U.S. patent application number 12/396586 was filed with the patent office on 2009-07-30 for kits for isolating nucleic acids using multifunctional group-coated solid phase carriers.
This patent application is currently assigned to AGENCOURT BIOSCIENCE CORPORATION. Invention is credited to Adrianne D. Brand, Erik Gustafson, Kevin J. McKernan.
Application Number | 20090191566 12/396586 |
Document ID | / |
Family ID | 35677343 |
Filed Date | 2009-07-30 |
United States Patent
Application |
20090191566 |
Kind Code |
A1 |
McKernan; Kevin J. ; et
al. |
July 30, 2009 |
Kits for Isolating Nucleic Acids Using Multifunctional Group-Coated
Solid Phase Carriers
Abstract
The present invention is directed to a method of isolating a
target species (e.g., target nucleic acid species) from a mixture.
In the methods of the present invention, the mixture is combined
with solid phase carriers having a surface comprising multiple
functional groups one of which reversibly and selectively binds the
target species. In a particular embodiment, the mixture is combined
with solid phase carriers having a first functional group which
reversibly binds nucleic acids and a second functional group which
selectively and reversibly binds the target nucleic acid species,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the nucleic
acids to the first functional group and binding of the target
nucleic acid species to the second functional group. The solid
phase carriers are separated from the first combination, and
combined with an agent (e.g., buffer) that selectively removes
(e.g., elutes) either the nucleic acid from the first functional
group or the target nucleic acid species from the second functional
group of the solid phase carriers, thereby isolating a target
nucleic acid species from a mixture comprising a plurality of
nucleic acid species.
Inventors: |
McKernan; Kevin J.;
(Marblehead, MA) ; Gustafson; Erik; (Norwood,
MA) ; Brand; Adrianne D.; (Wenham, MA) |
Correspondence
Address: |
Michael C. Schiffer - Legal Dept.;BECKMAN COULTER, INC.
4300 N. HARBOR BOULEVARD, Mail Code A-42-C
FULLERTON
CA
92834-3100
US
|
Assignee: |
AGENCOURT BIOSCIENCE
CORPORATION
Beverly
MA
|
Family ID: |
35677343 |
Appl. No.: |
12/396586 |
Filed: |
March 3, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11194072 |
Jul 29, 2005 |
7527929 |
|
|
12396586 |
|
|
|
|
60592917 |
Jul 30, 2004 |
|
|
|
Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12N 15/1013 20130101; C12Q 1/6869 20130101; C12Q 1/6806 20130101;
C12Q 1/6834 20130101; C12Q 1/6806 20130101; C12Q 2527/125 20130101;
C12Q 1/6869 20130101; C12Q 2527/125 20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A kit comprising a solid phase carrier having a surface
comprising heterologous first and second functional groups and a
cell lysis buffer.
2. A kit comprising a solid phase carrier having a surface
comprising heterologous first and second functional groups and a
cell lysis buffer, wherein the solid phase carrier comprises a
magnetic microparticle, and wherein the first functional group
comprises a COOH functional group.
3. The kit of claim 2, wherein the second functional group
comprises an oligo dT functional group, or a derivative
thereof.
4. The kit of claim 2, wherein the second functional group
comprises an oligonucleotide comprising a sequence that is
complementary to a beta globin RNA sequence.
5. A kit comprising a solid phase carrier having a surface
comprising heterologous first and second functional groups, wherein
the solid phase carrier comprises a magnetic microparticle, and
wherein the first functional group comprises a COOH functional
group.
6. The kit of claim 5, wherein the second functional group
comprises a streptavidin group.
7. The kit of claim 6, comprising a buffer that is selected from
the group consisting of a lysis buffer, a binding buffer, a wash
buffer, an elution buffer, or a combination thereof.
8. The kit of claim 5, wherein the second functional group
comprises a polymyxin B functional group or a Limulus anti-LPS
factor functional group or a derivative thereof.
9. The kit of claim 8, wherein the Limulus anti-LPS factor
functional group, or a derivative thereof, is native or
recombinant.
10. The kit of claim 5, wherein the second functional group
comprises polyethyleneimine or poly-L-lysine or a derivative
thereof.
11. The kit of claim 5, wherein the second functional group
comprises an oligo dT functional group, or a derivative thereof.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 11/194,072 filed Jul. 29, 2005, which claims the benefit of
U.S. Provisional Application No. 60/592,917, filed Jul. 30, 2004.
The entire teachings of the above applications are incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Many molecular biology applications, such as capillary
electrophoresis, nucleotide sequencing, reverse transcription
cloning and gene therapy protocols, which contemplate the
transfection, transduction or microinjection of mammalian cells,
require the isolation of high quality nucleic acid and peptide
preparations. A need exists for methods which produce such high
quality nucleic acid and peptide preparations.
SUMMARY OF THE INVENTION
[0003] The present invention is directed to a method of selectively
isolating a target species of nucleic acid molecule present in a
mixture, comprising combining the mixture with solid phase carriers
having a surface comprising a first functional group which binds
nucleic acids and a second functional group which selectively binds
the target species of nucleic acid, thereby producing a first
combination. The first combination is maintained under conditions
appropriate for binding of the nucleic acids to the first
functional group. The solid phase carriers are removed from the
first combination, and combined with an agent that removes (elutes)
the nucleic acid from the first functional group of the solid phase
carriers and promotes (allows, causes) binding of the target
species of nucleic acid to the second functional group of the solid
phase carriers, thereby producing a second combination. The solid
phase carriers are separated from the second combination, thereby
isolating the target species of nucleic acid present in the mixture
comprising nucleic acids.
[0004] The present invention is also directed to a method of
selectively isolating a target species of nucleic acid molecule
present in a mixture comprising nucleic acids, comprising combining
the mixture with solid phase carriers having a surface comprising a
first functional group which binds nucleic acids and a second
functional group which selectively binds the target species of
nucleic acid, thereby producing a first combination. The first
combination is maintained under conditions appropriate for binding
of the nucleic acids to the first functional group. The solid phase
carriers are removed from the first combination, and combined with
a first agent that removes the nucleic acid from the first
functional group of the solid phase carriers, and a second agent
that allows binding of the target species of nucleic acid to the
second functional group of the solid phase carriers, thereby
producing a second combination. The solid phase carriers are
separated from the second combination, thereby isolating the target
species of nucleic acid present in the mixture comprising nucleic
acids.
[0005] In one embodiment, the invention is directed to a method of
isolating mRNA present in a mixture comprising nucleic acids,
comprising combining the mixture with solid phase carriers having a
surface comprising a first functional group which binds nucleic
acids and a second functional group which selectively binds mRNA,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the nucleic
acids to the first functional group. The solid phase carriers are
separated from the first combination and combined with an agent
that removes the nucleic acid from the first functional group of
the solid phase carriers and binds the mRNA to the second
functional group of the solid phase carriers, thereby producing a
second combination. The solid phase carriers are removed from the
second combination, thereby isolating mRNA present in a mixture
comprising nucleic acids.
[0006] The present invention is also directed to a method of
isolating mRNA present in a mixture comprising nucleic acids,
comprising combining the mixture with solid phase carriers having a
surface comprising a first functional group which binds nucleic
acids and a second functional group which selectively binds mRNA,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the nucleic
acids to the first functional group. The solid phase carriers are
removed from the first combination and combined with a first agent
that removes the nucleic acid from the first functional group of
the solid phase carriers, and a second agent that allows binding of
the mRNA to the second functional group of the solid phase
carriers, thereby producing a second combination. The first and
second agent can be added simultaneously or sequentially. The solid
phase carriers are separated from the second combination, thereby
isolating mRNA present in a mixture comprising nucleic acids.
[0007] A method of separating globin RNA from nucleic acid present
in a mixture is also encompassed by the present invention. The
method comprises combining the mixture with solid phase carriers
having a surface comprising a first functional group which binds
nucleic acids and a second functional group which selectively binds
globin RNA, thereby producing a first combination. The first
combination is maintained under conditions appropriate for binding
of the nucleic acids to the first functional group. The solid phase
carriers are separated from the first combination and combined with
at least one agent that removes the nucleic acid from the first
functional group of the solid phase carriers and binds the globin
RNA to the second functional group of the solid phase carriers,
thereby producing a second combination. The solid phase carriers
are separated from the second combination, thereby isolating globin
RNA present in a mixture comprising nucleic acids. In a particular
embodiment, the solid phase carriers are combined with a first
agent that removes the nucleic acid from the first functional group
of the solid phase carriers, and a second agent that allows binding
of the globin RNA to the second functional group of the solid phase
carriers, thereby producing the second combination. The first and
second agent can be added simultaneously or sequentially. In
another embodiment, the first functional group is COOH and the
second functional group is an oligonucletoide comprising a sequence
that is complementary to globin RNA sequence.
[0008] In a particular embodiment, the invention is directed to a
method of separating globin RNA from nucleic acid present in a
mixture, comprising combining the mixture with biotin labeled
oligonucleotides comprising sequences that are complementary to
globin RNA sequences present in the mixture, thereby producing a
first combination. The first combination is maintained under
conditions in which hybridization occurs between the
oligonucleotides and the globin RNA, and combined with solid phase
carriers having a first functional group that binds nucleic acid
and a second functional group that selectively binds biotin,
thereby producing a second combination. The second combination is
maintained under conditions in which the nucleic acid binds to the
first functional groups and the oligonucleotides which are
hybridized to the globin RNA, bind to the second functional group
of the solid phase carriers. The solid phase carriers are separated
from the second combination and combined with an agent that elutes
the nucleic acid from the first functional group, thereby
separating globin RNA from nucleic acid present in the mixture.
[0009] The present invention is also directed to a method of
separating endotoxin from nucleic acid in a mixture, comprising
combining the mixture with solid phase carriers having a surface
comprising a first functional group which binds nucleic acids and a
second functional group which selectively binds endotoxin, thereby
producing a first combination. The first combination is maintained
under conditions appropriate for binding of the nucleic acids to
the first functional group and binding of endotoxin to the second
functional group. The solid phase carriers are separated from the
first combination and combined with at least one agent that removes
the nucleic acid from the first functional group of the solid phase
carriers, thereby producing a second combination. The solid phase
carriers, to which the endotoxin is still bound, are separated from
the second combination, thereby separating endotoxin from nucleic
acid present in the mixture. In a particular embodiment, the first
functional group is COOH and the second functional group is
selected from the group consisting of: polymyxin B, native Limulus
anti-LPS factor (LALF) and recombinant LALF.
[0010] In a particular embodiment, the invention is directed to a
method of separating endotoxin from nucleic acid in a mixture
comprising combining the mixture with solid phase carriers having a
surface comprising a first functional group which binds nucleic
acids and a second functional group which selectively binds
endotoxin, thereby producing a first combination. The first
combination is maintained under conditions appropriate for binding
of endotoxin to the second functional group. The solid phase
carriers are separated from the first combination and combined with
solid phase carriers having a surface comprising a functional group
which binds nucleic acids with the first combination, thereby
producing a second combination. The second combination is
maintained under conditions appropriate for binding of nucleic acid
to the functional group of the solid phase carriers and the solid
phase carriers are separated from the second combination, thereby
separating endotoxin from nucleic acid present in the mixture.
[0011] The present invention is also directed to a method of
isolating nucleic acid of an organism comprising combining the
organism with solid phase carriers having a surface comprising a
first functional group which binds the organism and a second
functional group that binds nucleic acid, thereby producing a first
combination. The first combination is maintained under conditions
in which the organism binds to the first functional group. The
solid phase carriers are separated from the first combination and
combined with an agent that lyses the organism and binds the
nucleic acid of the organism to the second functional group,
thereby producing a second combination. The second combination is
maintained under conditions in which the organism is lysed and the
nucleic acid of the organism binds to the second functional group,
thereby isolating the nucleic acid of the organism.
[0012] The present invention also relates to a method of separating
forward extension products and reverse extension products of a
sequencing reaction comprising combining a sequencing reaction
mixture which comprises forward extension products and reverse
extension products with solid phase carriers having a surface
comprising a first functional group which selectively binds the
forward extension products and a second functional group which
binds nucleic acid, thereby producing a first combination. The
first combination is maintained under conditions appropriate for
binding of the forward extension products to the first functional
group and binding of the reverse extension products to the second
functional group. The solid phase carriers are separated from the
first combination and combined with a buffer that selectively
elutes the reverse extension products from the second functional
group of the solid phase carriers, thereby producing a second
combination. The solid phase carriers are separated from the second
combination, thereby separating forward extension products and
reverse extension products of the sequencing reaction.
[0013] The present invention also relates to a method of separating
forward extension products and reverse extension products of a
sequencing reaction comprising combining a sequencing reaction
mixture which comprises forward extension products and reverse
extension products with solid phase carriers having a surface
comprising a first functional group which binds nucleic acid and a
second functional group that selectively binds the forward
extension products, thereby producing a first combination. The
first combination is maintained under conditions appropriate for
binding of the forward extension products to the second functional
group and binding of the reverse extension products to the first
functional group. The solid phase carriers are separated from the
first combination and combined with a buffer that selectively
elutes the reverse extension products from the first functional
group of the solid phase carriers, thereby producing a second
combination. The solid phase carriers are separated from the second
combination, thereby separating forward extension products and
reverse extension products of the sequencing reaction.
[0014] In a particular embodiment, the present invention relates to
a method of separating forward extension products and reverse
extension products of a sequencing reaction comprising combining a
sequencing reaction mixture which comprises forward extension
products and reverse extension products with solid phase carriers
having a surface comprising a first functional group which
selectively binds the forward extension products and a second
functional group which binds nucleic acid, thereby producing a
first combination. The first combination is maintained under
conditions appropriate for binding of the forward extension
products to the first functional group. The solid phase carriers
are separated from the first combination, thereby producing a
second mixture comprising the reverse extension products. The solid
phase carriers are combined with a buffer that selectively elutes
the forward extension products from the first functional group of
the solid phase carriers, thereby producing a second combination.
The solid phase carriers are separated from the second combination
and combined with the second mixture, thereby producing a third
combination. The third combination is maintained under conditions
appropriate for binding of the reverse extension products to the
second functional group and combined with a buffer that selectively
elutes the reverse extension products from the second functional
group of the solid phase carriers, thereby separating forward
extension products and reverse extension products of the sequencing
reaction.
[0015] The present invention is also directed to kits for use in
the methods of the present invention. In one embodiment, the kit
comprises bifunctional beads and a cell lysis buffer. In a
particular embodiment, the kit comprises bifunctional magnetic
microparticles comprising COOH groups and oligo dT groups and cell
lysis buffer. In another embodiment, the kit comprises bifunctional
magnetic microparticles comprising COOH groups and streptavidin
groups, and buffers such as binding and/or wash buffers. In another
embodiment, the kit comprises heterobifunctional magnetic
microparticles comprising COOH functional groups and polymyxin B
functional groups. In yet another embodiment, the kit comprises
heterobifunctional magnetic microparticles comprising COOH
functional groups and LALF functional groups. The kits of the
present invention can further comprise additional buffers such as
wash buffers and elution buffers.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawing(s) will be provided by the Office
upon request and payment of the necessary fee.
