U.S. patent application number 11/129218 was filed with the patent office on 2006-01-05 for solid phase technique for selectively isolating nucleic acids.
This patent application is currently assigned to Whitehead Institute for Biomedical Research. Invention is credited to Paul McEwan, Kevin McKernan, William Morris.
Application Number | 20060003357 11/129218 |
Document ID | / |
Family ID | 26772764 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060003357 |
Kind Code |
A1 |
McKernan; Kevin ; et
al. |
January 5, 2006 |
Solid phase technique for selectively isolating nucleic acids
Abstract
A method of isolating target nucleic acid molecules from a
solution comprising a mixture of different size nucleic acid
molecules, in the presence or absence of other biomolecules, by
selectively facilitating the adsorption of a particular species of
nucleic acid molecule to the functional group-coated surface of
magnetically responsive paramagnetic microparticles is disclosed.
Separation is accomplished by manipulating the ionic strength and
polyalkylene glycol concentration of the solution to selectively
precipitate, and reversibly adsorb, the target species of nucleic
acid molecule, characterized by a particular molecular size, to
paramagnetic microparticles, the surfaces of which act as a
bioaffinity adsorbent for the nucleic acids. The target nucleic
acid is isolated from the starting mixture based on molecular size
and through the removal of magnetic beads to which the target
nucleic acid molecules have been adsorbed. The disclosed method
provides a simple, robust and readily automatable means of nucleic
acid isolation and purification which produces high quality nucleic
acid molecules suitable for: capillary electrophoresis, nucleotide
sequencing, reverse transcription cloning the transfection,
transduction or microinjection of mammalian cells, gene therapy
protocols, the in vitro synthesis of RNA probes, cDNA library
construction and PCR amplification.
Inventors: |
McKernan; Kevin; (Cambridge,
MA) ; McEwan; Paul; (Cambridge, MA) ; Morris;
William; (Cleveland Hts., OH) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Whitehead Institute for Biomedical
Research
Cambridge
MA
02142
|
Family ID: |
26772764 |
Appl. No.: |
11/129218 |
Filed: |
May 13, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10346714 |
Jan 16, 2003 |
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11129218 |
May 13, 2005 |
|
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09311317 |
May 13, 1999 |
6534262 |
|
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10346714 |
Jan 16, 2003 |
|
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60085480 |
May 14, 1998 |
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60121779 |
Feb 26, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/270 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12Q 1/6806 20130101; C12Q 2527/137 20130101; C12Q 2563/143
20130101; C12Q 2523/308 20130101; C12N 15/1013 20130101; C12Q
1/6806 20130101 |
Class at
Publication: |
435/006 ;
435/270 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 1/08 20060101 C12N001/08 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a
National Human Genome Research Institute Grant, Grant Number 5P 50
HG00098-09, from the National Institutes of Health. The United
States Government has certain rights in the invention.
Claims
1. A kit for high through-put automated DNA template production
comprising: (a) reagents for preparing a host cell lysate; (b) an
aqueous solution of functional group-coated solid phase carriers;
and (c) at least one binding buffer formulated to comprise a
suitable salt and a suitable nucleic acid precipitating reagent,
wherein the salt and the nucleic acid precipitating reagent are
each present at a concentration appropriate for binding a nucleic
acid species characterized by a particular molecular size to the
solid phase carriers or reagents for the formulation of a suitable
buffer.
2. The kit of claim 1 wherein the kit additionally comprises
reagents for the formulation of a wash buffer and an elution
buffer, wherein the wash buffer dissolves impurities, but not
nucleic acids bound to solid phase carriers and the elution buffer
is a low ionic strength buffer.
3. The kit of claim 1 wherein the solid phase carriers are
paramagnetic particles.
4. The kit of claim 3 wherein the kit further comprises a magnetic
plate holder appropriate for applying a magnetic field of at least
about 1000 Gauss to the wells of a microtiter plate, wherein said
magnet comprises at least one N35 magnet.
5. The kit of claim 1 wherein the nucleic acid precipitating agent
is selected from the group consisting of: polyalkylene glycol and
alcohol.
6. The kit of claim 5 wherein the polyalkylene glycol is selected
from the group consisting of: polyethylene glycol and polypropylene
glycol, and the alcohol is selected from the group consisting of:
ethanol and isopropanol.
7. The kit of claim 1 wherein the reagent for preparing a host cell
lysate is a detergent.
8. The kit of claim 1 wherein the salt is selected from the group
consisting of: sodium chloride, magnesium chloride, calcium
chloride, potassium chloride, lithium chloride, barium chloride and
cesium chloride.
9. The kit of claim 1 wherein the solid phase carriers having a
functional group-coated surface that reversibly binds nucleic acid
molecules are selected from the group consisting of: amine-coated,
carboxyl-coated and encapsulated carboxyl group-coated solid phase
carriers.
Description
RELATED APPLICATION(S)
[0001] This application is a divisional of U.S. application Ser.
No. 10/346,714, filed Jan. 16, 2003, which is a continuation of
U.S. application Ser. No. 09/311,317, filed May 13, 1999, which
claims the benefit of U.S. Provisional Application No. 60/085,480,
filed May 14, 1998 and U.S. Provisional Application No. 60/121,779,
filed on Feb. 26, 1999. The entire teachings of the referenced
applications are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 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 preparations.
Quality is a particularly important factor for capillary
electrophoresis for all sequencing methods and for gene therapy
protocols. Quantity is an equally important consideration for some
applications, for example, large scale genomic mapping and
sequencing projects, which require the generation of hundreds of
thousands of high quality DNA templates.
[0004] Extension product quality is crucial to the success of
automated dye-labeled dideoxynucleotide sequencing methods, such as
those described in Maniatis, T., et al., Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratories,
Cold Spring Harbor, N.Y., Sanger, E., et al., Proc. Natl. Acad.
Sci. 74:5463-5467 (1977), and Mierendorf, R. and Pfeffer, D.
Methods Enzymol. 152:5556-562 (1987), and is a particularly
critical consideration for capillary electrophoresis protocols. The
isolation of high quality nucleic acid preparations from starting
mixtures of diverse composition and complexity is a fundamental
technique in molecular biology.
[0005] The advent of demanding molecular biology applications has
increased the need for high-throughput, and preferably readily
automatable, purification protocols capable of producing high
quality nucleic acid preparations. Although recent technological
advancements and the advent of robotics have facilitated the
automation of sequencing reactions and gel reading steps,
throughput is still limited by the availability of readily
automatable methods of nucleic acid purification.
SUMMARY OF THE INVENTION
[0006] The present invention is a method of separating different
species of nucleic acid molecules present in a mixture, which can
be a solution or a suspension, on the basis of differences in their
molecular size. Separation is accomplished by selectively adsorbing
target nucleic acid molecules (e.g., targeted or selected for
isolation or purification), present in a mixture to a magnetically
responsive solid phase carrier such as paramagnetic microparticles.
In addition to target nucleic acid molecules the mixture can
comprise other components, which include, but are not limited to,
other non-target nucleic acid molecules, proteins, cell components,
and reagents or chemicals used in methods in which the nucleic
acids are processed or used. That is, the mixture can comprise a
wide variety of types of molecules, from which target nucleic acid
molecules are separated, by the present method on the basis of
differences in molecular size. Thus, the invention discloses a
method useful for isolating a particular species of nucleic acid
molecule from a mixture. The nucleic acid to be isolated (e.g.,
separated or purified) (referred to as target nucleic acids or
target nucleic acid molecules) are separated from the rest of the
mixture based on molecular size and through the physical removal of
the solid phase carrier to which the target nucleic acid molecules
have been adsorbed according to the method described herein.
[0007] The present invention provides a method of selectively
isolating (e.g., purifying) a species of nucleic acid molecule,
based on its molecular size, from a mixture. This embodiment of the
invention involves selectively precipitating and facilitating the
adsorption of a target species of nucleic acid molecule to the
functional group coated surface of a solid phase carrier.
