001 of
NP-Mag-IE-glycidyl-histidine particles were added in step 8 after
the vortexing step, and followed by five seconds of vortexing,
prior to placement of the tube into the magnetic rack.
D. MagneSil-IE-glycidyl-histidine and isopropanol
[0244] The "Non-Porous-Mag-IE-glycidyl-histidine and Isopropanol"
method described above was also used with porous
Mag-E-glycidyl-histidine particles. The only changes in the
protocol were the use of 50 .mu.l of Mag-IE-glycidyl-histidine
instead of 100 .mu.l of Non-Porous-Mag-E-glycidyl-histidine
particles in step 3, and the use of porous
Mag-IE-glycidyl-histidine particles in step 8.
[0245] For the isolation of white blood cells by centrifugation,
followed by clearing of the lysate and isolation of DNA with
Mag-IE-glycidyl-histidine particles: Steps 2-4 were replaced by
centrifugation for 10 minutes at 800.times.g, followed by removal
of the lysed red blood cell debris, and vortexing the cell pellet
to resuspend the white blood cells. Additionally, 50 .mu.l of
Mag-IE-glycidyl-histidine particles were added in step 8 after the
vortexing step, and followed by five seconds of vortexing, prior to
placement of the tube into the magnetic rack.
E. Assay Results
[0246] The A.sub.260/A.sub.280 data and DNA yields were calculated
from UV spectrophotometry, except for the porous
Mag-IE-glycidyl-histidine particles white blood cell concentration
samples, where estimates taken from gel electrophoresis were used,
as denoted by "(gel)" below. These results are summarized in Table
4, below: TABLE-US-00004 TABLE 4 A.sub.260/ PARTICLES USED METHOD
USED A.sub.280 YIELD (.mu.g) Porous Mag-IE- Spin Cells, Clear
Lysate 1.77 11 glycidyl-histidine (salt wash) 1.79 7 Porous Mag-IE-
Concentrate Cells with 1.27 10 (gel) glycidyl-histidine Particles,
Clear Lysate 1.29 8 (gel) (salt wash) MagneSil .TM. Spin Cells,
Clear Lysate 1.75 12 (guanidine thiocyanate) 1.82 10 MagneSil .TM.
Concentrate with Particles, 1.75 8 Clear Lysate (guanidine 1.71 7
thiocyanate) Porous Mag-IE- Spin Cells, Clear Lysate 1.76 10
glycidyl-histidine (isopropanol) 1.78 15 Porous Mag-IE- Concentrate
with Particles, 1.71 9 glycidyl-histidine Clear Lysate
(isopropanol) 1.75 13 Non-Porous Mag-IE- Spin Cells, Clear Lysate
1.77 4 glycidyl-histidine (isopropanol) 1.78 5 Non-Porous Mag-IE-
Concentrate with Particles, 1.65 5 glycidyl-histidine Clear Lysate
(isopropanol) 1.57 7
* * * * *