[0017] FIG. 1 is a schematic of a solid phase based purification
procedure using two functional groups on magnetic beads which
separates a duplex sequencing reaction into its respective forward
and reverse primers.
[0018] FIG. 2 show the sequencing results of a duplex sequencing
reaction using the solid based purification procedure.
[0019] FIG. 3 is a gel showing that both carboxyl beads and
bi-functional oligo-dT carboxyl beads are able to isolate genomic
DNA and total RNA via the solid phase reversible immobilization
(SPRI) technique.
[0020] FIG. 4 show graphs of experimental data which illustrate
mRNA isolation from cells.
[0021] FIG. 5 shows three graphs of experimental data which
illustrate mRNA isolation from cells.
[0022] FIG. 6 is a schematic of the standard carboxyl bead and the
oligo-dT bead.
[0023] FIG. 7 is a schematic showing poly-A RNA hybridized to the
oligo-dT bead.
[0024] FIG. 8 is a schematic of direct mRNA preparation.
[0025] FIG. 9 is a graph showing PMX-B added to solution versus
PMX-B bound to beads.
[0026] FIG. 10 is a schematic of endotoxin removal from a plasmid
preparation.
[0027] FIG. 11 is a bar graph showing endotoxin removal with
adsorbed PMXB/CM beads.
[0028] FIG. 12 is a bar graph showing endotoxin removal with
PMXB/CM beads as a function of time.
[0029] FIG. 13 is a bar graph showing endotoxin removal using LALF
coupled CM beads.
[0030] FIG. 14 is a bar graph showing preparation of PMX-B
covalently coupled magnetic beads.
[0031] FIG. 15 is a schematic illustration of bi-functional PMXB-CM
beads.
[0032] FIG. 16 is a bar graph showing removal of endotoxin from
plasmid DNA with bifunctional PMXB-CM.
[0033] FIGS. 17A and 17B are bar graphs showing transfection of
endotoxin free DNA prepared with bi-functional beads.
[0034] FIG. 18 shows the structure of lipopolysaccharide (LPS).
[0035] FIG. 19 shows that structure of polymyxin B (PMXB).
[0036] FIG. 20 is a gel showing isolation of virus using
bifunctional solid phase carriers.
[0037] FIG. 21 shows an electropheregram of total RNA isolated from
0.3 mls human blood using this protocol.
[0038] FIG. 22 is a schematic of a heteromultifunctional solid
phase carrier.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is based, in part, on the discovery
that at least two molecules present in a mixture can be separated
using a solid phase carrier having a surface comprising at least
two, distinct (heterologous) functional groups (multifunctional
groups). Two separate binding events occur wherein each molecule
binds to a distinct functional group present on the solid phase
carrier, thereby providing for the separation of the molecules. The
binding events that occur on the solid phase carrier can occur
simultaneously (in one step) or sequentially (in more than one
step).
[0040] Accordingly, the present invention is directed to methods of
separating or isolating a (one or more) target species present in a
mixture of species using solid phase carriers having a surface
comprising at least two, distinct (heterologous) functional groups
(multifunctional groups), wherein the target species binds to one
of the functional groups and the species from which the target
species is being separated binds to another functional group that
is distinct from the group to which the target species is bound.
Examples of target species present in a mixture that can be
isolated using the methods of the present invention include nucleic
acids (e.g., DNA, RNA), peptides (e.g., polypeptide, protein),
saccharides (e.g., polysachharides, lipopolysaccharides), whole
organisms (e.g., virus) and contaminants. Examples of suitable
mixtures or starting material which comprise the target species
(e.g., a target nucleic acid species) for use in the methods of the
present invention include biological samples (e.g., blood, tissue,
tissue lysates, cells, cell lysates), and the products of nucleic
acid manipulations (e.g., sequencing reactions) used for molecular
diagnostics, expression profiling, genotyping and transfection.
[0041] The methods are particularly suited for separating or
isolating a target nucleic acid species in a mixture from other
species or components present in the mixture, using solid phase
carriers having a surface comprising at least two, distinct
functional groups, wherein the target nucleic acid species binds to
one of the functional groups and the one or more component(s) from
which the target species is being separated binds to another
functional group that is distinct from the functional group to
which the target nucleic acid species is bound. The other
components in the mixture from which the target nucleic acid
species can be separated include such components as other nucleic
acid species, peptides, saccharides, whole organisms and
contaminants.
[0042] The multifunctional solid phase carriers of the present
invention can be used to separate or isolate nucleic acid species
and peptide species present in a mixture comprising a plurality of
nucleic acid species and peptide species. In one embodiment, the
present invention is directed to a method of isolating a target
nucleic acid species from a mixture comprising a plurality of
nucleic acid species and peptide species. In another embodiment,
the present invention is directed to a method of isolating a target
nucleic acid species from a mixture comprising a plurality of
nucleic acid species. The mixture is combined with solid phase
carriers having a surface comprising multiple functional groups
which reversibly bind nucleic acid and peptides.
[0043] In a particular embodiment, the present invention is
directed to a method of separating a (one or more) nucleic acid
species in a mixture from a (one or more) peptide species in the
mixture. The mixture is combined with solid phase carriers having a
first functional group which reversibly binds nucleic acids and a
second functional group which reversibly binds the peptide species,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the nucleic
acids to the first functional group and binding of the peptide
species to the second functional group. The solid phase carriers
are separated from the first combination, and combined with an
agent (e.g., buffer) that selectively removes (e.g., elutes) either
the nucleic acid from the first functional group or the peptide
species from the second functional group of the solid phase
carriers, thereby separating a nucleic acid species in a mixture
from one or more peptide species in the mixture.
[0044] In another embodiment, the present invention is directed to
a method of isolating a target nucleic acid species from a mixture
comprising a plurality of nucleic acid species. The mixture is
combined with solid phase carriers having a first functional group
which reversibly binds nucleic acids and a second functional group
which selectively and reversibly binds the target nucleic acid
species, thereby producing a first combination. The first
combination is maintained under conditions appropriate for binding
of the nucleic acids to the first functional group and binding of
the target nucleic acid species to the second functional group. In
a particular embodiment, the second functional group has a higher
affinity for the target nucleic acid species than the first
functional group, and thus, the target nucleic acid species
preferably binds to the second functional group. The solid phase
carriers are separated from the first combination, and combined
with an agent (e.g., buffer) that selectively removes (e.g.,
elutes) either the nucleic acid from the first functional group or
the target nucleic acid species from the second functional group of
the solid phase carriers, thereby isolating a target nucleic acid
species from a mixture comprising a plurality of nucleic acid
species.
[0045] The solid phase carriers can be separated from the first
combination, and combined with an agent (e.g., buffer) that
selectively removes (e.g., elutes) the target nucleic acid species
from the second functional group of the solid phase carriers,
thereby isolating a target nucleic acid species from a mixture
comprising a plurality of nucleic acid species. That is, combining
the solid phase carriers with the agent results in removal of the
target nucleic acid species from the second functional group but
not removal of nucleic acid bound to the first functional group. In
the presence of the agent, the nucleic acid remains bound to the
first functional group on the solid phase carriers.
[0046] Alternatively, the solid phase carriers can be separated
from the first combination, and combined with an agent that
selectively removes the nucleic acid species from the first
functional group of the solid phase carriers, while the target
nucleic acid remains bound to the second functional group on the
solid phase carriers. That is, combining the solid phase carriers
with the agent results in removal of the nucleic acid from the
first functional group but not removal of the target nucleic acid
species bound to the second functional group. The agent (e.g., a
buffer; an enzyme) can result in, for example, either elution or
degradation of the nucleic acid bound to the first functional
group. The solid phase carriers to which are bound the target
nucleic acid species are then removed, thereby isolating the target
nucleic acid species from a mixture comprising a plurality of
nucleic acid species. The target nucleic acid species can be eluted
from the second functional group on the solid phase carriers.
[0047] In a particular embodiment, the present invention is
directed to a method of isolating a target nucleic acid species
present in a mixture comprising nucleic acids. The mixture is
combined with solid phase carriers having a surface comprising a
first functional group which reversibly binds nucleic acids and a
second functional group which selectively and reversibly binds the
target species of nucleic acid, thereby producing a first
combination. The first combination is maintained under conditions
appropriate for binding of the nucleic acids to the first
functional group. In this embodiment, the nucleic acid in the
sample, including the target nucleic acid species, bind to the
first functional group. The solid phase carriers are separated from
the first combination and can be combined with an agent that
removes the nucleic acid from the first functional group of the
solid phase carriers and promotes (allows) binding (selective
binding) of the target nucleic acid species to the second
functional group on the solid phase carriers, thereby producing a
second combination. In the second combination, the target species
nucleic acid is bound to the second functional group, while other
nucleic acid remain in solution. Alternatively, the solid phase
carriers which have been separated from the first combination can
be combined with an agent that removes the nucleic acid from the
first functional group, and a second agent that promotes (causes)
the selective binding of the target species of nucleic acid to the
second functional group of the solid phase carriers is added,
thereby producing a second combination. The solid phase carriers
are separated from the second combination, thereby isolating the
target species of nucleic acid present in the mixture comprising
nucleic acids.
[0048] The multifunctional solid phase carriers described herein
can also be used to purify RNA from a mixture such as a cell or
cell lysate. For example, mRNA and/or globin RNA can be isolated or
separated from a mixture using the methods described herein.
[0049] In one embodiment, total nucleic acid present in the cell or
cell lysate is bound to solid phase carriers which comprise free
COOH groups and COOH groups to which oligo dT groups have been
covalently attached as a second functional group, thereby isolating
the nucleic acid in the mixture. Following nucleic acid isolation,
the solid phase carriers are combined with a buffer (e.g., a low
ionic strength buffer) which promotes elution of total nucleic acid
and subsequent binding of the poly-A of the mRNA to the oligo-dT
functional group on the solid phase carriers. The solid phase
carriers can be removed and the mRNA can be eluted from the solid
phase carriers, thereby isolating the mRNA. Prior to elution of the
mRNA, the solid phase carriers can be washed with a suitable wash
buffer.
[0050] In one embodiment, the invention is directed to a method of
isolating mRNA present in a mixture comprising nucleic acids. The
mixture is combined with solid phase carriers having a surface
comprising a first functional group which binds nucleic acids and a
second functional group which selectively binds mRNA, thereby
producing a first combination. The first combination is maintained
under conditions appropriate for binding of the nucleic acids to
the first functional group. The solid phase carriers are separated
from the first combination and combined with at least one agent
that removes the nucleic acid from the first functional group of
the solid phase carriers and allows binding of the mRNA to the
second functional group of the solid phase carriers, thereby
producing a second combination. The solid phase carriers are
removed from the second combination, thereby isolating mRNA present
in a mixture comprising nucleic acids. The mRNA can then be eluted
from the solid phase carriers in a suitable elution buffer. In a
particular embodiment, the solid phase carriers are combined with a
first agent that removes the nucleic acid from the first functional
group of the solid phase carriers, and a second agent that allows
binding of the mRNA to the second functional group of the solid
phase carriers, thereby producing the second combination.
[0051] The multifunctional solid phase carriers described herein
can also be used to separate globin RNA from nucleic acid present
in a mixture. Examples of globin RNA that can be isolated using the
methods described herein include alpha, beta, delta, gamma,
epsilon, theta and zeta globin RNA.
[0052] In one embodiment, the present invention relates to a method
of removing globin nucleic acid sequences present in a mixture of
nucleic acid, comprising combining he mixture with solid phase
carriers having a first functional group which binds nucleic acids
and a second functional group which selectively binds nucleic acids
containing globin specific sequences, thereby producing a first
combination. The first combination is maintained under conditions
appropriate for binding of the nucleic acids to the first
functional group. The solid phase carriers are separated from the
first combination and combined with at least one agent that removes
the nucleic acid from the first functional group of the solid phase
carriers and promotes (allows) binding of globin containing
sequences to the second functional group of the solid phase
carriers, thereby producing a second combination. The solid phase
carriers are removed from the second combination, thereby removing
globin specific sequence from the second combination.
[0053] In another embodiment, the method of removing globin nucleic
acid sequences present in a mixture of nucleic acid comprises
combining the mixture with solid phase carriers having a first
functional group which binds nucleic acids and a second functional
group which selectively binds nucleic acids containing globin
specific sequences, thereby producing a first combination. The
first combination is maintained under conditions appropriate for
binding of the nucleic acids to the first functional group. The
solid phase carriers are separated from the first combination and
combined with a first agent that removes the nucleic acid from the
first functional group of the solid phase carriers. A second agent
that allows selective binding of globin containing sequences to the
second functional group of the solid phase carriers is added,
thereby producing a second combination. The solid phase carriers
are removed from the second combination, thereby removing globin
specific nucleic acid sequence(s) from the second combination.
[0054] In another embodiment, the method of removing globin nucleic
acid sequences present in a mixture of nucleic acid sequences
comprises combining a mixture of nucleic acid sequences with biotin
labeled oligonucleotides complementary in sequence to globin
sequences under conditions that promote hybridization between the
biotin labeled oligonucleotides and the globin sequences thereby
producing a first combination. The first combination is combined
with solid phase carriers having a first functional group that
binds nucleic acid and a second functional group which selectively
binds biotin, producing a second combination. This second
combination is maintained under conditions appropriate for binding
of the hybrids formed between the biotin labeled oligonucleotides
and the globin sequences. An agent which promotes binding of the
remaining nucleic acid to the first functional group is added,
thereby producing a third combination. The solid phase carriers are
removed from the third combination, washed, and combined with an
agent that elutes nucleic acid bound to the first functional group,
thereby separating globin sequences from the nucleic acid
mixture.
[0055] The multifunctional solid phase carriers described herein
can also be used to separate contaminants from nucleic acid in a
mixture or sample. Introduction of high-throughput DNA preparation
methods has fueled the growth of large-scale sequencing efforts and
has resulted in the generation of a vast collection of genomic and
expressed gene sequences. Further characterization of these
sequences in vitro may be applied using high-throughput analysis of
expressed genes for the evaluation of mammalian cell function
(Ziauddin, J., et al., Nature, 411:107-110 (2001). Moreover, gene
therapy studies in vivo using plasmid and BAC DNA have been widely
applied in animal models for both the characterization of disease
states and for the evaluation of potential therapeutic intervention
(Nabel, G. J., et al., Proc. Natl. Acad. Sci., USA, 90:11307-11311
(1993)). Additionally, the increased understanding of genetic
immunization using naked DNA vaccines has also held great promise
as a novel therapeutic deliverable (Lewis, P. J., et al., Adv.
Virus Res., 54:129-188 (1999); Liu, M., J. Intern. Med.,
253:402-410 (2003)). Unfortunately, such applications and therapies
are very sensitive to contaminants typically present in nucleic
acid preparations. Standard methods of plasmid DNA isolation from
bacteria, including alkaline lysis, high pressure (French Press)
boiling, and the use of lysozyme or detergents will induce the
release of lipopolysaccharide (LPS), or endotoxin, from the outer
membrane of the bacteria along with plasmid DNA. The LPS will then
form micelles with physical characteristics (density, size, and
charge distribution) similar to plasmid DNA, and as a consequence,
be carried through the purification steps along with the plasmid
DNA. Contaminating endotoxins from E. coli host typically used to
prepare DNA molecules has been shown to induce apoptosis during
culture of mammalian cell in vitro (Kuwabara, T., Apoptosis,
9:467-474 (2004)), as well as toxic shock, sepsis and a variety of
related clinical complications in vivo (DiPiro, J. T., Am. J. Hosp.