Purification of the target nucleic acids is accomplished by
applying an external force which results in the removal (e.g.,
separation) of the solid phase carrier from the mixture in which
the carriers have been suspended. In a preferred embodiment, the
solid phase carrier is a paramagnetic microparticle and separation
is accomplished by magnetic means.
[0008] The present invention is useful to isolate, from a mixture
from which at least one species of nucleic acid molecule has been
selectively removed, one or more additional (e.g., a second, third,
fourth etc.) species of nucleic acid molecules which are of a
smaller molecular size than the one or more target nucleic acid
species which have already been removed from an initial (or
starting) mixture by the method described herein. The additional
species of nucleic acid molecule targeted for isolation in this
additional embodiment remained soluble in the presence of the PEG
and salt concentrations used to isolate the larger nucleic acid
molecule and, therefore, will still be present in the mixture from
which the first target nucleic acid molecule has been removed.
[0009] In an alternative embodiment of the instant invention, two
or more species of nucleic acid molecule present in the same
mixture, which differ in molecular size from each other by at least
a factor of two, are separated from each other. The method
described herein is used to isolate a particular species (e.g., a
target species) of nucleic acid molecules of virtually any size,
present in a wide variety of sources, from other nucleic acid
molecules which are also present in the mixture. For example, the
method disclosed herein can be used to isolate recombinant nucleic
acid species, produced in host cells, including selective RNA
precipitations based on molecular size, or replicative form DNA
produced by a virus during lytic replication from endogenous host
cell nucleic acid species. The method can also be used to isolate a
particular species of nucleic acid from a solution resulting from a
restriction enzyme digestion or an agarose solution containing
nucleic acid. Alternatively, the method disclosed herein provides a
size selection purification scheme suitable for use after a DNA
shearing process (e.g., hydroshearing or sonication), thereby
providing an alternative to the more traditional method of gel
electrophoresis and band excision which are conventionally used to
isolate a species of nucleic acid molecule targeted for
purification. The disclosed method also finds utility as a method
of separating multiplex PCR products, or as a sequencing reaction
detemplating protocol. For example, using the method disclosed
herein solid phase magnetically responsive paramagnetic
microparticles can be used to selectively remove sequencing
products and DNA templates from sequencing samples.
[0010] The present invention is also useful to isolate, from a
mixture from which at least one species of nucleic acid molecule
has been selectively removed, one or more additional (e.g., a
second, third, fourth etc.) species of nucleic acid molecules which
are of a smaller molecular size than the one or more target nucleic
acid species which have already been removed from an initial (or
starting) mixture by the method described herein. The additional
species of nucleic acid molecule targeted for isolation in this
additional embodiment remained soluble in the presence of the PEG
and salt concentrations used to isolate the larger nucleic acid
molecule and, therefore, will still be present in the mixture from
which the first target nucleic acid molecule has been removed.
[0011] One embodiment of the instant invention provides a method of
selectively isolating a target species of nucleic acid molecule
from a host cell lysate which is a mixture of target nucleic acid
molecules, non-target nucleic acid molecules and other cellular
components. The presence of these other components (e.g., cellular
components, reagents or biomolecules) could have an adverse effect
on the downstream molecular biology application for which the
target nucleic acids are being prepared. For example, the present
method is useful to selectively isolate exogenous nucleic acid from
endogenous host cell nucleic acid molecules. More specifically, the
present method is useful to selectively isolate recombinant DNA,
produced by the replication of exogenous DNA in an appropriate host
cell, from endogenous host cell DNA and from other host cell
components. Exogenous DNA (e.g., recombinant or plasmid DNA) can be
introduced into suitable host cells, or their ancestors, by a
vector, such as a bacterial artificial chromosome (BAC), a yeast
artificial chromosome (YAC), a phage artificial chromosome (PAC), a
P1, a cosmid (plasmids with .lamda. phage packaging sites) or a
bacterial plasmid. The exogenous DNA replicates in the host cell
and can be isolated from host cell DNA using the method described
herein. Initially, host cell (endogenous) DNA is removed through
the use of appropriate concentrations of PEG and salt.
Subsequently, exogenous nucleic acid molecules targeted for
isolation are removed, again through the use of appropriate
concentrations of PEG and salt. In this embodiment, as well as in
all others described herein, nucleic acid molecules of varying
sizes can be removed from a mixture sequentially. That is, all
nucleic acid molecules above a certain size cutoff can be removed
through the use of appropriate PEG and salt concentrations, with
the result that smaller nucleic acid molecules remain in the
mixture. This process can be repeated to remove progressively
smaller nucleic acid molecules. According to this embodiment,
exogenous nucleic acid molecules are selectively isolated from
endogenous host cell nucleic acid molecules and other biomolecules
present in a host cell lysate. More specifically, this embodiment
comprises combining functional group-coated paramagnetic
microparticles and suitable concentrations of a precipitating
reagent, for example, a polyalkylene glycol, and a salt to result
in the facilitated adsorption of endogenous nucleic acid (e.g.,
host cell genomic DNA) present in the mixture, to the surfaces of
the microparticles suspended therein, and the subsequent removal,
such as by magnetic means, of the microparticles from the resulting
combination. The removal of the microparticle having host cell
genomic DNA (e.g., endogenous DNA) adsorbed to its surfaces from
the combination results in the concomitant separation of the target
nucleic acid selected for isolation from other relatively
smaller-sized nucleic acids (e.g., exogenous nucleic acids), which
remain in solution, thereby producing a mixture that is enriched
for exogenous nucleic acid. The resulting PEG-facilitated
adsorption of the precipitated host cell DNA to the functional
group-coated surfaces of the paramagnetic microparticles (which can
be considered a pre-clearance of host cell DNA) is rapid; it is
generally complete within thirty seconds. Exogenous target nucleic
acid molecules present in the mixture enriched for relatively
smaller-sized nucleic acids can subsequently be isolated from the
mixture by performing a second PEG-induced precipitation, according
to the method provided herein.
[0012] The present invention further relates to a method of
isolating an exogenous DNA template (e.g., a plasmid DNA template)
suitable for use in either manual or a high-throughput automated
sequencing methods. In general terms this method comprises:
treating host cells which contain exogenous DNA (e.g., plasmid DNA)
with an alkali and detergent (e.g., sodium dodecyl sulphate (SDS))
combination, thus producing a lysed host cell suspension;
suspending paramagnetic microparticles which bind DNA (e.g., have
an affinity for DNA) in the lysed host cell suspension in the
presence of sufficient concentrations of a DNA precipitating
reagent, for example, polyethylene glycol, and a salt to
selectively precipitate and adsorb host cell DNA (e.g., genomic
DNA), but not exogenous DNA, to the surfaces of the paramagnetic
microparticles; removing the microparticles having host cell DNA
bound thereto from the suspension, preferably by magnetic means,
thereby producing a plasmid DNA-enriched supernatant; combining
additional paramagnetic microparticles having an affinity for DNA
with the resulting plasmid DNA-enriched supernatant; and adjusting
the precipitating reagent and/or salt concentration of this
supernatant to suitable levels to result in the selective
precipitation and adsorption of exogenous (e.g., plasmid) DNA to
the microparticles suspended therein. As a result, exogenous (e.g.,
plasmid) DNA is bound to the microparticles, thereby producing
microparticle-bound exogenous DNA.
[0013] The purity of the microparticle-bound exogenous DNA can be
improved by washing the particle-bound nucleic acid molecules to
remove other host cell biomolecules by contacting the
microparticles with a high ionic strength wash buffer which
dissolves, for example impurities (e.g., proteins, reagents or
chemicals) adsorbed to the paramagnetic microparticles, but does
not solubilize the adsorbed DNA. As a result, the exogenous DNA
targeted for isolation remains adsorbed to the solid phase carrier
surface. The washed, particle-bound exogenous DNA template can
subsequently be removed from the solid phase carrier by contacting
the washed microparticles with an elution buffer which solubilizes
the adsorbed DNA, thereby preparing plasmid DNA suitable for use as
a DNA nucleotide sequencing template.