Pharm., 47:S6-10 (1990)). The transfection efficiency of
endotoxin-containing DNA in mammalian cells such as HeLa, Huh7,
COS7, and LNH is reduced significantly compared to endotoxin-free
DNA (Weber, M., et al., BioTechniques, 19:930-940 (1995)).
[0056] Endotoxins are constituents of the outer-membrane of
Gram-negative bacteria that contribute to the organization and
stability of the outer membrane. First termed by R. Pfeiffer
(1858-1945), endotoxin characterizes a class of lipopolysaccahrides
(LPS) that have since been well characterized both structurally and
chemically (Rietschel, et al., 1994). The general structure of all
endotoxins is a polar heteropolysaccharide chain, covalently linked
to a non-polar moiety (lipid A). As dominant bacterial membrane
structures, endotoxins participate in the interaction of the
bacterial cell with its surroundings. Endotoxins do not act
directly against cells or organs but through activation of the
immune system (Anspach 2001). When gram-negative bacteria gains
access to a mammalian host, the presence of endotoxin activates the
host's immune system and has been shown to be involved in the
pathogenesis of inflammation and septic shock in the host. Small
quantities of endotoxin have been shown to alter phenotypes of
various cell types (Gould et al. 1984), particularly mononuclear,
endothelial, smooth muscle cells and polymorphonuclear granulocytes
and monocytes (Galanos and Freudenberg 1993; Galanos et al. 1992).
These cell types respond to endotoxin presence by producing
bioactive lipids, reactive oxygen species and various peptide
inflammatory mediators (Rietschel et al., 1994). It is this type of
cellular response that causes endotoxins to produce striking
pathophysiological reactions when introduced into animals including
high fever, vasodilation, diarrhea and, in extreme cases, fatal
shock (Morrison, D. C., Ann. Rev. Med., 38:417-432 (1987)).
[0057] In addition to toxic effects on cells from in vivo
introduction of gram negative bacteria, endotoxin has been shown to
exert toxicity on mammalian cells in vitro. Transfection efficiency
of endotoxin contaminated DNA is hindered due to toxic effects of
endotoxin on mammalian cells such as HeLa, Huh7, COS7 and LNH
(Weber, M., et al., Biotechniques, 19:930-939, 1995). This toxicity
is seen when introducing DNA using either adenovirus, glycerol or
cationic lipid based trasnfections and is attributed to the lipid A
component of endotoxin. Lipid A itself has no consequences, but the
introduction of endotoxin into the vesicular system, cytoplasm, or
nucleus of cells during transfection leads to an apoptotic pathway
(Cotton and Saltik, 1997). As could be expected, endotoxins also
exert toxicity during introduction of DNA in vivo in processes such
as microinjection and gene therapy research (Weber et al., 1995;
Vukajlovich, S. W. et al., 1987; Schleef, M. 1999). The adverse
reactions make it imperative to remove endotoxin from drugs,
injectables and other biological and pharmaceutical products as
well as from plasmid DNA preparations. Studies on humans using such
products have resulted in strict guidelines by the FDA which
require that nucleic acids used for any therapeutic application
have less than 300 EU/mg or 300 IU/mg (U.S. Pat. No.
6,297,371).
[0058] A number of peptide, proteins and receptor motifs interact
strongly with endotoxins, and thus, can be used, in the methods of
the present invention. Some of these include lipopolysaccharide
binding protein (LBP), bactericidal/permeability-increasing protein
(BPI) (Beamer, L. J., Protein Sci., 7:906-914 (1998)), polymyxin
and polymyxin analogs (Jacobs, D. M., et al., J. Immunol.,
118:21-27)1997)), amyloid P component (de Haas, C. J., et al.,
Infect. Immun., 67:2790-2796 (1999)), cationin protein 18 (de Haas,
C. J., et al., Biochem. Biophys. Res. Comm., 252:492-496 (1998)),
MD-2 and Toll-like receptor (TLR) (Shimazu, R., et al., J. Exp.
Med., 189:1777-1782 (1999)), TLR2 (Sabroe, I., et al., J. Immunol.,
168:4701-4710 (2002)), CD14 (Soler-Rodriguez, A. M., et al., J.
Immunol., 164:2674-2683 (2000)), Bac7 (About, S., et al., Cancer
epidemiology, biomarkers and prevention, 11:1130-1133 (2002), Liu,
M. A., J, Intern. Med., 253:402-410 (2003)), a synthetic peptide
derived from a protein found in bovine neutrophils (Ghiselli, R.,
et al., Shock, 19:577-581 (2003)), limulus factor-C and synthetic
peptides derived from Sushi3 domain thereof (Li. C., et al.,
Protein Eng., 116:629-635 (2003)) and antibodies raised against the
lipid A component of endotoxin (Helmerhorst, E. M., et al., Infect.
Immun., 66:870873 (1998); Holy, R. A., et al., Science, 298:129-149
(2002)).
[0059] One molecule that has been extensively used as an endotoxin
absorbent is polymyxin B (PMXB), a cyclic cationic polypeptide
antibiotic. PMXB binds stoiciometrically to the lipid A moiety of
endotoxin molecules, primarily through the hydrophobic interactions
(Srimal, S., et al., Biochem. J., 315:679-686 (1996)).
Specifically, PMXB and endotoxin associate primarily due to
interactions of a hydrophobic patch at one side of the peptide and
Lipid A component of endotoxin (Srimal, et al. 1996).
[0060] Another particular molecule that can be used in the methods
of the present invention is Limulus anti-LPS factor (LALF) isolated
from the American Horseshoe crab (Limulus polyphemus). This is the
protein used in the LAL assay to determine endotoxin levels. In the
methods of the present invention, LALF native protein and
recombinant protein (e.g., LALF expressed in Pichia pastoris; LALF
expressed in Saccharomyces cerevisiae) can be used.
[0061] A protein or molecule which binds endotoxin can be adsorbed
onto carboxy containing solid phase carrier via charge and
hydrophobicity interactions. PXMB carries a net positive charge at
pH<10, and readily associates with carboxy solid phase carriers
by simple incubation of the molecule in the presence of MES buffer
and a solid phase carrier solution (1% solids) for 24 hours.
[0062] In a particular embodiment, covalent coupling is used.
Covalent coupling of a molecule which binds endotoxin (e.g., PMXB
molecule) to a carboxy group on a solid phase carrier allows for a
heterobifunctional solid phase carrier which comprises two distinct
functional groups (e.g., COOH and PMXB) on the solid phase carrier
surface each designed to target a different biochemical molecule.
For example, by covalently coupling PMXB to carboxylated solid
phase carriers, it is possible to drive endotoxin to the PMXB
functional group on the solid phase carrier under low salt
conditions and subsequently drive nucleic acid to the COOH
functional group on the same solid phase carrier under different
conditions. Once the solid phase carriers are separated from the
mixture, and the supernatant removed, the DNA can be eluted from
the COOH functional groups on the solid phase carrier under
conditions in which the endotoxin remains attached to the PMXB
functional groups on the same solid phase carrier.
[0063] In one embodiment, the invention relates to a method of
removing endotoxin contamination from a nucleic acid solution by
simultaneously binding nucleic acid and endotoxin to bi-functional
solid phase carriers comprising an endotoxin binding group, such as
PMXB or LALF proteins, and a nucleic acid binding group such as
carboxyl groups. The solid phase carriers are added to a solution
containing endotoxin and nucleic acid, such as a bacterial cleared
lysate, under conditions (e.g., buffer conditions) in which binding
of the nucleic acid to the carboxyl functional group and binding of
endotoxin to the endotoxin binding functional group occurs. The
solid phase carriers are removed from the solution and contacted
with a buffer (e.g., a buffer of low ionic strength) which causes
selective elution of nucleic acids from the solid phase carriers.
The solid phase carriers, to which are still bound the endotoxin,
are removed leaving a solution of purified (substantially pure)
nucleic acid.
[0064] In another embodiment, the invention relates to a method of
removing endotoxin contamination from a nucleic acid solution by
simultaneously binding nucleic acid and endotoxin to bi-functional
solid phase carriers comprising an endotoxin binding functional
group, such as PMXB or LALF proteins, and a nucleic acid binding
functional group such as carboxyl groups. The bi-functional solid
phase carriers are added to a solution containing endotoxin and
nucleic acid, such as a bacterial cleared lysate, in buffer
conditions creating a first mixture which causes binding of the
endotoxin to the endotoxin binding functional groups. The buffer
conditions in the first mixture are adjusted to provide conditions
in which nucleic acid binds to the nucleic acid binding functional
group and endotoxin remains bound to the endotoxin binding
functional group. The solid phase carriers are removed from the
solution and mixed with a buffer (e.g., of low ionic strength)
which causes selective elution of nucleic acids from the solid
phase carriers. The solid phase carriers, to which the endotoxin
remains bound, are removed leaving a solution of purified nucleic
acid.
[0065] In yet another embodiment, the invention relates to a method
of removing endotoxin contamination from a nucleic acid solution by
adding bifunctional solid phase carriers containing an endotoxin
binding functional group, such as PMXB or LALF proteins, and a
nucleic acid binding functional group such as carboxyl groups, to a
solution of nucleic acids and endotoxin such as a bacterial lysate,
creating a first mixture in which endotoxin is bound to the solid
phase carriers through the endotoxin binding functional group. The
solid phase carriers are removed from solution leaving a second
mixture of nucleic acids essentially purified from endotoxin.
Bifunctional solid phase carriers are added to the second mixture
under conditions which cause binding of the nucleic acid to the
carboxy functional group creating a third mixture. Alternatively,
solid phase carriers comprising a carboxy group can be added to
create the third mixture. The solid phase carriers are removed from
the third mixture and mixed with a buffer of low ionic strength to
elute purified nucleic acid from the solid phase carriers.
[0066] The methods described herein can also be used to concentrate
a virus from a solution and subsequently isolate viral nucleic
acid. A polycationic polymer, such as the polymer polyethyleneimine
(PEI) or poly-L-lysine (PLL), is covalently coupled to solid phase
carriers containing a carboxyl functional group. Carboxyl groups
are first activated by carbodiimide
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and then
reacted with free amino groups on the polymer. This produces a
solid phase carrier with two functional groups, a polycation
polymer for virus binding, and carboxyl groups for nucleic acid
binding. The polymer modified bi-functional solid phase carriers
are added to a volume of virus containing serum or lysate and mixed
well creating a first mixture in which viral particles in the serum
are concentrated on the surface of the solid phase carriers via
interaction with the polymer. The solid phase carriers are
separated and the serum supernatant is removed. To the solid phase
carriers are added a lysis buffer containing, for example, 20 mM
Tris pH 7.0, 1% Triton-X-100, 2% SLS, 10 mM DTT, isopropanol and an
RNase inhibitor. This results in a second mixture in which virus is
lysed, and upon lysis, viral nucleic acid becomes bound to the
solid phase carriers via interaction with the carboxyl groups. The
solid phase carriers are separated, and can be washed in a wash
buffer and/or 70% ethanol. The solid phase carriers can be dried
and the purified viral nucleic acid is eluted in a low ionic
strength buffer.
[0067] In one embodiment, the present invention is directed to a
method of isolating the nucleic acid of an organism (e.g, a
pathogen, such as a virus, bacteria, fungus, parasite) comprising
combining the organism with solid phase carriers having a surface
comprising a first functional group which binds nucleic acids and a
second functional group which selectively binds the organism,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the organism
to the second functional group; and the solid phase carriers are
separated from the first combination. The solid phase carriers are
combined with an agent that lyses the organism, and upon lysis, the
nucleic acid of the organism is released and becomes bound to the
first functional group on the solid phase carriers. The solid phase
carriers are separated and the organism's nucleic acid can then be
eluted from the solid phase carriers, thereby isolating the nucleic
acid of the oraganism.
[0068] As used herein the term "isolating" is intended to mean that
the material in question exists in a physical milieu distinct from
that in which it occurs in nature and/or has been completely or
partially separated, isolated or purified from other nucleic acid
molecules.
[0069] As used herein the terms "nucleic acid" and "nucleic acid
molecule" are used synonymously with the term polynucleotides and
they are meant to encompass DNA (e.g., single-stranded,
double-stranded, covalently closed, relaxed circular forms, genomic
DNA, cDNA, plasmid DNA), RNA (e.g., single-stranded and
double-stranded, mRNA), cRNA, antisense RNA, micro RNA, RNA/DNA
hybrids and polyamide nucleic acids (PNAs).
[0070] A "solid phase carrier" is an entity that has, or to which
can be added, a functional group (one or more) that reversibly
binds the target species. The solid phase carrier is essentially
insoluble under conditions in which a target species can be
precipitated onto (can bind to) the solid phase carrier. Suitable
solid phase carriers for use in the methods of the present
invention have sufficient surface area to permit efficient binding
of the target species to the functional group(s) on the carriers,
and are further characterized by having surfaces which are capable
of reversibly binding the target species. Suitable solid phase
carriers include, but are not limited to, microparticles (e.g.,
beads), fibers and supports which have an affinity for a target
species, such as nucleic acid, and which can embody a variety of
shapes, that are either regular or irregular in form, and
preferably have a shape that maximizes the surface area of the
solid phase, and embodies a carrier which is amenable to microscale
manipulations. In one embodiment, the solid phase carrier is a
magnetic microparticle (e.g., a paramagnetic (magnetically
responsive) microparticle).
[0071] As used herein, "paramagnetic microparticles" refer to
microparticles which respond to an external magnetic field (e.g., a
plastic tube or a microtiter plate holder with an embedded rare
earth (e.g., neodymium) magnet) but which demagnetize when the
field is removed. Thus, the paramagnetic microparticles are
efficiently separated from a solution using a magnet, but can be
easily resuspended without magnetically induced aggregation
occurring. Particular paramagnetic microparticles comprise a
magnetite rich core encapsulated by a pure polymer shell. In one
embodiment, suitable paramagnetic microparticles comprise about
20-35% magnetite/encapsulation ratio. For example, magnetic
particles comprising a magnetite/encapsidation ration of about 23%,
25%, 28% 30% 32% or 34% are suitable for use in the present
invention. Magnetic particles comprising less than about a 20%
ratio are only weakly attracted to the magnets used to accomplish
magnetic separations. Depending on the nature of the mixture used
in the methods of the present invention, paramagnetic
microparticles comprising a higher percentage of magnite should be
considered. The use of encapsulated paramagnetic microparticles,
having no exposed iron, or Fe.sub.30.sub.4, on their surfaces,
eliminates the possibility of iron interfering with polymerase
function in certain downstream manipulations of the isolated
nucleic acid. However the larger the magnetite core the higher the
chance of encapsulation leakage (e.g., release of iron oxides).