[0014] In one embodiment, the invention is a readily automatable
method of isolating a plasmid DNA template for nucleotide
sequencing. The use of paramagnetic microparticles having
functional group-coated surfaces as a bioaffinity adsorbent for
nucleic acid molecules affords an alternative, and readily
automatable, means of molecular separation useful in the design of
a solid phase technique for the selective isolation of nucleic acid
molecules targeted for isolation. A particular advantage of the
disclosed method is that it obviates the necessity of
centrifugation and filtration steps and the use of organic
solvents. In addition, the ample surface area provided by suitable
solid phase carriers allows the volume of the reactions to be
reduced, which not only facilitates automation, but which also
eliminates the need for subsequent concentration steps which
conventionally require the use of a solvent whose presence in the
final preparation can adversely effect the quality of the isolated
DNA. Thus, the invention offers purification methods which are
simple, cost effective, robust and readily amenable to
automation.
[0015] The present invention also relates to a kit comprising
magnetically responsive microparticles having a functional
group-coated surface that reversibly binds nucleic acid molecules,
at least one binding buffer, a suitable nucleic acid precipitating
reagent and salt at concentrations suitable for reversibly binding
nucleic acids onto the surface of the microparticle. The kit may
additionally comprise preformulated solutions of a host cell lysis
buffer, or reagents for the preparation of such buffer, a wash
buffer and an elution buffer. The exact compositions of the buffers
may vary with the nature of the starting material and the purpose
(e.g., the molecular biology application) for which the nucleic
acid preparation is being isolated. The kit may further include a
magnetic microtiter plate holder specifically designed to optimize
the field strength applied to remove the paramagnetic
microparticles from the resulting combinations and solutions.
DETAILED DESCRIPTION OF THE INVENTION
[0016] As described herein, Applicants have shown that different
species of nucleic acid present in a solution can be isolated, on
the basis of their molecular size, through the use of appropriate
concentrations of a nucleic acid precipitating reagent, preferably
a polyalkylene glycol, and a salt to result in the selective
precipitation and facilitated adsorption of a particular nucleic
acid species to the functional group-coated surfaces of a suitable
magnetically responsive solid phase carrier which functions as a
bioaffinity adsorbent for a species of nucleic acid molecule
targeted for isolation.
[0017] According to the instant invention, one embodiment of the
method comprises selectively adsorbing (e.g., non-covalently
binding) target nucleic acid molecules in a reversible manner to
paramagnetic microparticles having a functional group coated (e.g.,
carboxyl-coated) surface by preparing a combination comprising a
mixture of nucleic acid molecules, polyethylene glycol, salt, and
solid phase carriers having a functional group-coated surface that
reversibly binds nucleic acid molecules wherein the polyethylene
glycol and salt are present in sufficient concentrations to
selectively precipitate the target species of nucleic acid
molecules. The combination (first combination) is maintained under
conditions appropriate for adsorption of the precipitated nucleic
acid molecules to the functional group-coated surfaces of the solid
phase carriers, thereby producing solid phase carriers having the
target species of DNA bound thereto. Isolation of the target
species of nucleic acid molecules is accomplished by removing the
nucleic acid-coated carriers from the first combination. The solid
phase carriers (e.g., paramagnetic microparticles) can be recovered
from the first combination, for example, by applying a magnetic
field to draw down the paramagnetic microparticles. More
specifically, 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; for example, vacuum filtration
or centrifugation can be used. The remaining solution can then be
removed; leaving paramagnetic microparticles having a particular
nucleic acid species adsorbed to their surface. Once separated from
the mixture, the isolated nucleic acid species adsorbed to the
solid phase carrier can be recovered by contacting the
microparticles with a suitable elution buffer. As a result, a
solution comprising the target nucleic acid molecules and
paramagnetic microparticles is produced. Using appropriate means,
for example, magnetic means, the microparticles are subsequently
removed from the solution whereby the target species of nucleic
acid molecule is isolated from the mixture and a second mixture is
produced.
[0018] A suitable elution buffer can be water or any aqueous
solution in which the salt concentration and polyalkylene
concentration are below the concentrations required for binding of
DNA onto magnetic microparticles, as discussed above. For example,
useful buffers include, but are not limited to, TRIS-HCl,
Tris-acetate, sucrose (20%) and formamide (100%) solutions. Elution
of the DNA from the microparticles occurs quickly (e.g., in thirty
seconds or less) when a suitable low ionic strength elution buffer
is used. Once the bound DNA has been eluted, the magnetic
microparticles are separated from the elution buffer.
[0019] Optionally, impurities (e.g., host cell components,
proteins, metabolites or cellular debris) can be removed by washing
the paramagnetic microparticles with target nucleic acid bound
thereto (e.g., by contacting the microparticles with a suitable
wash buffer solution) before separating the microparticle-bound
nucleic acid from the solid phase magnetically responsive
microparticle. The composition of the wash buffer is chosen to
ensure that impurities either bound directly to the microparticle,
or associated with the adsorbed DNA are dissolved. 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 exemplifies a preferred wash
buffer useful to remove excess PEG and salt. The magnetic
microparticles with bound DNA can also be washed with more than one
wash buffer solution. The paramagnetic microparticles 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 DNA. 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 bound to the magnetic microparticles does not elute off of the
microparticles, but remains bound. 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 DNA 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); and 0.5M NaCl. A preferred 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).
[0020] In an alternative embodiment the method provides a method of
selectively isolating an additional species of nucleic acid
molecules from the second mixture (produced by the removal of
nucleic acid-coated microparticles from the first combination)
wherein the additional species of nucleic acid molecule is of a
smaller molecular size than the first target species isolated from
the first combination. More specifically, this method comprises the
steps of producing a second combination by adding to the second
mixture solid phase carriers having a functional group-coated
surface that reversibly binds nucleic acid molecules, polyethylene
glycol and salt, wherein the polyethylene glycol and salt are
present in sufficient concentrations to precipitate the additional
target species of nucleic acid molecules. The second combination is
maintained under conditions appropriate for the absorption of the
additional target species to the functional group-coated surfaces
of the solid phase carries, thereby producing solid phase carriers
having the additional target species of nucleic acid molecule bound
thereto. Isolation is accomplished by removing the solid phase
carriers having the additional species of nucleic acid molecules
absorbed thereto from the second combination and eluting the
additional target species into a suitable low ionic strength
solution.
[0021] 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 (single-stranded, double-stranded,
covalently closed, and relaxed circular forms), RNA
(single-stranded and double-stranded), RNA/DNA hybrids and
polyamide nucleic acids (PNAs).
[0022] The term "species" as it used herein to refer to nucleic
acid molecules means a particular subclass, family or type of
nucleic acid molecule defined on the basis of a characteristic
size. Thus, the members of a "species of nucleic acid molecules"
are all of approximately equivalent molecular size within a small
range of molecule sizes.
[0023] As used herein the term "isolated" 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 or purified from other nucleic acid
molecules.
[0024] Appropriate starting material includes, but is not limited
to, lysates prepared from cells obtained from either mammalian
tissue or body fluids, nucleic acid samples eluted from agarose or
polyacrylamide gels, solutions containing multiple species of DNA
molecules resulting either from a poymerase chain reaction (PCR)
amplification or from a DNA size selection procedure and solutions
resulting from a post-sequencing reaction. Suitable starting
solutions typically are mixtures of biomolecules (e.g. proteins,
polysacchardies, lipids, low molecular weight enzyme inhibitors,
oligonucleotides, primers, templates) and other substances such as
agarose, polyacrylamide, trace metals and organic solvents, from
which the target nucleic acid molecule must be isolated.
[0025] For example, a suitable starting material can be host cells
containing an exogenous nucleic acid (e.g., recombinant DNA,
bacterial DNA or replicative form DNA) which is targeted for
isolation from host cell chromosomal DNA and other host cell
biomolecules. According to the current method, host cells are lysed
using known methods, thereby preparing a mixture suitable for use
with the method of the instant invention. An alternative starting
material appropriate for use with the current invention is an
agarose solution. For example, a mixture of nucleic acid can be
separated, according to methods known to one skilled in the art
(e.g., gel electrophoresis), such as by agarose gel
electrophoresis. A plug of agarose containing nucleic acid on
interest can be excised from gel and combined with an appropriate
buffer, into which the nucleic acid is released by heating the
combination to dissolve the agarose plug. The method of the instant
invention can also be used to separate a particular species of DNA
present in a post-shearing procedure mixture; or to remove a
template and primers from a sequencing reaction or to separate PCR
primers from the reaction product of a PCR amplification
protocol.
[0026] As used herein the terms "selective" and "selectively" refer
to the ability to isolate a particular species of DNA molecule, on
the basis of molecular size (e.g., host cell chromosomal DNA or
exogenous plasmid DNA), from a combination which includes or is a
mixture of species of DNA molecules, such as a host cell lysate and
other host cell components. The selective isolation of a particular
species is accomplished through the use of an appropriate
precipitating reagent (e.g., polyalkylene glycol salt) to result in
the precipitation and facilitated adsorption of a particular DNA
species (e.g., characterized on the basis of size) to the surfaces
of paramagnetic microparticles.
[0027] Suitable precipitating reagents include ethanol, isopropanol
and polyalkylene glycols. Appropriate polyalkylene glycols include
polyethylene glycol (PEG) and polypropylene glycol. Generally, PEG
is used. 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 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 soltution. The advantages of using PEG which
is a nondenaturing water soluble polymer, rather than an organic
precipitating reagent (e.g., ethanol, isoproponal or phenol), are
attributed to its benign chemical properties. According to the
current invention, the PEG-induced nucleic acid precipitates are
adsorbed to the surfaces of magnetically responsive microparticles
which can be physically manipulated to facilitate the isolation of
essentially pure species of nucleic acid molecules from complex
solutions comprising mixtures of nucleic acids, in the presence or
absence of other host cell biomolecules. Although numerous
biological macrostructures (bacteriophage, ribosomes, plant and
animal viruses, proteins and nucleic acids) are precipitable with
PEG, the threshold concentration required varies for each
macrostructure (Lis, Methods in Enzymology, 1980). This observation
makes it possible to use the instant method to isolate nucleic acid
molecules, not only from other nucleic acid molecules having a
different molecular size, but also from other host cell
biomolecules and biological macrostructures, each of which will
have a distinct PEG threshold concentration at which it will
precipitate.
[0028] 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 a preferred 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.
[0029] As used herein, "facilitated adsorption" refers to a process
whereby a precipitating reagent, (e.g., a poly-alkyelene glycol) 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. The resulting reversible
interaction is distinct from, for example, an interaction between
two binding partners (e.g., streptavidin/biotin, antibody/antigen
or a sequence-specific interaction), which are conventionally
utilized for the purpose of isolating particular biomolecules based
on their composition or sequence.
[0030] Suitable magnetically responsive paramagnetic microparticles
have sufficient surface area to permit efficient binding and are
further characterized by having surfaces which are capable of
reversibly binding nucleic acids. Suitable solid phase carriers
include, but are not limited to, other particles, fibers, beads and
or supports which have an affinity for DNA and which can embody a
variety of shapes, that are either regular or irregular in form,
provided that the shape maximizes the surface area of the solid
phase, and embodies a carrier which is amenable to microscale
manipulations. Generally, paramagnetic microparticles are used.
[0031] As used herein, "paramagnetic microparticles" refers 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.
Preferred paramagnetic microparticles comprise a magnetite rich
core encapsulated by a pure polymer shell. 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 host cell, the viscosity of the cell
growth and the nature of the vector (e.g. high or low copy)
paramagnetic microparticles comprising a higher percentage of
magnite should be considered. The use of encapsulated paramagnetic
microparticles, having no exposed iron, or Fe.sub.3O.sub.4 on their
surfaces, eliminates the possibility of iron interfering with
polymerase function in certain downstream manipulations of the
isolated DNA. However the larger the magnetite core the higher the
chance of encapsulation leakage (e.g., release of iron oxides).
Suitable paramagnetic microparticles for use in the instant
invention can be obtained for example from Bangs Laboratories Inc.,
Fishers, Ind. (e.g., estapor.RTM. carboxylate-modified encapsulated
magnetic microspheres).
[0032] 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 should not be so large that their
surface area is minimized or that they are not suitable 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.
[0033] As used herein, the term "functional group-coated surface"
refers to a surface which is coated with moieties which reversibly
bind nucleic acid (e.g., DNA, RNA or polyamide nucleic acids
(PNA)). One example 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 or 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 polyalkylene glycol precipitated DNA. In one
embodiment, the functional group is a carboxylic acid. 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 paramagnetic
microparticle. 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.
[0034] One embodiment of the instant invention is a method of
selectively isolating a target species of nucleic acid molecule, on
the basis of its molecular size, from a solution comprising a
mixture of the target nucleic acid species in the presence or
absence of other species of nucleic acid molecules and other
biomolecules. As described herein, the method comprises preparing a
combination comprising a mixture of nucleic acids in the presence
of polyethylene glycol (PEG) and salt, wherein the PEG and salt
concentrations are sufficient to selectively precipitate a
particular species of nucleic acid molecule, which has been
targeted for isolation. The PEG and salt should be present at
levels which are sufficient to precipitate the targeted nucleic
acid species, but insufficient to precipitate relatively smaller
sized nucleic acid molecules or other host cell biomolecules. The
precipitated nucleic acid species targeted for isolation is removed
from the solution by adding a solid phase carrier, such as a
magnetically responsive microparticle, which has a functional
group-coated surface that reversibly binds nucleic acid molecules
to its surfaces. The nucleic acid-coated carrier represents a solid
phase product which can subsequently be removed from the starting
solution by the application of an external force (e.g.,
centrifugation, filtration or magnetic field).
[0035] The removal of the solid phase microparticle from the
solution, results in the isolation of a target species of nucleic
acid molecule, characterized by a particular molecular size, which
is essentially free of other host cell biomolecules and as a
consequence produces a solution from which nucleic acids
characterized by a particular molecular size have been removed. An
additional species of nucleic acid molecule (e.g., a second, third,
fourth, etc.) characterized by having a relatively smaller
molecular size can subsequently be isolated from the resulting
solution by adding solid phase carriers, having a functional
group-coated surface that reversibly binds nucleic acid molecules,
to the solution (from which nucleic acid molecules of relatively
higher molecular weight have been removed) in the presence of
sufficient polyethylene glycol and salt to precipitate the
relatively smaller species of nucleic acid molecule subsequently
targeted for isolation. The resulting combination is maintained
under conditions which favor the adsorption of the second nucleic
acid species, but not other host cell biomolecules present in the
solution, to the surfaces of the microparticles, thereby producing
a second solid phase product. The removal of the second solid phase
product from the solution results in the isolation of an additional
species of nucleic acid molecule that is essentially free of other
species of nucleic acid molecules, characterized by different
molecular sizes, and of other biomolecules present in the starting
solution.
[0036] As used herein a "host cell" is any cell into which
exogenous DNA can be introduced, producing a host cell which
contains exogenous DNA, in addition to host cell DNA. As used
herein the terms "host cell DNA" and "endogenous DNA" refer to DNA
species (e.g., genomic or chromosomal DNA) that are present in a
host cell as the cell is obtained. As used herein, the term
"exogenous" refers to DNA other than host cell DNA; exogenous DNA
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 DNA species which is exogenous to a
particular host cell is a DNA species which is non-endogenous (not
present in the host cell as it was obtained or an ancestor of the
host cell). Appropriate host cell include, but are not limited to,
bacterial cells, yeast cells, plant cells and mammalian cells.
[0037] 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, 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.