[0072] Suitable paramagnetic microparticles should be of a size
that their separation from solution, for example by magnetic means
or by filtration, is not difficult. In addition, preferred
paramagnetic microparticles are those that are not so large that
their surface area is minimized or that they are unsuitable for
microscale manipulation. Suitable sizes range from about 0.1.mu.
mean diameter to about 100.mu. mean diameter. A preferred size is
about 1.0.mu. mean diameter. Suitable magnetic microparticles for
use in the instant invention can be obtain, for example, from
Agencourt Biosciences, Polysciences, Bioclone, Seradyne, Bangs
Laboratories Inc., Fishers, and IN (e.g., estapor.RTM.
carboxylate-modified encapsulated magnetic microspheres).
[0073] In one embodiment, the target species in the mixture binds
non-specifically to at least one functional group on the solid
phase carrier. "Non-specific binding" refers to binding of
different target species molecules (e.g., different species of
nucleic acid, such as nucleic acid which differ in size) with
approximately similar affinity to the functional groups on the
solid phase carriers, despite differences in the structure (e.g.,
nucleic acid sequence) or size of the different target species
molecules. The binding can occur, for example, via facilitated
adsorption. As used herein, "facilitated adsorption" refers to a
process whereby a precipitating reagent (e.g., a poly-alkylene
glycol, ethanol, isopropanol) is used to promote the precipitation
and subsequent adsorption of a species of DNA molecules, which were
initially in mixture, onto the surface of a solid phase
carrier.
[0074] In another embodiment, the target species in the mixture
binds specifically (selectively) to at least one functional group
on the solid phase carrier. "Specific binding" or "selective
binding" refers to binding of, for example, particular nucleic acid
molecules (e.g., a target nucleic acid species) to one or more
functional groups on the solid phase carriers to the exclusion of
other nucleic acid species in a mixture. In this embodiment, the
functional group has a greater affinity for particular nucleic acid
molecules (e.g., the target nucleic acid species) than other
functional groups on the solid phase carrier. Such reversible
interactions include an interaction between two binding partners.
For example, the interaction can be between two binding partners
which are conventionally utilized for the purpose of isolating
particular biomolecules based on their composition or sequence
(e.g., streptavidin/biotin, antibody/antigen, ligand receptor or a
sequence-specific interaction such as hybridization of
complementary sequences).
[0075] The solid phase carriers used in the methods of the present
invention have a functional group coated surface. In particular,
the surface of the solid phase carriers for use in the methods of
the present invention comprise multiple (at least two), distinct
functional groups. As used herein, the term "functional
group-coated surface" refers to a surface of a solid phase carrier
that is coated with functional groups or moieties which reversibly
bind a target molecule present in a mixture, such as nucleic acid
(e.g., DNA, RNA or polyamide nucleic acids (PNA)), peptides,
saccharides, whole organisms, and contaminants, either directly
(the functional group binds the nucleic acid or peptides) or
indirectly (the functional group (e.g., streptavidin) binds a group
that is linked to the nucleic acid (e.g., biotin) or peptides).
[0076] Methods for coating solid phase carriers with functional
groups, either directly or indirectly, are known in the art. For
example, the functional groups (e.g., COOH) can coat a solid phase
carrier during formation of the solid phase carrier. See, for
example, U.S. Pat. No. 5,648,124 which is incorporated herein by
reference. In additional, solid phase carriers can be coated with
functional groups by covalently coupling a functional group (one or
more) to a COOH group (one or more) on the solid phase carrier. A
particular example of a functional group coated surface is a
surface which is coated with moieties which each have a free
functional group which is bound to the amino group of the amino
silane of the microparticle; as a result, the surfaces of the
microparticles are coated with the functional group containing
moieties. The functional group acts as a bioaffinity adsorbent for
precipitated nucleic acid (e.g., polyalkylene glycol precipitated
DNA) or peptides.
[0077] In one embodiment, at least one of the functional groups is
a carboxylic acid (COOH). A suitable moiety with a free carboxylic
acid functional group is a succinic acid moiety in which one of the
carboxylic acid groups is bonded to the amine of amino silanes
through an amide bond and the second carboxylic acid is unbonded,
resulting in a free carboxylic acid group attached or tethered to
the surface of the solid phase carrier. Carboxylic acid-coated
magnetic particles are commercially available from, for example,
Polysciences, Inc. Carboxy groups play a key role in effective
elution of nucleic acid from a solid phase carrier. Carboxy groups
have a pKa of 4.7 so they are negatively charged at neutral pH.
Nucleic acid, such as DNA, is negatively charged, and in the
absence of any crowding reagents or salt, nucleic acid repels
itself from the microparticles at neutral pH.
[0078] Suitable solid phase carriers having a functional group
coated surface that reversibly binds nucleic acid molecules are for
example, magnetically responsive solid phase carriers having a
functional group-coated surface, such as, but not limited to,
amino-coated, carboxyl-coated and encapsulated carboxyl
group-coated paramagnetic microparticles.
[0079] In a particular embodiment, other functional groups can be
coupled to the solid phase carriers through carboxyiimide coupling
to carboxy groups on the surface of the solid phase carrier. Solid
phase carriers having a high density of carboxyl groups on the
surface can be contacted with another functional group (e.g.,
oligo-dT) that binds to some but not all of the carboxy groups
through carbodiimide coupling. Sufficient carboxy functional groups
remain (which can be used, for example, to bind nucleic acid)
following carboiimide coupling to a distinct functional group
resulting in a solid phase carrier having dual functionality
wherein binding of nucleic acid to the carboxy groups and a binding
of a separate moiety to the second functional group can occur.
Thus, the solid phase carriers can be used to remove or retain
another target molecule.
[0080] Functional groups that bind target species, such as nucleic
acids and peptides, are well known in the art (e.g., see Hermanson,
G. T., Bioconjugate Techniques, Academic Press, San Diego, Calif.
(1996) which is incorporated herein by reference). Functional
groups that bind nucleic acid and peptides directly include, for
example, metal ions, an amine group, a carboxyl group, an
encapsulated carboxyl group, silica (SiOH), diethyl aminoethyl
(DEAE), and a group which hybridizes to a nucleic acid sequence in
the mixture.
[0081] A functional group which hybridizes to a nucleic acid
sequence can be a nucleic acid sequence that is complementary to
all or a portion of a nucleic acid in the mixture (e.g.,
complementary to all or a portion of the nucleic acid sequence of
the target nucleic acid sequence to be isolated). In a particular
embodiment, the nucleic acid sequence that is complementary is a
sequence that is specific to (characteristic of) the nucleic acid
species to be isolated so that substantially all the nucleic acid
(the majority of nucleic acid species) in the mixture the bind the
complementary sequence comprise the target nucleic acid species,
while other nucleic acid sequences present in the mixture do not
bind to the complementary sequence. For example, the group can be
an oligodeoxythymidine (oligo dT) group which is a polymer of
deoxyribothymidine and is complementary to the adenine nucleotide
polymer (polyadenylate (poly A) tail) at the 3' end of messenger
RNA (mRNA), and is a sequence that is characteristic of mRNA. Oligo
dT groups can be a polymer of from about 3 to about 100 thymidines,
from about 5 to about 75 thymidines, from about 8 to about 60
thymidines, from about 10 to about 50 thymidines, from about 15 to
about 40 thymidines or from about 20 to about 30 thymidines.
Modified oligo dT groups can also be used in the methods of the
present invention. For example, an oligo dT wherein the last two 3'
nucleotides are N or an oligo dT wherein the last two 3'
nucleotides are VN, where "N" is adenine (A), cytosine (C),
thymidine (T) or guanidine (G), and "V" is A, C or G can be used.
In the method of isolating or separating globin RNA form nucleic
acid present in a mixture, the functional group can be an
oligonucleotide having a sequence complementary to all or a portion
(a portion that distinguishes the sequence as a beta globin
sequence) of the sequence of the beta globin being isolated.
[0082] In one embodiment, the functional group is a (one or more)
transition metal ion which binds peptides (proteins) directly. For
example, Immobilized Metal Ion Affinity Chromatography (IMAC) which
was described by Porath et. al. (Nature, 258:598-599) is a
well-known technique used for separation of proteins based on
affinity between amino acid side chains and immobilized transition
metal ions. Immobilized transition metals can form a reversible
coordination complex with electron donor groups on the surface of
proteins. In particular the side chains of histidine, cysteine, and
tryptophan show affinity to transition metals including Cu, Zn, Co,
and Ni. The metal ions are immobilized by way of metal chelators
that are chemically coupled to an immobilized surface such as
agarose, polyacrylimide, and silica. Common chelators include
iminodiacetic acid (IDA), nitriloacetic acid (NTA), carboxymethyl
aspartic acid (CM-Asp), and trsicarboxymethyl ethylene diamine
(TED). Numerous others have been described (e.g., see U.S. Pat. No.
5,047,513 and U.S. Pat. No. 6,623,655; and US Published Application
No. 2002/0019496 A1 which are incorporated herein by reference).
The complexes can be used to effectively separate proteins, which
due to the reversible nature of the binding, can be eluted using
non-denaturing or denaturing conditions. Though IMAC can be used to
separate native proteins based on differing affinities to the metal
complexes, the greatest commercial use has been through the
introduction of a polyhistidine fusion tag to recombinant proteins
and their subsequence purification via immobilized Ni complexes,
generally IDA and NTA coupled to agarose (U.S. Pat. No. 5,284,933
and Schmitt et. al. Molecular Biology Reports, vol. 18, pp.
223-230, 1998, both of which are incorporated herein by reference).
In addition, coupling of metal chelating complexes to microspheres
(Lauer S A and Nolan J P. Cytometry 48:136-145, 2002) and water
soluble polymers have also been described (U.S. Pat. No. 6,703,498,
which is incorporated herein by reference).
[0083] In the methods of separating endotoxins from nucleic acid
present in a mixture, the functional group can be PMXB, LALF,
lipopolysaccharide binding protein (LBP),
bactericidal/permeability-increasing protein (BPI), polymyxin and
polymyxin analogs, amyloid P component, cationin protein, MD-2 and
Toll-like receptor (TLR), CD14, Bac7, a synthetic peptide derived
from a protein found in bovine neutrophils, limulus factor-C and
synthetic peptides derived from Sushi3 domain thereof and
antibodies raised against the lipid A component of endotoxin.
[0084] Groups that bind target species such as nucleic acid or
peptides indirectly bind to a moiety, such as a label or tag, that
is attached to the nucleic acid or peptide. Therefore, nucleic acid
or peptides comprising a tag that can bind to a functional group on
the solid phase carrier can be isolated using the methods of the
present invention. Such groups include, for example, groups that
interact with a binding partner. For example, the functional groups
can be a binding partner which is conventionally used to isolate
particular biomolecules based on their composition or sequence.
Examples of such functional groups for use in the methods of the
present invention include avidin, streptavidin, biotin, an
antibody, an antigen, a sequence-specific interaction (a
hybridizable tag), DNA specific binding protein (e.g., finger
domains, transcription factors) and derivatives thereof.
[0085] In a particular embodiment, the functional group is biotin
or a molecule that comprises biotin. Biotin, a water-soluble
vitamin, is used extensively in biochemistry and molecular biology
for a variety of purposes including macromolecular detection,
purification and isolation, and in cytochemical staining (see,
e.g., U.S. Pat. No. 5,948,624; the entire teachings of which are
incorporated herein by reference). Biotin also has important
applications in medicine in the areas of clinical diagnostic
assays, tumor imaging and drug delivery, and is used extensively in
the field of affinity cytochemistry for the selective labeling of
cells, subcellular structures and proteins. The utility of biotin
arises from its ability to bind strongly to the tetrameric protein
avidin, found in egg white and the tissues of birds, reptiles and
amphibians, or to its chemical cousin, streptavidin, which is
slightly more specific for biotin than avidin. The biotin
interaction with avidin is among the strongest non-covalent
affinities known, exhibiting a dissociation constant of about
1.3.times.10.sup.-15 M (Hermanson, G .T., Bioconjugate Techniques,
Academic Press, San Diego, Calif. (1996), p. 570). In other
embodiments, the functional group is biocytin and/or a biotin
analog (e.g., biotin amido caproate-hydroxysuccinimide ester,
biotin-PEO.sub.4N-hydroxysuccinimide ester, biotin 4-amidobenzoic
acid, biotinamide caproyl hydrazide) and biotin derivatives (e.g.,
biotin-dextran, biotin-disulfide-N-hydroxysuccinimide ester,
biotin-6 amido quinoline, biotin hydrazide, d-biotin-N
hydroxysuccinimide ester, biotin maleimide, d-biotin p-nitrophenyl
ester, biotinylated nucleotides, biotinylated amino acids such as
N.epsilon.- biotinyl-1-lysine) (see, e.g., U.S. Pat. No.
5,948,624).
[0086] In another embodiment, the functional group is avidin or is
a molecule that comprises avidin (avidinylated). Avidin is a
glycoprotein found in egg whites that contains four identical
subunits, each of which possesses a binding site for biotin
(Hermanson, G. T., Bioconjugate Techniques, Academic Press, San
Diego, Calif. (1996), p. 570). Streptavidin and other avidin
analogs can also be used in the methods of the present invention.
Such avidin analogs include, e.g., avidin conjugates, streptavidin
conjugates, highly purified and/or fractionated species of avidin
or streptavidin, non or partial amino acid variants of avidin or
streptavidin (e.g., recombinant or chemically synthesized avidin
analogs with amino acid or chemical substitutions which still allow
for high affinity, multivalent or univalent binding of the avidin
analog to biotin). Streptavidin is another biotin-binding protein
that is isolated from Streptomyces avidinii (Hermanson, supra).
[0087] The functional group can also be an antibody. As used
herein, the term "antibody" encompasses both polyclonal and
monoclonal antibodies (e.g., IgG, IgM, IgA, IgD and IgE
antibodies). The terms polyclonal and monoclonal refer to the
degree of homogeneity of an antibody preparation, and are not
intended to be limited to particular methods of production. Any
antibody or antigen-binding fragment can be used in the methods of
the invention. For example, single chain antibodies, chimeric
antibodies, mammalian (e.g., human) antibodies, humanized
antibodies, CDR-grafted antibodies (e.g., primatized antibodies),
veneered antibodies, multivalent antibodies (e.g., bivalent) and
bispecific antibodies are encompassed by the present invention and
the term "antibody". Chimeric, CDR-grafted or veneered single chain
antibodies, comprising portions derived from different species, are
also encompassed by the present invention and the term "antibody".
The various portions of these antibodies can be joined together
chemically by conventional techniques, or can be prepared as a
contiguous protein using genetic engineering techniques. For
example, nucleic acids encoding a chimeric or humanized chain can
be expressed to produce a contiguous protein. See, e.g., Cabilly et
al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent No.
0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397; Boss et al.,
European Patent No. 0,120,694 B1; Neuberger, M. S. et al., WO
86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276
B1; Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No.
0,239,400 B1; Queen et al., European Patent No. 0 451 216 B1; and
Padlan, E. A. et al., EP 0 519 596 A1. See also, Newman, R. et al.,
BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody,
and Ladner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al.,
Science, 242: 423-426 (1988)) regarding single chain
antibodies.
[0088] Alternatively, the functional group can be an antigen. As
used herein, the term "antigen", "immunogen" or "epitope" (e.g., T
cell epitope, B cell epitope) refer to a substance for which an
antibody or antigen-binding fragment has binding specificity. The
antibodies and antigen-binding fragments for use in the methods of
the invention have binding specificity for a variety of immunogens
(e.g., polypeptides).