Optionally, RNase can be added to the host cell lysate to degrade
host cell RNA, thereby allowing DNA to bind to the magnetic
microparticles 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 PEG-induced precipitation that is being performed. For
example, if the conditions selected for isolation are appropriate
for isolating nucleic acids comprising at least 4000 base pairs,
then it is unlikely that RNA species will be an appreciable
contaminant.
[0038] In one embodiment, the present invention provides a method
of selectively isolating a species of nucleic acid molecule present
in a mixture from other nucleic acid molecules, and from other
biomolecules, biological macrostructures, or reagents possibly
present in the starting material. More specifically, this
embodiment of the method involves: combining a mixture which
comprises the target species of nucleic acid molecules to be
isolated admixed with other nucleic acid molecules, biomolecules,
biological macrostructures or reagents; solid-phase carriers (e.g.,
particles) having a functional group-coated surface, which acts as
a bioaffinity absorbent for nucleic acids; a suitable concentration
of a nucleic acid precipitating reagent(s) (e.g., polyethylene
glycol (PEG) and salt, or an alcohol, such as ethanol or
isopropanol) to result in the facilitated absorption of the target
species of nucleic acid molecules, but not of smaller-sized species
of nucleic acid molecules or other biomolecules, biological
macrostructures or reagents present in the starting material.
Separation of the target species of nucleic acid molecule is
accomplished by applying an external force (e.g., magnetic field,
centrifigation, filtration) suitable to remove the solid phase
carrier having the selectively precipitated nucleic acid bound
thereto from the combination. In a preferred embodiment the solid
phase carrier is a paramagnetic microparticle and separation is
accomplished by applying a magnetic field of appropriate strength.
In a further embodiment the solid phase carrier is a paramagnetic
microparticle and separation is accomplished by applying a magnetic
field of at least 1000 Gauss. This embodiment of the invention is
useful for example to isolate a restriction enzyme digest fragment
having a particular molecular size from smaller fragments present
in the same digest; for isolating a single PCR product from a
multiplex PCR reaction; for the selection of DNA fragments having a
homogenous sized distribution resulting from a shearing procedure
(e.g., nebulizer, sonicator, hydroshear); for removing a nucleic
acid template from a sequencing reaction or for selectively
precipitating the extension products (e.g., Sanger Sequencing
products) from a detemplated sequencing reaction prior to capillary
electrophoresis. For example, the production of shattered DNA
libraries for large scale sequencing experiments requires a
size-selection step to minimize the deviation in size of the DNA
inserts selected for cloning. The ability to produce a library
comprising sheared DNA fragments, characterized by a narrow size
distribution improves an investigator's ability to construct a map
of the original pre-sheared DNA molecule. Using the method
described herein an investigator can preselect a cut off size and
formulate a binding buffer appropriate to precipitate and
selectively adsorb a homogeneous population of DNA fragments. This
embodiment can also be used to isolate extension products from a
detemplated sequencing reaction mixture. The adsorbed nucleic acid
molecules (e.g., Sanger sequencing products) can be thoroughly
washed free of salts (e.g., reagent) and excess terminals whose
presence will interfere with the electrophoretic injection of the
sample to be sequenced.
[0039] In a second embodiment, the method disclosed herein can be
used to isolate two different species, for example, endogenous host
cell nucleic acids and exogenous nucleic acid molecules, present in
the starting material, by first isolating the relatively higher
molecular weight host cell DNA, and subsequently isolating the
relatively smaller-sized exogenous nucleic acid molecules. Thus, an
alternative embodiment of the instant invention further provides a
means for the selective removal of endogenous host cell DNA from a
lysed host cell suspension by performing a first step designed to
precipitate and promote the adsorption of host cell DNA chromosomal
to the functional group-coated surfaces (e.g., a
carboxyl-group-coated surface) of a suitable solid phase carrier
(e.g., microparticle surfaces). The removal of the solid phase
carrier (to which the host cell DNA is bound) from the resulting
mixture results in the removal of the relatively larger-sized host
cell DNA.
[0040] As described above, high quality exogenous DNA can
subsequently be isolated from an exogenous DNA enriched supernatant
by selectively precipitating and adsorbing the relatively lower
molecular weight exogenous DNA to the surfaces of additional
paramagnetic microparticles which are introduced resulting into the
supernatant.
[0041] This alternative embodiment, which results in isolation of
both endogenous host cell nucleic acid and exogenous nucleic acid
(separately) comprises: combining functional group-coated
paramagnetic microparticles and suitable concentrations of a
precipitating reagent, for example, a polyalkylene glycol, and a
salt to promote the facilitated adsorption of precipitated
endogenous host cell nucleic acid (e.g., chromosomal DNA) and
subsequently of exogenous nucleic acid molecules (e.g., bacterial
or viral nucleic acids), each species being characterized by a
particular molecular size, to the surfaces of the microparticles
suspended therein; and the removal, such as by magnetic means, of
the nucleic acid-coated microparticles from the resulting first
combination. The removal of the microparticle having endogenous
host cell nucleic acid adsorbed to its surfaces from the first
combination results in the concomitant separation of host cell
nucleic acid from both exogenous nucleic acid species and from
other host cell biomolecules present in the sample. Exogenous
nucleic acid present in the same sample can subsequently be
isolated by producing a second combination by adding paramagnetic
microparticles which have a functional group-coated surface and a
sufficient quantity of a nucleic acid precipitating reagent to
increase the concentration of the precipitating reagent to a level
sufficient to result in the adsorption of exogenous nucleic acid to
the microparticles suspended therein, thereby producing a third
combination comprising exogenous nucleic acid bound to the
microparticles; removing the paramagnetic microparticles from the
third combination. Thus, exogenous nucleic acid bound to the
microparticles is isolated from other host cell biomolecules
present in the starting solution. Thus, the present invention also
provides a method of selectively separating exogenous nucleic acids
from relatively larger species of endogenous host cell nucleic
acids present in the same sample.
[0042] The selective precipitation of endogenous host cell DNA
(e.g., chromosomal or genomic DNA is mediated by concentrations of
PEG as low as about 1% (w/v) and as high as about 4% (w/v)
depending upon the size of the host cell DNA and the ionic strength
of the solution. In a preferred embodiment, the concentration of
PEG is preferably adjusted to about 3% (weight/volume). The
subsequent selective precipitation of exogenous plasmid DNA is
accomplished by adjusting the PEG concentration to a level which
has been empirically determined to be optimal to promote the
precipitation of a DNA species of a specified macromolecular size
range. For example, exogenous DNA produced from the replication of
a bacterial plasmid in a suitable strain of E. coli would be
isolated by adjusting the PEG concentration of the second
precipitation reaction to about 10% (weight/volume).
[0043] At high salt concentrations (e.g., synonymous with high
ionic strengths) suitable paramagnetic microparticles will adsorb
DNA fragments of all sizes. The term "high salt concentration"
refers to salt concentrations greater than about 0.5 M. At "low
salt concentrations" (or low ionic strengths), which as used herein
connotes concentrations less than about 0.2 M, essentially no DNA,
of any size, will be precipitated even in the presence of a PEG
concentration that is as high as 12% (weight/volume) (Lis, John T,
Methods in Enzymology 65: 437-353 (1980). At intermediate salt
concentrations (e.g., ranging from about 0.3M to about 0.45M) the
characteristic macromolecular size of the DNA species precipitated
by a particular PEG concentration is a function of the interaction
between ionic strength and PEG concentration and reflects a
relationship between macromolecular size and requisite threshold
PEG concentration required to precipitate DNA molecules of a given
size. In general, smaller fragments of DNA will interact with the
functional group-coated surfaces with a lower affinity than larger
DNA fragments in the presence of relatively low concentrations of
salt. To maximize yield and efficiency, sodium chloride
concentration is preferably adjusted to about 0.55 M for the
selective removal of host cell DNA from a lysed host cell
suspension. Yields of bound DNA decrease if the salt concentration
is adjusted to less than about 0.5 M or greater than about 5.0 M.