[0089] Any number of heterologous (distinct) functional groups
(e.g., heterobifunctional, heterotrifunctional,
heteromultifunctional) can be present on the surface of the solid
phase particles as long as the presence of the functional groups do
not interfere (e.g., chemically, sterically) with the reversible
binding of the target species. In the methods of the present
invention, at least two distinct functional groups are present on
the solid phase carriers. In one embodiment, there can be a
functional group from about every 2 A.sup.2 up to about 200
A.sup.2. In a particular embodiment, there is a functional group
every 50.5 A.sup.2 on the solid phase carrier. In another
embodiment, there is a functional group every 13.8 A.sup.2 on the
solid phase carrier.
[0090] A person of skill in the art can envision any number of
groups on the heteromultifunctional beads for use in the present
invention. The number of distinct functional groups on a solid
phase carrier can be from about 2 to about 100 distinct groups. In
a particular embodiment, the solid phase carrier has two distinct
functional groups (bifunctional solid phase carrier, such as
bifunctional magnetic microparticles). In another embodiment, the
solid phase carrier has two groups, one of which binds nucleic acid
present in a mixture, and the other binds one or more non-nucleic
acid components or moieties (e.g., endotoxin, an organism) present
in the mixture. In yet another embodiment, the solid phase carrier
has two groups, one of which binds al nucleic acid present in a
mixture, and the other binds a particular target nucleic acid
species (e.g., mRNA) present in the mixture.
[0091] In a particular embodiment, at least one of the functional
groups is a streptavidin group or a derivative thereof.
[0092] In other embodiments, the solid phase carriers comprise two,
distinct functional groups wherein the first functional group is a
COOH group and the second functional group is a streptavidin group
or derivative thereof; wherein the first functional group is a COOH
group and the second functional group is an olig dT group or
derivative thereof; first functional group is a COOH group and the
second functional group is a PMXB group or derivative thereof;
first functional group is a COOH group and the second functional
group is a LALF group or derivative thereof; first functional group
is a COOH group and the second functional group is a
polyethyleneimine (PEI) group or derivative thereof; and first
functional group is a COOH group and the second functional group is
a poly-L-lysine (PLL) group or derivative thereof.
[0093] In the methods of the present invention, the combination of
the mixture comprising the target species and the solid phase
carriers are maintained under conditions appropriate for binding of
the target species to the functional groups on the carriers. The
methods and agents (reagents) described herein can be used together
with a variety of purification techniques (e.g., nucleic acid
and/or peptide purification techniques) which involve binding of
nucleic acid to solid phase carriers, including those described in
U.S. Pat. No. 5,705,628 (Hawkins); U.S. Pat. No. 5,898,071
(Hawkins); U.S. Pat. No. 6,534,262 (McKernan et al.), PCT Published
Application No. WO 99/58664, U.S. Published Application No.
20020094519 A1, U.S. Pat. No. 5,047,513, U.S. Pat. No. 6,623,655
and U.S. Pat. No. 5,284,933 the contents of which are herein
incorporated by reference.
[0094] As described herein, one or more agents (e.g., buffers,
enzymes) are used to bind or remove the target species from the
solid phase carriers. The components of the agents needed to bind
and/or remove the target species from the solid phase carriers can
be present in one agent or in multiple agents (e.g., a first agent,
a second agent, a third agent, etc.). Accordingly, when more than
one agent is used in the methods of the present invention, the
agents can be used simultaneously or sequentially. Depending on the
purpose for which the methods described herein are used, one of
skill in the art can determine the number and order of agents to be
used in the methods of the present invention.
[0095] In one embodiment, the agent is used in the methods of the
present invention to cause the target species in the mixture to
precipitate or absorb onto the functional groups on the surface of
the solid phase carriers (a nucleic acid precipitating agent). In
one embodiment, a nucleic acid or peptide precipitating agent is
used at a sufficient concentration to precipitate the nucleic acid
of the mixture onto the solid phase carrier.
[0096] A "nucleic acid precipitating reagent" or "nucleic acid
precipitating agent" is a composition that causes the nucleic acid
of a cell to go out of solution. Suitable precipitating agents
include alcohols (e.g., short chain alcohols, such as ethanol or
isopropanol) and a poly-OH compound (e.g., a polyalkylene glycol).
The nucleic acid precipitating reagent can comprise one or more of
these agents. The nucleic acid precipitating reagent is present in
sufficient concentration to nonspecifically and reversibly bind the
nucleic acid of the cell onto the solid phase carriers. Such
nucleic acid precipitating agents can be used, for example, to bind
nucleic acids non- specifically, or target nucleic acid species
specifically, depending on the concentrations used, to solid phase
carriers comprising COOH as a functional group.
[0097] In one embodiment, carboxy-based magnetic beads are used
which involve binding nucleic acids to carboxyl coated solid phase
carriers (e.g., magnetic microparticles) using various nucleic acid
precipitating reagents (crowding reagents) such as alcohols,
glycols (e.g., alkylene, polyalkylene glycol, ethylene,
polyethylene glycol) and Polyvinyl Pyrrolidinone-40. The molecular
weights of these crowding reagents can be optimized to produce low
viscosity solutions with substantial precipitating power. Size
specific nucleic acid isolation can be performed by either
adjusting the concentration of the crowding reagent, the molecular
weight of the crowding reagent or adjustment to salt, pH, polarity
or hydrophobicity of the solution. Large nucleic acid molecules
will be crowded out of solution at low concentrations of salt and
crowding reagent whereas the smaller size species required higher
concentrations of crowding reagents. See, for example, U.S. Pat.
No. 5,705,628; U.S. Pat. No. 5,898,071; U.S. Pat. No. 6,534,262 and
U.S. Published Application No. 2002/0106686, all of which are
incorporated herein by reference.
[0098] Suitable "peptide precipitating reagents" or "peptide
precipitating agents" include any suitable affinity binding buffer
(e.g,. U.S. Pat. Nos. 5,047,513; 6,623,655; 5,284,933; and US
Published Application No. 2002/0019496, all of which are
incorporated herein by reference).
[0099] Appropriate alcohol (e.g., ethanol, isopropanol)
concentrations (final concentrations) for use in the methods of the
present invention are from about 5% to about 100%; from about 40%
to about 60%; from about 45% to about 55%; and from about 50% to
about 54%.
[0100] Appropriate polyalkylene glycols include polyethylene glycol
(PEG) and polypropylene glycol. Suitable PEG can be obtained from
Sigma (Sigma Chemical Co., St. Louis Mo., Molecular weight 8000,
Dnase and Rnase fee, Catalog number 25322-68-3) The molecular
weight of the polyethylene glycol (PEG) can range from about 250 to
about 10,000, from about 1000 to about 10,000, from about 2500 to
about 10,000, from about 6000 to about 10,000, from about 6000 to
about 8000, from about 7000 to about 9000, from about 8000 to about
10,000. In a particular embodiment PEG with a molecular weight of
about 8000 is used. In general, the presence of PEG provides a
hydrophobic solution which forces hydrophilic nucleic acid
molecules out of solution. In one embodiment, the PEG concentration
is from about 5% to about 20%. In other embodiments, the PEG
concentration ranges from about 7% to about 18%; from about 9% to
about 16%; and from about 10% to about 15%.
[0101] Optionally, salt may be added to the reagent to cause
precipitation of the nucleic acid and/or peptide in the mixture
onto the solid phase carriers. Suitable salts which are useful for
facilitating the adsorption of nucleic acid molecules targeted for
isolation to the magnetically responsive microparticles include
sodium chloride (NaCl), lithium chloride (LiCl), barium chloride
(BaCl.sub.2), potassium (KCl), calcium chloride (CaCl.sub.2),
magnesium chloride (MgCl.sub.2) and cesium chloride (CsCl). In one
embodiment, sodium chloride is used. In general, the presence of
salt functions to minimize the negative charge repulsion of the
nucleic acid molecules. The wide range of salts suitable for use in
the method indicates that many other salts can also be used and
suitable levels can be empirically determined by one of ordinary
skill in the art. The salt concentration can be from about 0.005M
to about 5M, from about 0.1M to about 0.5M; from about 0.15M to
about 0.4M; and from about 2M to about 4M.
[0102] Additional components may be added to the reagent. In one
embodiment, RNAse is added to the nucleic acid precipitating
agent.
[0103] In the embodiment in which the functional group is a
sequence which is complementary, and thus hybridizes, to a nucleic
acid species in the mixture, a hybridizing buffer can be used for
binding. Suitable buffers for use in such a method are known to
those of skill in the art. An example of a suitable buffer is a
buffer comprising NaCl (e.g., about 0.1M to about 0.5M), Tris-HCl
(e.g., 10 mM), EDTA (e.g., 0.5 mM), sodium chcloride sodium citrate
(SSC) and combinations thereof.
[0104] Depending on the functional group used (e.g., a streptavidin
functional group which is used to bind a nucleic acid comprising a
biotin label or tag), those of skill in the art will be able to
determine the agent to use to either bind the nucleic acid and/or
peptide to, or elute the nucleic acid and/or peptide from, the
functional group on the solid phase carrier.
[0105] In the methods of the present invention, a mixture
comprising a plurality of nucleic acids and/or peptide species are
combined with solid phase carriers. Any mixture comprising a
plurality of nucleic acids and or peptides can be used in the
methods. Examples, of appropriate starting material include
biological samples such as blood, tissue, tissue lysates, cells
(intact or whole cells such as buccal cells), and cell lysates
(cells in growth or culture media). Additional appropriate starting
materials include assay samples comprising nucleic acid.
Appropriate starting material also include cells obtained from
either mammalian (i.e., human, primate, equine, canine, feline,
bovine, murine) tissue or body fluids and lysates prepared from
such cells. Examples of cells for use in the methods of the present
invention include, but are not limited to, mammalian cells (e.g.,
blood cells, such as whole blood cells), bacterial cells (e.g., E.
Coli such as DH5.alpha., DH10B, DH12S, C600 or XL-1 Blue), yeast
cells, plant cells, tissue cells (cells from, for example, C.
elegans, mouse tails, human biopsies) and host cells containing
exogenous nucleic acid (e.g., recombinant DNA, bacterial DNA or
replicative form DNA) and/or peptides which are targeted for
isolation from host cell chromosomal DNA and other host cell
biomolecules. Alternatively, the starting material can be lysates
prepared from such cells.
[0106] As used herein a "host cell" is any cell into which
exogenous nucleic acid and/or peptide can be introduced, thereby
producing a host cell which contains exogenous nucleic acid and/or
peptide, in addition to host cell nucleic acid and peptides. As
used herein the terms "host cell nucleic acid", "endogenous nucleic
acid", "host cell peptides", and "endogenous peptides" refer to
nucleic acid species (e.g., genomic or chromosomal nucleic acid)
and peptide species that are present in a host cell as the cell is
obtained. As used herein, the term "exogenous" refers to nucleic
acid and peptides other than host cell nucleic acid (e.g., plasmid)
and peptides; exogenous nucleic acid and peptides can be present
into a host cell as a result of being introduced in the host cell
or being introduced into an ancestor of the host cell. Thus, for
example, a nucleic acid species (peptide species) which is
non-endogenous, is not present in the host cell as it was obtained
or an ancestor of the host cell. Appropriate host cells include,
but are not limited to, bacterial cells, yeast cells, plant cells
and mammalian cells.
[0107] As used herein, a "lysate" is a solution in which the cells'
membranes have been disrupted by any means with the result that the
contents of the cell, including the nucleic acid therein, are in
solution. A "cleared lysate" is a lysate in which the chromosomal
or genomic nucleic acid, proteins and membranes of the cell have
been removed such as by chemical treatment or centrifugation of the
lysate. Cells are lysed using known methods, thereby preparing a
mixture suitable for use with the method of the instant invention.
For example, cells can be lysed using chemical means (e.g., alkali
or alkali and anionic detergent treatment, nonionic detergent
(e.g., Triton X)), cationic detergent, isotonic shock, or physical
disruption (e.g., homogenization).
[0108] The term "lysed host cell suspension", as used herein,
refers to a suspension comprising host cells whose membranes have
been disrupted by any means (e.g., chemical, such as alkali or
alkali and anionic detergent treatment, nonionic detergent,
cationic detergent, isotonic shock, or physical disruption by
homogenization); such a suspension is a mixture of host cell
biomolecules, cellular components and disrupted membrane debris. In
one embodiment, a lysed host cell suspension suitable for use in
the instant invention is prepared by contacting host cells with an
alkali and anionic detergent (e.g., sodium dodecyl sulphate (SDS))
solution (e.g., 0.2 N NaOH, 1% SDS). Optionally, lysozyme could be
included in the lysis buffer. The presence of an anionic detergent
in the lysing solution functions to produce an anti-protein
environment by neutralizing the effective charge of the proteins,
thereby minimizing their attraction to the surfaces of the
functional group-coated paramagnetic microparticles. In one
embodiment, the lysed host cell suspension is non-neutralized.
[0109] According to the methods of the present invention, in one
embodiment, a cell is combined with solid phase carriers and a
reagent, wherein the reagent causes the nucleic acids of the cell
to bind non-specifically and reversibly to the solid phase
carriers. As described above, in the embodiment in which the
starting material is a cell the agent(s) used in the methods of the
present invention can be formulated to cause the lysis of a cell. A
variety of lysis components can be used to cause the disruption of
a membrane (such as alkali, alkali and anionic detergent treatment,
or isotonic shock). In one embodiment, the lysis component of the
reagent is an alkali (NaOH) and/or an anionic detergent (e.g.,
sodium dodecyl sulphate (SDS)) solution (e.g., final concentration
of 0.2 N NaOH, 1% SDS when added to a cell). Optionally, lysozyme
could be included in the lysis component of the first reagent. The
presence of an anionic detergent in the lysis component functions
to produce an anti-protein environment by neutralizing the
effective charge of the proteins, thereby minimizing their
attraction to the surfaces of the solid phase carrier (e.g., a
functional group-coated paramagnetic microparticle). In one
embodiment, an RNA lysis buffer is used. In a particular
embodiment, the RNA lysis buffer is 20 mM Citrate buffer, pH 4.5,
2% sodium lauryl sarcosine, 10 mM EDTA, 1 mM Aurin tricarboxylic
acid, 1% triton-x-100, 1M LiCl, 30% isopropanol, 0.05% sodium
azide). In another embodiment, the RNA lysis buffer is 50 mM
Citrate buffer, pH 7.0, 2% sodium lauryl sarcosine, 10 mM EDTA, 1
mM Aurin tricarboxylic acid, 1% triton-x-100, 1M LiCl, 30%
isopropanol, 0.05% sodium azide
[0110] According to the methods of the invention, the isolation of
the target species in a mixture is accomplished by removing the
nucleic acid-coated solid phase carrier from the combination. The
solid phase carrier (e.g., a paramagnetic microparticle) can be
recovered from the first combination, for example, by vacuum
filtration, centrifugation, or by applying a magnetic field to draw
down the solid phase carrier (e.g., a paramagnetic microparticle).
Paramagnetic microparticles are preferably separated from solutions
using magnetic means, such as applying a magnet field of at least
1000 Gauss. However, other methods known to those skilled in the
art can be used to remove the magnetic microparticles from the
supernatant (e.g., vacuum filtration or centrifugation). The
remaining solution can then be removed, leaving solid phase
carriers having the nucleic acid of the cell adsorbed to their
surface.
[0111] As described herein agents which can be used to remove
target species, such as nucleic acid and/or peptides, from the
solid phase carriers include buffers, such as elution buffers. A
suitable "elution buffer" for use in the methods of the present
invention is a buffer that elutes (e.g., selectively) target
species such as nucleic acid and/or peptides from the functional
group(s) of the solid phase carriers. In one embodiment, a suitable
elution buffer for use in the present invention can be water or any
aqueous solution. For example, useful buffers include, but are not
limited to, TRIS-HCl (e.g., 10 mM, pH 7.5), Tris acetate, sucrose
(20%), EDTA and formamide (100%) solutions. In one embodiment, the
elution buffer is a buffered salt solution comprising a monovalent
(one or more) cation such as sodium, lithium, potassium, and/or
ammonium (e.g., from about 0.1M to about 0.5M). Elution of nucleic
acid or peptides from the solid phase carrier can occur quickly
(e.g., in thirty seconds or less) when a suitable low ionic
strength elution buffer is used. Once the bound target nucleic acid
and/or peptide species have been eluted, the solid phase carrier,
to which is bound non-target nucleic acid and/or peptide species,
is separated from the elution buffer.
[0112] Optionally, the agent can comprise a component that degrades
nucleic acid (e.g., an enzyme) or peptides (e.g., proteinases, such
as proteinase K). For example, DNase (e.g., DNase I) can be added
to degrade DNA (e.g., host cell DNA), thereby allowing RNA to bind
to the solid phase carriers free, or essentially free of DNA.
Alternatively, RNAse can be added to degrade RNA (e.g., host cell
RNA), thereby allowing DNA to bind to the solid phase carriers
free, or essentially free, from RNA. Alternatively, RNAse (e.g.,
1.75 ng/ul RNAse/ddH.sub.2O) can be added to the lysis component to
degrade host cell RNA, thereby allowing DNA to bind to the solid
phase carrier free, or essentially free, from RNA. The necessity of
including a RNAse step will largely be determined by the size of
the nucleic acid species that is targeted for isolation in the
particular nucleic acid precipitation that is being performed. For
example, if the conditions selected for isolation are appropriate
for isolating nucleic acids comprising at least 4,000 base pairs,
then it is unlikely that RNA species will be an appreciable
contaminant.
[0113] In addition, impurities (e.g., host cell components,
proteins, metabolites, chemicals or cellular debris) can be removed
from the solid phase carriers by washing the solid phase carriers
with target species bound thereto (e.g., by contacting the solid
phase carriers with a suitable wash buffer solution) before
separating the solid phase carrier-bound target species from the
solid phase carriers. As used herein, a "wash buffer" is a
composition that dissolves or removes impurities either bound
directly to the microparticle, or associated with the adsorbed
nucleic acid, but does not solubilize the target species absorbed
onto the solid phase. The pH and solute composition and
concentration of the wash buffer can be varied according to the
types of impurities which are expected to be present. For example,
ethanol (e.g., 70%) exemplifies a preferred wash buffer useful to
remove excess PEG and salt. In one embodiment, the wash buffer
comprises NaCl (e.g., 0.1M), Tris (e.g., 10 mM) and EDTA (e.g., 0.5
mM). The solid phase carriers with bound nucleic acid and/or
peptide can also be washed with more than one wash buffer solution.
The solid phase carriers can be washed as often as required (e.g.,
three to five times) to remove the desired impurities. However, the
number of washings is preferably limited to in order to minimize
loss of yield of the bound target species. A suitable wash buffer
solution has several characteristics. First, the wash buffer
solution must have a sufficiently high salt concentration (a
sufficiently high ionic strength) that the nucleic acid and/or
peptide bound to the solid phase carriers does not elute off of the
solid phase carriers, but remains bound to the microparticles. A
suitable salt concentrations is greater than about 0. 1 M and is
preferably about 0.5M. Second, the buffer solution is chosen so
that impurities that are bound to the nucleic acid or
microparticles are dissolved. The pH and solute composition and
concentration of the buffer solution can be varied according to the
types of impurities which are expected to be present. Suitable wash
solutions include the following: 0.5.times.5 SSC; 100 mM ammonium
sulfate, 400 mM Tris pH 9, 25 mM MgCl.sub.2 and 1% bovine serum
albumin (BSA); 1-4M guanidine hydrochloride (e.g., 1M guanidine HCL
with 40% isopropanol and 1% Triton X100); and 0.5M NaCl. In one
embodiment, the wash buffer solution comprises 25 mM Tris acetate
(pH 7.8), 100 mM potassium acetate (KOAc), 10 mM magnesium acetate
(Mg.sub.2OAc), and 1 mM dithiothreital (DTT). In another
embodiment, the wash solution comprises 2% SDS, 10% Tween and/or
10% Triton.
[0114] The components of the agents used in the methods of the
present invention can be contained in a single agent (reagent) or
as separate components. In the embodiment in which separate
components of the agent(s) are used, the components can be combined
simultaneously or sequentially with the mixture. Depending on the
particular embodiment, the order in which the elements of the
combination are combined may not necessarily be critical. The
nature and quantity of the components contained in the reagent are
as described in the methods above. The reagent may formulated in a
concentrated form, such that dilution is required to obtain the
functions and/or concentrations described in the methods
herein.
[0115] The methods described herein can also be used to separate a
duplex sequencing reaction into its respective forward and reverse
Sanger extension products. Duplex sequencing reactions ensure
impeccable read pairing and cut thermal cycler demand in half. For
example, using bi-functional magnetic particles comprising
streptavadin and carboxy functional groups, biotinylated forward
reads can be bound to magnetic particles through
biotin-streptavadin interaction while (simultaneously or
subsequently) binding the reverse reads to carboxy functionalized
beads in a reaction vessel (see FIG. 1). In a particular
embodiment, biotinylated forward reads can be bound to magnetic
particles through biotin-streptavadin interaction while
subsequently binding the reverse reads to carboxy functionalized
beads in a second reaction vessel. In this embodiment one standard
streptavadin purification can be performed to capture most of the
forward product which is eluted for sequencing with formamide. The
supernatant is then moved to a new vessel and bifunctional
streptavadin/carboxy beads are added followed by a two step binding
reaction. Because the streptavadin/biotin interaction cannot be
dissociated with water alone, the residual biotin labeled forward
product remains on the bead mixture while the unlabelled reverse
product is eluted from the carboxy groups and transferred to a new
plate for capillary sequencing.
[0116] In another embodiment, the invention relates to a method of
simultaneously or subsequently amplifying and sequencing a template
DNA in the same reaction vessel using a biotinlylated forward
primer and a reverse primer and separating the forward and reverse
extension products. A template DNA, a biotinlylated forward primer,
a reverse primer and a non-proofreading DNA polymerase are present
in a polymerase chain reaction (PCR) comprising deoxynucleotide
triphospahte (dNTP) (e.g., 2-10.times. concentration) dependent
upon the size of the amplicon, and labeled dideoxynucleotide
triphosphate (ddNTP) terminators. In a particular embodiment,
exponential amplification of the template occurs during the first
few cycles of PCR. After a certain level of dNTP has been consumed,
linear sequencing, consuming both dNTP's and labeled ddNTP's is the
predominate reaction in the mixture producing forward and reverse
extension products. Using bi-functional magnetic particles
comprising streptavadin and carboxy functional groups, biotinylated
forward reads can be bound to magnetic particles through
biotin-streptavadin interaction while (simultaneously or
subsequently) binding the reverse reads to carboxy functionalized
beads in a reaction vessel. In a particular embodiment,
biotinylated forward reads can be bound to magnetic particles
through biotin-streptavadin interaction while subsequently binding
the reverse reads to carboxy functionalized beads in a second
reaction vessel. Streptavadin purification can be performed to
capture most of the forward product which is eluted for sequencing
with formamide. The supernatant is then moved to a new vessel and
bifunctional streptavadin/carboxy beads are added followed by a two
step binding reaction. Because the streptavadin/biotin interaction
cannot be dissociated with water alone, the residual biotin labeled
forward product remains on the bead mixture while the unlabelled
reverse product is eluted from the carboxy groups.
[0117] Accordingly, the invention relates to a method of separating
forward extension products and reverse extension products of a
sequencing reaction. The method comprises combining a sequencing
reaction mixture which comprises forward extension products and
reverse extension products with solid phase carriers having a
surface comprising a first functional group which selectively binds
the forward extension products and a second functional group which
binds nucleic acid, thereby producing a first combination. The
first combination is maintained under conditions appropriate for
binding of the forward extension products to the first functional
group and binding of the reverse extension products to the second
functional group. The solid phase carriers are removed from the
first combination and combined with a buffer that selectively
elutes the reverse extension products from the second functional
group of the solid phase carriers, thereby producing a second
combination. The solid phase carriers are removed from the second
combination, thereby separating forward extension products and
reverse extension products of the sequencing reaction.
[0118] In one embodiment of separating forward extension products
and reverse extension products of a sequencing reaction, the method
comprises combining a sequencing reaction mixture which comprises
biotinylated forward extension products and reverse extension
products with solid phase carriers having a surface comprising a
(one or more) streptavidin functional group which selectively binds
the forward extension products and a (one or more) COOH functional
group which binds nucleic acid, thereby producing a first
combination. The first combination is contacted with an agent that
promotes binding of the biotinylated forwards to the streptavidin
functional group (e.g., salt) and that promotes binding of nucleic
acid to the COOH group (e.g., ethanol, polyethylene glycol).
[0119] Contacting the first combination with an agent that promotes
binding of the biotinylated forwards to the streptavidin functional
group and that promotes binding of nucleic acid to the COOH group,
can be performed in one or more steps (e.g., one step, two steps,
three steps, etc.) using one or more agents (e.g., a single agent,
two agents, three agents, etc.). In one embodiment, the first
combination is contacted with a first agent that promotes binding
of the biotinylated forwards to the streptavidin functional group
(e.g., salt) and then contacted with a second agent that promotes
binding of nucleic acid to the COOH group (e.g., ethanol,
polyethylene glycol). In another embodiment, the first combination
is contacted with a first agent that promotes binding of nucleic
acid to the COOH group (e.g., ethanol, polyethylene glycol) and
then contacted with a second agent that promotes binding of the
biotinylated forwards to the streptavidin functional group (e.g.,
salt).
[0120] The solid phase carriers are removed from the first
combination and combined with a buffer that selectively elutes the
reverse extension products from the COOH functional group of the
solid phase carriers (e.g., water), thereby producing a second
combination. Alternatively, the solid phase carriers can be
combined with a buffer that selectively elutes the forward
extension products from the streptavidin group, thereby producing a
second combination. The solid phase carriers are removed from the
second combination, thereby separating forward extension products
and reverse extension products of the sequencing reaction. This
method can be also performed wherein the reverse extension
products, rather than the forward extension products, are
biotinylated.
[0121] In another embodiment of separating forward extension
products and reverse extension products of a sequencing reaction,
the method comprises combining a sequencing reaction mixture which
comprises biotinylated forward extension products and reverse
extension products with solid phase carriers having a surface
comprising a (one or more) streptavidin functional group which
selectively binds the forward extension products and a (one or
more) COOH functional group which binds nucleic acid, thereby
producing a first combination. The first combination is contacted
with an agent that promotes binding of the biotinylated forwards to
the streptavidin functional group (e.g., salt) and that promotes
binding of nucleic acid to the COOH group (e.g., ethanol,
polyethylene glycol). This step can be performed in one or more
steps (e.g., one step, two steps) using one or more agents (e.g., a
single agent, two agents). The solid phase carriers are removed
from the first combination and combined with a buffer that
selectively elutes the reverse extension products from the COOH
functional group of the solid phase carriers (e.g., water), thereby
producing a second combination. Alternatively, the solid phase
carriers can be combined with a buffer that selectively elutes the
forward extension products from the streptavidin group, thereby
producing a second combination. The solid phase carriers are
removed from the second combination, thereby separating forward
extension products and reverse extension products of the sequencing
reaction. This method can be also performed wherein the reverse
extension products, rather than the forward extension products, are
biotinylated.
[0122] In a particular embodiment, the present invention relates to
a method of separating forward extension products and reverse
extension products of a sequencing reaction. In this embodiment,
the method comprised combining a sequencing reaction mixture which
comprises forward extension products and reverse extension products
with solid phase carriers having a surface comprising a first
functional group which selectively binds the forward extension
products and a second functional group which binds nucleic acid,
thereby producing a first combination. The first combination is
maintained under conditions appropriate for binding of the forward
extension products to the first functional group. The solid phase
carriers are removed from the first combination thereby producing a
second mixture comprising the reverse extension products. The solid
phase carriers are combined with a buffer (e.g., formamide) that
selectively elutes the forward extension products from the first
functional group of the solid phase carriers, thereby producing a
second combination. The solid phase carriers are separated from the
second combination and combined with the second mixture, thereby
producing a third combination. The third combination is maintained
under conditions appropriate for binding of the reverse extension
products to the second functional group. The solid phase carriers
are removed from the third combination and combined with a buffer
that selectively elutes the reverse extension products from the
second functional group of the solid phase carriers, thereby
separating forward extension products and reverse extension
products of the sequencing reaction.
[0123] In another embodiment, the methods described herein can also
be used to separate a nucleic acid species and a peptide species.
For example, using bifunctional magnetic beads comprising carboxy
and immobilized metal ion functional groups, DNA or RNA can be
bound to the particles via interaction with carboxy groups while,
simultaneously or subsequently, peptides can be bound to the
particles via interaction with immobilized metal ion complexes. In
one particular embodiment, cells recombinantly expressing a
polyhistidine fusion protein can be lysed and both the recombinant
protein and recombinant DNA species which expresses the recombinant
protein can be bound and isolated by the bifunctional
microparticles. Such a technique is useful for isolating protein
and the recombinant DNA clone that expresses the protein in the
iterative process of directed evolution or in screening expression
constructs from a cDNA library. In another embodiment, mRNA and
protein can be isolated by bi-functional beads containing carboxy
or oligo-dT functional groups to bind mRNA and immobilized metal
ion functional groups to bind protein. Such a technique is useful
for co-purifying protein and mRNA for quantitative co-assessment of
gene transcriptional and translational expression levels for
proteomic applications.
[0124] In the methods of the present invention, the isolated target
species can be subjected to further analysis, such as sequence
analysis (e.g., by polyacrylamide gel or capillary
electrophoresis). Nucleic acids isolated by the disclosed method
can be used for molecular biology applications requiring high
quality nucleic acids (e.g., the preparation of DNA sequencing
templates; the microinjection, transfection or transformation of
mammalian cells; the in vitro synthesis of RNA probes; reverse
transcription cloning; cDNA library construction; PCR
amplification; or gene therapy research; as well as for other
applications with less stringent quality requirements including,
but not limited to, transformation; restriction endonuclease or
microarray analysis; selective RNA precipitations; in vitro
transposition; separation of multiplex PCR amplification products;
in vitro siRNA; RNAi hairpins; preparation of DNA probes and
primers and detemplating protocols).