Purity (e.g. quality) of recombinant DNA isolated during the second
precipitation reaction decreases if the sodium chloride
concentration exceeds about 0.55 M.
[0044] Another embodiment of the instant invention provides a
method by which recombinant nucleic acid molecules expressed in
host cells can be selectively isolated from host cell lysates
comprising a mixture of nucleic acid molecules and other host cell
biomolecules. The following is a description of this embodiment
with reference to nucleic acid molecules as exemplified by DNA. It
is to be understood that the instant embodiment is also useful for
separation of other nucleic acids in a similar manner. This
embodiment of the invention comprises the steps of: preparing a
first combination comprising a lysed host cell solution prepared
from cells expressing a recombinant nucleic acid; encapsulated
carboxyl group-coated paramagnetic microparticles, and low
percentage PEG and low molarity salt. According to the method
disclosed herein, the PEG and salt are present at sufficient
concentrations that high molecular weight host cell DNA is
precipitated and reversibly binds (adsorbs) to the encapsulated
carboxyl group-coated paramagnetic microparticles, thereby
producing paramagnetic microparticles having host cell DNA bound
thereto. The DNA-coated microparticles (and, thus, the
microparticle-adsorbed endogenous DNA) are removed from the first
combination, thereby producing a recombinant DNA-enriched
supernatant. A second combination is produced by adding carboxyl
group-coated paramagnetic microparticles to the recombinant
DNA-enriched supernatant and sufficient polyethyleme glycol to
result in the selective precipitation and adsorption of the
relatively smaller sized recombinant DNA to the surfaces of the
micro-particles, thereby producing paramagnetic microparticles
having recombinant DNA bound thereto; and removing the paramagnetic
microparticles (and thus, the adsorbed recombinant DNA), whereby
recombinant DNA is selectively isolated from host cell DNA.
[0045] Examples of recombinant DNA which can be introduced into a
host cell include, but are not limited to, bacterial artificial
chromosomes (BACs), yeast artificial chromosomes (YACs), PACs, P1s,
cosmids and bacterial plasmids. The exogenous DNA may be directly
introduced into a host cell, or an ancestor thereof, by means well
known to one of ordinary skill in the art, such as transformation
or transfection methods. Alternatively, plasmid DNA may be
indirectly introduced into a host cell, or its ancestor by use of a
phage into which exogenous DNA has been packaged. Suitable plasmid
DNAs which can be packaged into a phage include a cosmid or P1
vector. Suitable host cells include bacterial cells, yeast cells,
plant cells and mammalian cells. For example, suitable strains of
E. coli bacteria include but are not limited to: DH5.alpha., DH1,
DH10B, DH12S, C600 or XL-1 Blue. As used herein the term "plasmid"
refers to double stranded circular DNA species which originate from
an exogenous source (e.g., are introduced into a host cell) and
which are capable of self-replication independent of host
chromosomal DNA. Thus, the term encompasses cloned DNA produced
from the replication of any of the above-mentioned vectors.
Suitable vectors are well known in the art and include, for example
high copy vectors, selected from, but not limited to the group
consisting of pUC, pOT, pBluescript, pGEM, pTZ, pBR322, pSC101,
pACYC, SuperCos and pWE15.
[0046] BACs are particularly difficult to separate and purify from
cleared lysates due to their low concentrations in the lysates,
which is attributable to their low copy number presence in the host
cell. However, BAC DNA (e.g., up to 180 kb in size) is readily
isolated by the method of the present invention. Cosmids are
particularly difficult to isolate from expression host cells using
commercially available chromatography-based methods because of
their relatively large size (e.g., 35 to 40 kb). However, cosmids
are readily separated by the methods of the present invention.
[0047] Thus, the method of the present invention is also useful to
separate recombinant DNA resulting from the replication of an
exogenous vector from a host cell lysate containing an admixture of
host cell biomolecules, including host cell DNA and exogenous
cloned DNA produced by the host cell. Yields of recombinant DNA
following elution typically approach 100% when the magnetic
microparticles are used in excess. High copy plasmid DNA templates
have been prepared according to the disclosed method
characteristically result in sequences which were read with 99% of
unedited accuracy. The simplicity and robust nature of the
disclosed method makes it particularly useful for the preparation
of DNA sequencing templates for automated nucleotide
sequencing.
[0048] Another embodiment of the present invention relates to a
method of isolating a nucleic acid molecule suitable for use as a
template for nucleotide sequencing using either manual or
high-throughput automated sequencing methods. This embodiment
comprises: treating host cells with an alkali and anionic detergent
combination, thereby producing a lysed host cell suspension;
suspending paramagnetic microparticles which bind DNA in the lysed
host cell suspension in the presence of low percentage polyethylene
glycol and low molarity salt, which promotes the precipitation and
selective adsorption of host cell DNA to the surfaces of the
microparticles; removing the paramagnetic microparticles having
host cell DNA bound thereto from the suspension, thereby producing
a plasmid-enriched supernatant; combining additional paramagnetic
microparticles which bind DNA with the plasmid-enriched supernatant
and adjusting the polyethylene glycol percentage and salt
concentration of the supernatant to levels which result in binding
of plasmid DNA to the microparticles; removing the microparticles
having plasmid DNA bound thereto from the supernatant; washing the
microparticles with a wash buffer to remove impurities adsorbed to
the microparticles, thereby producing a purified template; and
contacting the microparticle-bound purified template with an
elution buffer, whereby the plasmid DNA template is released from
the microparticles and is dissolved in the elution buffer, thereby
isolating a purified plasmid DNA template suitable for nucleotide
sequencing.
[0049] The present invention also further relates to a kit
comprising reagents for preparing a host cell lysate which is
appropriate for automated processing according to a system which
has been optimized for high through-put DNA templates preparation;
and an aqueous solution of paramagnetic microparticles having a
functional group-coated surface capable of reversibly binding
nucleic acid molecules. In addition the kit may comprise either at
least one binding preformulated binding buffer comprising
polyalkylene glycol and salt, at concentrations which been
empirically determined to be appropriate to reversibly bind nucleic
acid molecules characterized by a particular molecular size range
onto the functional group-coated surfaces of the paramagnetic
microparticles, or reagents for the formulation of a binding
buffer. Additionally, the kit may comprise at least one
preformulated high ionic strength buffer suitable for use as a wash
buffer, or reagents for preparing such buffer; a preformulated
elution buffer or reagents for its preparation; and a magnetic
microtiter plate holder designed to optimize features of the
magnetic field known to be crucial to the efficiency of automated
processing. The design of the magnetic plate holder is instrumental
in producing a magnetic field having the requisite uniformity and
field strength to maximize the efficiency achievable with automatic
processing. Field Strength of up to and over 1600 Gauss can be
achieved with the use of N35 rare earth magnets configured with
alternating North and South polar spaced 9 mm apart. Since the
magnetically responsive microparticles are paramagnetic they will
attract to either pole.
[0050] 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
isolating and purifying nucleic acid molecules are now available.
Nucleic acids isolated by the disclosed method can be used for
molecular biology applications requiring high quality nucleic
acids, for example, 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, preparation of DNA probes and
primers and detemplating protocols.
[0051] The following Examples are offered for the purpose of
illustrating the present invention and are not to be construed to
limit the scope of this invention. The teachings of all references
cited herein are hereby incorporated, in their entirety, by
reference.
General Methodology
Beads
[0052] The paramagnetic microparticles used in the following
examples were polymer p (S/V-COOH)Mag/Encapsulated paramagnetic
microspheres (brown, mean diameter 1.12 .mu.m, 10% solids; catalog
number ME03N) from Bangs Laboratories, Inc., (Fishers, Ind.). The
particles were stored in phosphate buffered saline (PBS) at a
concentration of 20 mg/ml.
Bead Pretreatment
[0053] Prior to use in any of the separation protocols exemplified
herein, all beads are washed four times with 10 mM Tris Acetate pH
7.8, and diluted tenfold in the 10 mM Tris Acetate buffer. More
specifically, all beads are washed and diluted according to the
following protocol:
[0054] 1. Place 4 mls of beads into a tube.