[0125] The isolation of high quality nucleic acid preparations from
starting solutions of diverse composition and complexity is a
fundamental technique in molecular biology. Thus, as a result of
the work described herein, novel and readily automatable methods of
separating nucleic acid molecules are now available. In one
embodiment, the reagent is added to the cell by a multisample
transfer device. In another embodiment, the first reagent is added
simultaneously to a plurality of samples, e.g., at least 6, 12, 24,
96, 384, or 1536 samples, each sample containing one or more cells.
In another embodiment, the first reagent is sequentially delivered
to a plurality of samples (e.g., at least 6, 12, 24, 96, 384, or
1536 samples) each sample containing, for example, one or more
cells. The invention includes methods of analyzing a plurality of
nucleic acid samples. The methods include providing a plurality of
nucleic acid samples isolated by a method described herein and
analyzing the samples, e.g., performing sequence analysis on the
samples.
[0126] The present invention is also directed to kits for use in
the methods of the present invention. In one embodiment, the kit
comprises heteromultifunctional (heterobifunctional) solid phase
carriers and a cell lysis buffer. The kits of the present invention
can further comprise additional buffers (e.g., lysis buffers, wash
buffers and elution buffers) enzymes for nucleic acid degradation
and instructions for use. In particular embodiments, the kit
comprises bifunctional magnetic microparticles comprising COOH
groups and oligo dT groups, and optionally, a cell lysis buffer;
bifunctional magnetic microparticles comprising COOH groups and
streptavidin groups; bifunctional magnetic microparticles
comprising a COOH group and an olig dT group or derivative thereof;
bifunctional magnetic microparticles comprising a COOH group and a
PMXB group or derivative thereof; bifunctional magnetic
microparticles comprising a COOH group and a LALF group or
derivative thereof; bifunctional magnetic microparticles comprising
a COOH group and a polyethyleneimine (PEI) group or derivative
thereof; and bifunctional magnetic microparticles comprising a COOH
group and a poly-L-lysine (PLL) group or derivative thereof.
EXAMPLE 1
Separation of Duplex Sequencing Reactions Using Two Functional
Groups on Magnetic Beads
[0127] Methods/Materials
[0128] A mixture containing the following reagents and amounts per
reaction was made.
TABLE-US-00001 Concentration Reagent Amount, ul in reaction BigDye
terminator sequencing mix v2 1 1/8x Biotin-M13 forward primer 0.04
0.2 umol M13 reverse primer 0.025 1.0 umol pGEM template DNA 0.7
140 ng water 3.255 Total 5
[0129] The mixture was placed mixture in 96 well thermocycling
plate and cycled sequenced using the following parameters:
TABLE-US-00002 Step 1. 95.degree. C. for 10 s Step 2. 50.degree. C.
for 5 s Step 3. 60.degree. C. for 150 s Step 4. Repeat steps 1-3 40
times Step 5. 4.degree. C. hold
[0130] Procedure A. Streptavidin Magnetic Bead Cleanup (Forward
Sequence) [0131] 1. To the 5 uL sequencing reaction there was added
2.5 uL of magnetic streptavadin beads and 7.5 uL of 5M NaCl and the
reaction was mixed well. [0132] 2. The magnetic beads containing
the bound biotin-forward sequencing fragments were separated from
solution by placing on a magnet plate for 5 mins. [0133] 3. The
supernatant containing reverse sequencing fragments was aspirated
and dispensed into a new well for reverse sequence clean-up
(Procedure B). [0134] 4. Washed strepavidin beads 2.times. with 40
uL ddH2O, resuspending beads during each wash. [0135] 5.
Resuspended beads in 2 uL Hi-Dye formamide, spun down, and
incubated at 90.degree. C. for 10 minutes to elute the forward
sequencing fragments.
[0136] Procedure B. Carboxyl Bead Cleanup of Supernatant (Reverse
Sequence): [0137] 1. Added 5 uL of diluted carboxyl coated magnetic
beads and 23 uL 85% ethanol per well to the supernatant sample from
Step 3, Procedure A and mixed well. [0138] 2. The magnetic beads
containing the bound reverse sequencing fragments were separated
from solution by placing on a magnet plate for 5 minutes. [0139] 3.
Aspirated and discard supernatant. [0140] 4. Washed beads 2.times.
with 40 uL 85% ethanol. [0141] 5. Resuspended beads in 20 uL ddH20
to elute the reverse sequencing fragments. Forward and reverse
sequencing fragments were analyzed, separately, on an ABI 3700.
[0142] Results/Discussion:
[0143] Successful separation and sequencing of the two fragment
populations is demonstrated using magnetic beads with two different
functional groups. See FIG. 1. Analysis of both the forward and
reverse sequencing reactions is shown in FIG. 2. An effective
separation of the forward and reverse sequencing fragments from the
same reaction is demonstrated by a clean signal indicated by the
Phred 20 quality scores (P20).
EXAMPLE 2
Isolation of Total RNA Using Bi-Functional Oligo-dT/Carboxyl
Beads
[0144] To test oligo-dT modified carboxyl beads for the ability to
perform SPRI purification of total RNA.
[0145] Procedure
[0146] Started with 1.75.times.10e7 293T cells pelleted in 15 ml
conical tubes. Resuspended the two pellets in 3 mls RNA lysis
buffer (20 mM Citrate buffer, pH4.5, 2% sodium lauryl sarcosine, 10
mM EDTA, 1 mM Aurin tricarboxylic acid, 1% triton-x-100, 1M LiCl,
30% isopropanol, 0.05% sodium azide, 0.03% magnetic carboxyl or
oligo-dT magnetic beads) in a 15 ml conical. Put on magnet for 15
minutes. Beads were washed by resuspending in 5 mls 4 M
Guanidine-HCL/40% Isopropanol buffer then placing on magnet. Second
and third washes were with 1 ml 90% ethanol. The beads were
transferred to 1.5 ml eppendorf tube. Wash 4 and 5 were also with 1
ml 90% ethanol. Let the beads dry and eluted with 100 uL DEPC
water. Pelleted in microcentrifuge and recovered 45 uL.
[0147] Results
[0148] The bi-functional oligo-dT/carboxyl beads are able to
function in the SPRI process to isolate both genomic DNA and total
RNA as indicated by comparison to the same isolation performed with
the standard carboxyl beads. The gel in FIG. 3 shows total RNA 28,
18 and 5.8/5S ribosomal bands and genomic DNA bands. Both standard
carboxyl beads and bi-functional oligo-dT/carboxyl beads give
approximately equivalent yields of RNA as determined by Ribogreen
analysis of the samples.
TABLE-US-00003 Quantitated with RiboGreen- Total amount of ng/ul
RNA, ug Oilgo-dT bead total RNA Purification 403.8 18.2 Carboxyl
bead total RNA Purification 391.5 17.6
EXAMPLE 3
Direct mRNA Isolation from Cells
[0149] Direct Isolation of mRNA from 1.times.10.sup.6 3638C cells
using bi-functional Oligo-dT/carboxyl beads.
[0150] Procedure: [0151] 1. Had 1.1.times.10.sup.7 3638C cells
frozen in -80.degree. C. Thawed and resuspended in 10 mls total 100
mM NaCl, 10 mM Tris to give about 1.times.10.sup.6 cells per ml.
[0152] 2. Pelleted 2.times. 1 ml cells in epi tubes at 14K.times.g,
RT. [0153] 3. Resuspended pellets in either 0.5 mls RNA Lysis
buffer pH 7.0 with 0.03% bi-functional oligo-dT beads or std
carboxyl beads. [0154] 4. Incubated at RT 5 minutes. [0155] 5.
Incubated on magnet for 5 minutes. [0156] 6. Resuspended each
pellet in 0.5 mls Wash Buffer pH 7.3 (1M G-HCl, 1% TX-100, 25 mM
NaCitrate, 30% iso) [0157] 7. Placed on magnet 5 minutes. [0158] 8.
Washed pellets x4 with 70% ethanol, 500 uL each, 30 sec. Let dry
for 5 minutes. At this point, the beads have total nucleic acid
bound to them. [0159] 9. To preferentially isolate the mRNA, the
beads were resuspended in 160 uL 65.degree. C.
1.times.hybridization buffer (0.5M NaCl, 10 mM Tris-HCl, pH 7.5,
0.5 mM EDTA). Let cool at room temperature for 5 minutes, placed on
magnet 2 minutes, then repeated the resuspension in 160 uL 65 C
1.times.Hyb buffer and cooled at room temperature 5 minutes. Placed
on magnet 2 minutes. [0160] 10. Resuspended beads in 160 uL
hybridization buffer, placed on magnet for 2 minutes. [0161] 11.
Resuspended beads in 80 uL Wash Buffer (0.1M NaCl, 10 mM Tris-HCl,
pH 7.5, 0.5 mM EDTA), moved to a fresh tube and placed on magnet
for 2 minutes. [0162] 12. Resuspended beads in 20 uL 65 C DEPC
RNAse free water, placed on magnet 2 minutes, then moved to fresh
tube. Stored at -20.degree. C.
[0163] Results
[0164] Ran 1 uL on a Bioanalyer 2100 using a Pico RNA chip. The top
electropherogram in FIG. 4 shows the RNA ladder. The bottom
electropherogram in FIG. 4 shows a typical mRNA profile with a
range of mRNA size fragments that show up as a broad peak
indicating successful isolation of mRNA with the bi-functional
oligo-dT/carboxyl beads.
EXAMPLE 4
Direct mRNA Isolation from Cells
[0165] Purpose
[0166] Direct Isolation of mRNA from 5.times.10.sup.5 3638C cells
using bi-functional oligo-dT/carboxyl beads and standard carboxyl
beads.
[0167] Procedure [0168] 1. Had 9.9.times.10.sup.6 3638C cells
frozen in -80.degree. C. Thawed and resuspended in 10 mls total 100
mM NaCl, 10 mM Tris to give about 1.times.10e6 cells per ml. [0169]
2. Pelleted 2.times.0.5 ml cells in eppendorf tubes at 14K.times.g,
RT. [0170] 3. Resuspended pellets in either 0.5 mls RNA Lysis
buffer pH 7.0 with 0.03% oligo-dT/carboxyl beads or std carboxyl
beads. [0171] 4. Incubated at RT 5 minutes. [0172] 5. Incubated on
magnet for 5 minutes. [0173] 6. Removed supernatant and resuspended
each pellet in 0.5 mls Wash Buffer pH 7.3 (1M G-HCl, 1% TX-100, 25
mM NaCitrate, 30% iso) [0174] 7. Placed on magnet 5 minutes. [0175]
8. Washed pellets x4 with 70% ethanol, 500 uL each, 30 sec. Let dry
for 5 minutes. At this point, the beads have total nucleic acid
bound to them. [0176] 9. To preferentially isolate the mRNA, the
beads were resuspended in 80 uL 65.degree. C. 1.times.hybridization
buffer (0.5M NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA). Let cool
at room temperature for 5 minutes, placed on magnet 2 minutes, then
repeated the resuspension in 80 uL 65.degree. C. hybridization
buffer and cooled at room temperature 5 minutes. Placed on magnet 2
minutes. [0177] 10. Resuspended beads in 40 uL Wash Buffer (0.1M
NaCl, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA) moved to a fresh tube
and placed on magnet for 2 minutes. [0178] 11. Resuspended beads in
10 uL 65.degree. C. water, placed on magnet 2 minutes, then moved
to fresh tube. Stored at -20.degree. C.
[0179] Results
[0180] Ran 1 uL of each on a Bioanalyer 2100 using a Pico RNA chip.
The top electropherogram in FIG. 5 shows the RNA ladder. The middle
shows successful isolation of mRNA with oligo-dT/carboxyl beads as
indicated by the broad peak. The bottom electropherogram indicates
that the standard carboxyl beads do not isolate mRNA as indicated
by the absence of a mRNA peak.
EXAMPLE 5
Adsorption of Polymyxin-B with Carboxy Beads
[0181] Since PXMB carries a net positive charge at pH<10, it
should associate readily with carboxy beads. In the next
experiment, we investigated the adsorption of PMXB onto carboxy
beads for preparation of an endotoxin removal reagent.
[0182] Procedure: Protein can be adsorbed onto carboxy beads easily
in the presence of MES buffer at pH 6.1 using standard procedures
(Seradyn Microparticle Reagent Optimization Manual). The
association is due to both charge and hydrophobicity interactions.
The amount of protein that can be loaded onto the carboxy beads is
determined empirically for each protein. To determine the maximal
level of PMXB adsorption, increasing amounts of PMXB were added to
a 1% carboxy bead solution in 25 mM MES buffer pH 6.1 and mixed
gently on a rotating platform for 24 hours. The amount of bound and
unbound protein was determined by BCA assay as described above. To
determine the optimal amount of carboxy beads required, a similar
titration experiment was performed with carboxy bead concentrations
as the variable.
[0183] Results: Ten micrograms of PMXB was sufficient to saturate a
0.1 ml solution of 1% carboxy beads, with the remainder of the
protein being found in the supernatant (SN) (FIG. 9).
EXAMPLE 6
Endotoxin Removal Using Bi-Functional Beads
[0184] Endotoxin free DNA Preparation with bi-functional beads made
by adsorption of Polymyxin-B to magnetic beads.
[0185] An endotoxin removal reagent (ERR) consisting of polymyxin-B
(PMXB) adsorbed onto carboxy modified (CM) beads was prepared as
follows: 0.1 mg/mL PMXB dissolved in 25 mM MES buffer (pH 6.1) and
mixed with CM beads (1% final). The solution was equilibrated for a
minimum of 24 hours on a roller platform at 4C. The ERR reagent was
tested for the ability to remove endotoxin from plasmid
preparations in conjunction with Agencourt's CosMcPrep DNA
purification kit as illustrated in FIG. 10. Briefly, to each 240 uL
of cleared lysate (FIG. 10, step 4), 100 uL ERR was added
(containing 10 ug PMXB). Following incubation and magnetic removal
of the ERR beads, plasmid DNA in the supernatant was purified via
SPRI (FIG. 10, steps 7-10).
[0186] The amount of endotoxin in plasmid DNA preparations was
determined by a photometric Limulus Amoebocyte Lysate (LAL) assay
from BioWhittaker. This assay conforms to the United States Food
and Drug Administration published guidelines for establishing
endotoxin limits for pharmaceuticals and medical devices and for
validating the use of LAL as an end-product endotoxin test.
[0187] FIG. 10 shows CosMcPrep (Agencourt) DNA purification kit
modified to contain an endotoxin removal step.
[0188] Several reagents were tested for removal of endotoxin from
the cleared lysate in addition to the ERR reagent. These included
Magnesil silica magnetic beads (Promega), CM beads, PMXB agarose
(Pharmacia), CM beads mixed with PMXB (PMXB+CM beads non-absorbed),
and CM beads with PMXB adsorbed (PMXB/CM beads, aka ERR reagent).
The various bead reagents were mixed with cleared lysate for one
hour before proceeding to SPRI purification of the plasmid DNA.
Endotoxin levels were determined by the LAL assay. All results were
normalized to the amount of endotoxin per milligram DNA found in
untreated controls.
[0189] Results
[0190] FIG. 11 shows that PMXB absorbed to CM beads were found to
be an effective reagent for removal of endotoxin from plasmid DNA
preparations, with less than 1% of the amount of endotoxin
remaining in the plasmid DNA in this experiment compared to
control. Silica coated magnetic beads were intermediate in
effectiveness while CM beads alone or PMXB-agarose beads were much
less effective. The endotoxin removal was found to depend upon
adsorption of the PMXB to the CM beads, as CM beads with PMXB
added, but not adsorbed, was not effective in removing the
endotoxin. Endotoxin removal was also proportional to the duration
of exposure to the PMXB/CM beads, as summarized in FIG. 12. In this
experiment, after 30 minutes incubation of the cleared lysate with
ERR solution, the amount of endotoxin present in the plasmid DNA
was significantly less than 300 EU/mg DNA required for therapeutic
use. FIG. 10 shows endotoxin removal with adsorbed PMXB/CM beads.
FIG. 12 shows endotoxin removal with PMXB/CM beads as a function of
time.
TABLE-US-00004 DNA EU/mg % EU of Condition EU/mL mg/ml DNA Control
Untreated 628.2 0.2 2886.5 100.0 Magnesil Silica 550.9 0.5 1055.2
36.6 CM Beads 633.0 0.3 1857.7 64.4 PMXB-agarose 621.5 0.3 1979.1
68.6 PMXB + CM beads 612.7 0.2 2659.4 92.1 PMXB/CM beads 0.5 0.1
3.6 0.1 Untreated Control 629.2 0.38 1639.4 100 Magnesil silica
beads 521.0 0.54 968.4 59.1 CM Beads, 10 min 543.8 0.46 1182.8 72.1
PMXB/CM, 10 min 150.3 0.32 472.8 28.8 PMXB/CM, 30 min 25.9 0.15
178.5 10.9 PMXB/CM, 60 min 34.7 0.44 79.0 4.8
EXAMPLE 7
Endotoxin Free DNA Preparation with Bi-Functional Beads made by
Adsorption of Limulus Anti-LPS Factor to Magnetic Beads
[0191] PMXB has several advantages, including low cost and
availability in large quantities, but other endotoxin binding
proteins can be used in the method as well.
[0192] Procedure: Three preparations of the Limulus anti-LPS factor
(LALF) isolated from the American Horseshoe crab (Limulus
polyphemus) were examined for this purpose. The three preparations
include the LALF native protein isolate (Lp), a recombinant LALF
expressed in Pichia pastoris (Pp), and a recombinant LALF expressed
in Saccharomyces cerevisiae (Sc). The three proteins differ in
their ability to neutralize endotoxin in an in vitro assay
(Sc>Pp>Lp). The proteins were absorbed onto CM beads as
described for PMXB and tested in the Agencourt CosMcPrep protocol
modified to remove endotoxin (FIG. 1) using a 60 minute incubation
with the cleared lysate before SPRI purification of plasmid DNA.
Also included in the experiment were Magnesil silica beads and
adsorbed PMXB/CM beads as controls.
[0193] Results
[0194] The recombinant LALF's removed greater than 95% of the
endotoxin from the plasmid DNA compared to untreated control,
slightly better than the PMXB/CM beads in this experiment (FIG.
13). All three produced DNA containing less than 300 IU/mg plasmid
DNA. FIG. 13 shows endotoxin removal using LALF coupled CM
beads.
TABLE-US-00005 DNA EU/mg % EU of Treatment EU/mL mg/ml DNA Control
Control 2540.9 0.5 5286.1 100.0 Magnesil silica beads 1214.2 0.4
2844.3 53.8 PMX-B/CM beads, 1 hr 96.7 0.2 505.8 9.6 LALF Pp/CM
beads, 1 hr 58.8 0.3 178.8 3.4 LALF lP/CM beads, 1 hr 65 0.3 257.9
4.9 LALF Sc/CM beads, 1 hr 50.0 0.3 183.4 3.5
EXAMPLE 8
Covalent Coupling of PMXB to CM Beads
[0195] To prevent PMXB contamination using our endotoxin removal
scheme, PMXB is covalently coupled to CM beads.
[0196] For direct coupling, carboxyl groups on the surface of the
beads are first activated by carbodiimide
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC) and then
reacted with free amino groups on the protein. PMXB is titrated in
the coupling reaction to optimize the amount of protein coupled to
the surface. The amount of bound protein was determined by a copper
reduction/bicinchoninic acid reaction (BCA). To distinguish between
actual covalently attached protein and protein that is merely
adsorbed to the surface, non-covalently associated protein is
pre-eluted by treatment with a combination of base and detergent, a
process which completely removes any adsorbed protein. The BCA
protein assay is then used to measure the amount of protein
remaining on the beads, and in solution. FIG. 14 shows the results
of different coupling reaction using increasing amounts of PMX-B.
FIG. 15 is a schematic illustration of the beads. FIG. 14 shows
preparation of PMX-B covalently coupled magnetic beads. FIG. 15 is
a schematic illustration of bi-functional PMXB-CM.
EXAMPLE 9
Endotoxin Removal Using Bi-Functional PMX-B/CM Beads
[0197] Bi-functional PMX-B beads from Example 8 were tested for the
ability to remove endotoxin from plasmid preparations in
conjunction with Agencourt's CosMcPrep DNA purification kit as
illustrated in FIG. 10. Briefly, to each 240 uL of cleared lysate
(FIG. 10, step 4), 100 uL PMXB-CM beads was added (containing
differing amounts of PMXB). Following incubation and magnetic
removal of the bi-functional beads, plasmid DNA in the supernatant
was purified via SPRI (FIG. 10, steps 7-10). The amount of
endotoxin in plasmid DNA preparations was determined by a
photometric Limulus Amoebocyte Lysate (LAL) assay from
BioWhittaker. The PMXB-CM beads remove over 97% of the associated
endotoxin from the DNA preparation (FIG. 16). FIG. 16 shows removal
of endotoxin from plasmid DNA with bifunctional PMXB-CM.
TABLE-US-00006 PMX-B PMX-B PMX-B PMX-B PMX-B CM 0 ug/mg 3.3 ug/mg
6.8 ug/mg 10 ug/mg 11.2 ug/mg Beads CosMcPrep [DNA] in 386 405 344
338 406 626 536 ng/ul + ug/ml Total DNA in 19.32 20.25 17.21 16.88
20.30 31.28 26.80 ug EU LAL/ml 16580 1313 463 824 566 29092 31409
eluate EU LAL/ug 42.90 3.24 1.35 2.44 1.39 46.50 58.61 DNA EU
LAL/mg 42903 3242 1346 2440 1395 46497 58608 DNA
EXAMPLE 10
Transfection of Endotoxin Free DNA Prepared with Bi-Functional
Beads
[0198] One microgram of pEGFP expression plasmid purified with
PMX-B bifunctional beads were transfected into endotoxin
insensitive 293T cells or endotoxin sensitive Huh-7 cells using
Effectene reagent and Lipofectamine reagent respectively, according
to the manufacturer's instructions. Forty-eight hours post
transfection GFP fluorescence was measured with the Agilent
Bioanalyzer 2100 cell chip. As expected, the relatively easily
transfected 293 shows high expression among most of the preps (FIG.
17A). The difficult to transfect Huh-7 cells show lower expression
(FIG. 17B). In both cases though, the PMXB-CM beads produce DNA
that transfects as well as or better than Qiagen maxipreps or
Qiagen Endofree maxipreps, which are considered Gold Standard in
the industry.
[0199] FIG. 18 shows the structure of lipopolysaccharide (LPS);
FIG. 19 shows that structure of polymyxin B (PMXB).
EXAMPLE 11
Bi-Functional Bead Application for Virus Purification
[0200] In another embodiment, the methods described herein can also
be used to concentrate a virus from a solution and subsequently
isolate viral nucleic acid. A polycationic polymer, such as the
polymer polyethyleneimine (PEI) or poly-L-lysine (PLL), is
covalently coupled to magnetic beads containing a carboxyl
functional group. Carboxyl groups are first activated by
carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC)
and then reacted with free amino groups on the polymer. This
produces a bead with two functional groups, a polycation polymer
for virus binding, and carboxyl groups for nucleic acid binding.
The polymer modified bi-functional beads are added to a volume of
virus containing serum or lysate and mixed well creating a first
mixture in which viral particles in the serum are concentrated on
the surface of the beads via interaction with the polymer. The
beads are magnetically separated on a magnet and the serum
supernatant is removed. To the beads are added a lysis buffer
containing, for example, 20 mM Tris pH 7.0, 1% Triton-X-100, 2%
SLS, 10 mM DTT, isopropanol and an RNase inhibitor. This results in
a second mixture in which virus is lysed and viral nucleic acid
becomes bound to the bead via interaction with the carboxyl groups.
The beads are separated by a magnet, washed three times in a wash
buffer and/or 70% ethanol. The beads are dried and the purified
viral nucleic acid is eluted in a low ionic strength buffer.
EXAMPLE 12
PEI-Based Bead Virus Extraction
[0201] Procedure for concentrating and isolating viral nucleic acid
using PEI-carboxy bi-functional beads. [0202] 1. Added 50 .mu.l PEI
carboxy beads (1% solid) to 1 ml plasma containing 50 to 10,000 HBV
copies in an Eppendorf Tube. Mixed and incubated at room
temperature for 10 minutes. The beads were captured by the magnet
and the supernatant was discarded. The beads were resuspend beads
in 200 .mu.l water. [0203] 2. Added 400 .mu.l Viral Lysis Solution
(2 M GITC, 10 mM Tris pH 7.0, 1% NP-40, 10 mM DTT and 5 .mu.g of
poly A) to all samples. Mixed well. [0204] 3. Added 0.5 .mu.g/.mu.l
of 20 mg/ml Proteinase K (17.9 mg protein/ml; 907 units/ml: Sigma
P4850). Mixed and centrifuged briefly (.about.2 sec) to collect the
contents at the bottom of the tube. Incubate at 55 C for 20 min.
Kept on ice for 2 min. [0205] 4. Added 8 .mu.l 30% final
Isopropanol. Mixed the sample and incubated for 5 min. Centrifuged
briefly (.about.2 sec) to collect tube contents. [0206] 5.
Magnetically captured the beads, and discarded the supernatant.
[0207] 6. Washed the beads twice with 400 .mu.l Wash Buffer (14%
PEG, 1M NaCl, 6M Urea). [0208] 7. Washed the beads three times with
400 .mu.l 70% Ethanol and allowed the beads to dry at ambient
temperature for 10 minutes. [0209] 8. Eluted the viral nucleic acid
in 25 .mu.l water. Magnetically captured the beads as in the
previous steps and transferred the supernatant containing viral
nucleic acid to a nuclease-free container. [0210] 9. The nucleic
acid was amplified by nested PCR using gene specific primers.
[0211] Results
[0212] As shown in FIG. 20, the procedure was sensitive enough to
detect down to 50 copies per ml of HBV.
EXAMPLE 13
Bi-Functional Example of Globin Subtraction
[0213] Isolation of total RNA from human whole blood and removal of
beta-globin specific transcripts with bi-functional beads.
[0214] One third volume of lysis buffer containing 4M GITC, 2 mM
DTT, 2% Triton-X-100 and 0.4M sodium citrate is added to whole
blood and mixed well. To the mixture is added an equal volume
binding buffer comprising bi-functional magnetic beads and
isopropanol. The bi-functional beads comprise strepavidin and
carboxyl functional groups, to which a mixture of three
biotinlylated oligos complementary in sequences to human beta
globin mRNA transcript are attached via the biotin-strepavidin
interaction. The beads are separated by a magnet and the
supernatant is removed. The beads are washed with a first wash
buffer comprising 2M GITC, 1 mM DTT, 1% Triton-X-100 and 0.2M
sodium citrate and 30% isopropanol then three times by a second
wash buffer comprising 70% ethanol. The beads are dried and then
total RNA is eluted in a low ionic strength buffer. FIG. 21 shows
an electropheregram of total RNA isolated from 0.3 mls human blood
using this protocol. Total RNA was isolated with the method of the
invention from 2.5 mls of human blood collected into PaxGene
collection tubes.
[0215] To remove globin sequences, the elution buffer with the
beads is adjusted to a final concentration of 0.5 M LiCl, 10 mM
Tris, 1 mM EDTA, pH7.4, 0.1% LDS to promote binding of the
oligonucleotide sequences to the complementary beta-globin RNA
transcripts. The mixture is heated to 65 C for 5 minutes then
cooled on ice. The hybridization occurs for 30 minutes at ambient
temperature. The supernatant, which contains total blood RNA
depleted of globin transcripts, is removed and saved. The extent of
globin depletion was examined by performing quantitative RT-PCR on
the supernatant from anti beta-globin beads and control bead
hybridization that did not contain anti-globin oligos.
TABLE-US-00007 Sample Ct % reduction Negative Control Bead 22.3 5.7
anti-beta-globin bead 23.4 62.6 NTC Undetermined n/a total RNA
undiluted 21.8825 n/a 1:1 dilution total RNA 23.25393 n/a 1:5
dilution total RNA 24.65676 n/a 1:10 dilution total RNA 26.027063
n/a 1:20 dilution total RNA 26.56443 n/a
[0216] In this experiment, it was possible to remove 62% of
beta-globin sequence using an un-optimized mix of antisense
beta-globin primers.
EXAMPLE 14
Globin Removal
[0217] Total RNA, 20 ug, in 0.5M LiCl, 10 mM Tris, 1 mM EDTA pH7.4,
0.1% LDS was combined with 10 pmol of biotin labeled
oligonucleotides complementary to alpha and beta globin sequences
in a final volume of 50 uL. The mixture was incubated for 30
minutes at 50 C. Bi-functional beads, 100 ug, containing carboxy
and strepavidin groups were added to the mixture and incubated at
room temperature for 30 minutes. Isopropanol was added to a final
concentration of 30%. The beads washed three times with 70%
ethanol, were placed on a magnet and the supernatant removed. RNA,
minus globin sequences, was eluted with 20 mM LiCl. A globin
specific quantitative RT-PCR assay was used to determine the amount
of alpha and beta globin reduction compared to an internal
beta-actin control RNA.
[0218] Results
[0219] qRT-PCR results demonstrate that a majority of both the
alpha globin and beta globin transcripts were removed using this
protocol.
TABLE-US-00008 % % Total Concen .beta.-actin .beta.-globin
Reduction .alpha.-globin Reduction Sample (ug) (ng/ul) Ct Ct of
.beta.-globin Ct of .alpha.-globin Actin 27.1 15.5 15.23 Control 1
Sample 1 22 600 21.7 20.2 99.0% 17.28 99.4% Actin 26.8 15.4 15.15
Control 2 Sample 2 21 650 22.9 17.8 98.8% 15.72 95.5%
[0220] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
2179DNAUnknownsequencing results of a duplex sequencing reaction
1ttctacgctc ggtacccggg gatcctctag agtcgacctg caggcatgca agcttgagta
60ttctatagtg tcacctaaa 79274DNAUnknownsequencing results of a
duplex sequencing reaction 2ttttacaacg tcgtgactgg gaaaaccctg
gcgttaccca acttaatcgc cttgcagcac 60atcccccttt cgcc 74
* * * * *