[0055] 2. Add 26 mls of 10 mM Tris Acetate.
[0056] 3. Apply a magnet to the suspension, and pour off the wash
buffer.
[0057] 4. Repeat steps 2-3, 3 times.
[0058] 5. Resuspend the washed beads in a final volume of 40 mls of
10 mM Tris Acetate.
Lysis Buffer Preparation
[0059] Prepare 0.8 N NaOH/8% SDS lysis buffer by combining 160 ml
of 2 N NaOH, 160 mls of 20% SDS, and 80 mls of distilled water.
Immediately after mixing the buffer should be warmed to, and
maintained at a temperature of 60-70 degrees C. Lysis buffer should
be prepared fresh daily.
Sample Preparation
[0060] Pellet cells (e.g., centrifuge at 1645.times.g, for 10
mins.); decant culture medium; resuspend the pelleted cells in
water in a final volume that is appropriate based on the density of
the starting culture; vortex (e.g., 1200 rpm for 6 mins); and
transfer an aliquot of the resulting cell suspension to flat bottom
multiwell plates (e.g., 150 .mu.l in a 96-well plate).
Ethanol Wash Protocol
[0061] The paramagnetic microparticle bound nucleic acid species is
washed free of contaminants using three to five sequential washes
with 70% Ethanol, with the volume transfers either being
accomplished by hand or by using an automated plate washer (e.g.,
Tecan SLT/PW). A suitable wash protocol using an automated plate
washer would consist of the three to five repetitions of the
following cycle: add 250 ul ethanol, soak for 20 seconds, aspirate
for 7 seconds and repeat.
Sequencing
[0062] Suitable automated sequencing reactions can be performed
using Amersham ET primer Thermosequence 2 terminator chemistry, Big
Dye primer and terminator chemistries, NEN/Omnibase terminator
chemistries, or AB1 FS terminator chemistry using established
protocols that are well known to those of skill in the art.
Sequence results can be obtained using AB1377 Sequencers, MD
MegaBACE capillary or ABI 3700 capillary sequencers. All agarose
gels were run using 1% final agarose (U.S. Biochemical #32827) with
1.times.TBE buffers. The field strength was 10 V/cm with run times
from 40-60 minutes. The gels were post-stained with ethidium
bromide and visualized under UV.
EXAMPLE 1
Isolation of Plasmid DNA Using PEG-Induced Separation and
Paramagnetic Microparticles
[0063] This example provides a procedure, using the method
described herein, to simultaneously process 96 individual plasmid
miniprep samples from bacterial host cells comprising a pOT
plasmid. The teaching of the instant disclosure provides ample
guidance to allow an investigator of ordinary skill to modify this
example to perform routing experimentation to derive a modification
of this method that is capable of isolating exogenous DNA produced
by the expression of alternative vectors (e.g., cosmids, BACs, P1s
etc), in either high-copy- or low-copy-number, in numerous
alternative host cells. Plasmid (e.g., exogenous) DNA was purified
from the host cells by producing a mixture host cell lysate;
preclearing the lysate of high molecular weight endogenous (e.g.,
host cell genomic) DNA by selectively precipitating it under
conditions which promote its adsorption, but not the adsorption of
pOT plasmid DNA, to paramagnetic microparticles; removing the
endogenous DNA-coated microparticles from the sample; transferring
the resulting exogenous DNA (e.g., plasmid DNA) enriched solution
to new microtiter wells; selectively precipitating the plasmid DNA
to additional paramagnetic microparticles; removing the plasmid DNA
coated microparticles from the sample; and eluting the purified pOT
exogenous DNA from the microparticles. The following procedure was
used:
[0064] 1. Produce a Host Cell Lysate. [0065] Place 1.2 ml aliquots
of an overnight culture of E. coli host cells comprising the pOT
plasmid into each well of a 96 deep well microtiter plate. [0066]
Centrifuge the plate for 5 minutes to pellet the host cells. [0067]
Pour off, or aspirate, the resulting supernatant and resuspend the
host cell pellet in a 150 .mu.l volume of Tris-Acetate (pH 7.8), or
in an alternative volume appropriate for the capacity of the wells
of the assay plate being used for processing of the miniprep
samples. [0068] Transfer the resulting host cell 150 .mu.l host
cell suspension to the corresponding wells of a 96 shallow well
microtiter plate. [0069] Add 50 .mu.l of freshly prepared and
prewarmed Lysis Buffer (0.8 N NaOH, 4% SDS) and mix by shaking.
[0070] 2. Removal of High Molecular Weight Endogenous DNA [0071]
Add 20 .mu.l of prewashed carboxyl-coated paramagnetic
microparticles to the host cell lysate mixture. [0072] Add 55 .mu.l
of Precipitation Buffer (18% PEG 8000, 3.3 M NaCl) and mix by
shaking. [0073] Remove the high molecular DNA-coated microparticles
from the sample, preferably by magnetic means; thereby producing a
lysate which is precleared of high molecular weight DNA and
enriched for exogenous plasmid DNA.
[0074] 3. Selective Isolation of Plasmid DNA [0075] Transfer 210
.mu.l aliquots of the resulting plasmid DNA-enriched solution to
new wells of a second shallow well microtiter plate. [0076] Add 20
.mu.l of prewashed carboxyl-coated paramagnetic microparticles to
the host cell lysate mixture. [0077] Add 45 .mu.l of Binding Buffer
(40% PEG 8000) and mix by shaking. Remove the plasmid-DNA coated
paramagnetic microparticles from the sample and discard the
supernatant. For example, suitable magnetic means for the removal
of the microparticles comprise exposing the microtiter sample plate
to a magnetic field of about 1000 Gauss. [0078] Wash the plasmid
DNA coated microparticles three to four times with 250 .mu.l
volumes of Wash Buffer (70% Ethanol, 10 mM EDTA or 12%
Pyrilidinone). [0079] Air dry the washed microparticles. [0080]
Elute the purified plasmid DNA from the microparticle by
resuspending the washed microparticles in a small volume (e.g., 40
.mu.l) of a low ionic strength Elution Buffer, such as water or 10
mM Tris (pH 7.8). Results Electrophoresis: Electrophoretic analysis
of the nucleic acid eluted from the paramagnetic microparticles
used to adsorb the high molecular weight endogenous DNA indicates
the presence of genomic DNA and no RNA or exogenous plasmid DNA.
Electrophoretic analysis of the supernatant produced by the removal
of the endogenous DNA-coated microparticles reveals the presence of
RNA and relatively smaller molecular weight plasmid DNA.
Electrophoretic analysis of the nucleic acids eluted from the
plasmid DNA-coated microparticles obtained in the step (3),
indicates purified plasmid DNA in the absence of any other
detectable nucleic acids. Yields typically range from 10-20 .mu.gs
per 1.2 ml of host cell culture. Sequencing: Plasmid DNA isolated
according to the procedure described in this example was sequenced
using Taq FS polymerase and fluorescently labeled terminators. The
DNA sequence was then electrophoresed on an ABI 3700 sequencer. The
clarity of the data, the low frequency of ambiguous bases and the
average read length evidence DNA of high purity.
[0081] This example demonstrates that DNA obtained using the method
described herein is suitable for use in both manual and automated
nucleotide sequencing protocols; including methodologies which
employ cycle sequencing chemistries enabled by the use of DNA
polymerases having improved thermostability.
EXAMPLE 2
PEG-Induced Size Selection of Sheared DNA for Shotgun Library
Construction
[0082] Shortgun sequencing strategies enable the de novo
determination of an unknown nucleotide sequence. The method imposes
no limitation on the size of the starting DNA molecule whose
sequence is to be determined and requires no prior knowledge of the
nucleotide sequence of the DNA fragments selected as inserts for
cloning. According to protocols which are well-known to those of
skill in the art, the starting DNA molecule, whose sequence is to
elucidated, s fragmented, either by enzymatic digestion or by
physical shearing (e.g., using a nebulizer, sonicator or
hydroshearing) to produce a shattered DNA library typically
comprising 0.5-5 kb fragments. The shotgun strategy then requires
that a subfraction of these fragments characterized by a narrow
size range (e.g., 0.5-1.0 kb, 0.8-1.5 kb, 1.0-1.5 kb) be selected
for use as inserts into an appropriate DNA sequencing vector. The
nucleotide sequences of the resulting subclones are subsequently
determined from standard primer binding sites present in the
flanking DNA of conventional sequencing vectors. The ability to
selectively isolate high purity subfractions comprising DNA
fragments characterized by a narrow size range facilitates the
construction of a ungapped map comprising the complete sequence of
the starting DNA molecule which results from assembling the
resulting subclone sequences into contigs. As shown in this example
the method described herein enables an investigator of ordinary
skill to selectively precipitate DNA fragments characterized by a
narrow size range. Successful practice of the embodiment of the
invention requires only routine experimentation to empirically
determine the composition of a suitable binding buffer which
results in the precipitation of a population of subclones
characterized by a size range selected by the investigator. For
example, an investigator can chose to isolate all fragments larger
than a single cut off size which is determined by the percentage of
PEG and molarity of salt used to prepare the binding buffer.
Alternatively, an investigator may employ sequential PEG-Induced
precipitations, according to the method provided herein, to isolate
fragments characterized by a narrow size range defined by two
different molecular size cut offs which differ from each other by
at least a factor of two. For example, the following protocol has
been used to select fragments of sheared BAC DNA for
subcloning:
[0083] 1. Prepare a Shattered Fragment Library of the DNA to be
Sequenced. [0084] Place 5-10 .mu.g of BAC DNA suspended in 200
.mu.l of water and shatter by hydrodymic shearing using a
HydroShear.TM. (Genemachines) at speed 10.
[0085] 2. Isolation of DNA Fragments Characterized by a Narrow Size
Range Using PEG-Induced Size Selection [0086] Add 20 .mu.l of
prewashed carboxyl-coated paramagnetic microparticles to a 100
.mu.l aliquot of sheared DNA mixture. [0087] Add 110 .mu.l of a
Selective Binding Buffer formulated to precipitate and facilitate
the adsorption of DNA fragments larger than a particular size, and
mix by shaking. The composition of an appropriate Binding Buffer
should be empirically determined based on preliminary experiments
performed using Binding Buffers of varying compositions of PEG and
salt, combined with electrophoretic analysis of the precipitated
nucleic acids. The size range of the desired fragments selected for
precipitation will be dependent on the length of the starting DNA
sequence that is being elucidated and the expected average read
length of the subcloned sequences. For example, a binding buffer
comprising 18% PEG and 0.6 M NaCl can be used to isolate fragments
having the size range of 1.5 kb and greater. [0088] Incubate the
combination for a time sufficient to promote the precipitation and
facilitated adsorption of the target DNA fragments selected for
isolation to the paramagnetic microparticles. [0089] Remove the DNA
fragment coated paramagnetic microparticles from the sample. For
example, suitable magnetic means for the removal of the
microparticles comprise exposing the microtiter sample plate to a
magnetic field of about 1000 Gauss. [0090] Wash the microparticles
three times with 250 ul of wash solution (70% EtOH, 10 mM EDTA).
[0091] Air dry the washed microparticles [0092] Elute the purified
DNA fragments selected for subcloning by contacting the washed
microparticles with a suitable low ionic strength elution buffer,
such as water. Results Electrophoresis: Electrophoretic analysis of
the shattered fragment library resulting from hydroshearing
indicates sheared DNA fragments ranging in size from 500 base pairs
to 4.0 kb. Electrophoretic analysis of the nucleic acids eluted
from the paramagnetic microparticles used to adsorb the target DNA
fragments selected for isolation indicates fragments characterized
by a molecular size of 1.5 kb and greater thus reducing the
frequency of small inserts present in the library.
EXAMPLE 3
[0092] Use of PEG-Induced Size Selection to Prepare Post Nucleotide
Sequencing Reaction Extension Products for Capillary
Electrophoresis
[0093] A conventional sequencing reaction comprises a mixture of a
DNA template, numerous extension products, excess terminators or
primers and nucleotides (e.g., both deoxy- and dideoxynucleotides)
admixed with reagents (e.g., salts or alcohols). It is well known
that the quality of the DNA sequence data, as assessed by average
read length and unedited accuracy, is a direct correlate of the
purity of the extension products used for electrophoretic analysis.
Purity is an important factor for all sequencing methods, and is
particularly crucial to the success of automated dye-labeled
dideoxynucleotide sequencing methods. The following method has been
used to prepare extension products (e.g., Sanger sequencing
products) for capillary electrophoresis: [0094] Transfer a 20 ul
aliquot of detemplated fluorescently labeled dye terminator or dye
primer sequencing products into a shallow well microtiter plate.
The sequencing template can be removed using conventional methods
(e.g., filtration) or by an alternative embodiment of the method
described herein according to the method of Example 4. [0095] Add
20 ul of prewashed carboxyl coated magnetic microparticles to the
sequencing products. [0096] Add 80 ul of Binding Buffer formulated
to comprise PEG and salt concentrations which have been empirically
determined to be sufficient to precipitate the target species of
nucleic acid molecules present in the sequencing reaction. For the
purposes of this example the Binding Buffer was 15% PEG 8000, 20 mM
MgCl2. [0097] Mix by shaking and incubate for a time sufficient to
promote the precipitation and facilitated adsorption of the
extension products to the microparticles. [0098] Remove the
paramagnetic microparticles by exposing the assay plate to a
magnetic field of at least 1000 Gauss. [0099] Wash the
particle-bound extension products three times with 250 ul volumes
of a wash solution comprising 70% EtOH 10 mM EDTA. [0100] Air dry
the washed microparticles. [0101] Elute the purified sequencing
products by contacting the washed with a small volume (e.g., 10 ul)
of loading buffer (70% formamide, 10 mM EDTA or 12% pyrilidone).
Results Sequencing: Sequencing product which had been purified
using this procedure were then electrophoresed using an ABI 3700
capillary sequencer. For the dye terminator samples the
electropherograms exhibited efficient removal of excess dye
terminators and of short extension products below 25 bp. For dye
primer samples the electropherograms exhibited efficient removal of
excess dye primer and of short extension fragments below 25 base
pairs. In both instances the electropherograms showed more
normalized injection of different sized fragments than is exhibited
using standard EtOH precipitation with the electrophoretic
injection used in capillary electrophoresis. Results
Electrophoresis: Extension products purified according to this
procedure were then electrophoresed using an ABI 3700 capillary
sequencer. For the dye terminator samples the electropherograms
exhibited efficient removal of excess dye terminators and of short
extension products below 25 base pears. For dye primer samples the
electropherograms exhibited efficient removal of excess dye primer
and of short extension fragments below 25 base pears. In both
instances the electropherograms showed more normalized injection of
different sized fragments than is exhibited using standard EtOH
precipitation with the electrophoretic injection used in capillary
electrophoresis.
EXAMPLE 4
[0101] Use of PEG-Induced Precipitation and Paramagnetic
Microparticles to Remove a Template from a Nucleotide Sequencing
Reaction
[0102] Several capillary sequencers utilize electrophoretic
injection to selectively inject the relatively smaller components
of a sequencing reaction mixture. Therefore, it is imperative to
detemplate the sequencing reaction mixture in order to attain
effective injection of the extension products to be analyzed.
Conventional methods of template removal employ membranes and
consequently do not lend themselves to automation. Using the method
described herein, an investigator of ordinary skill in the art can
easily determine a protocol to selectively precipitate the DNA
template, but not the relatively smaller sized extension fragments,
under conditions which will facilitate the adsorption of the
template to the surfaces of paramagnetic microparticles introduced
into the mixture to facilitate the removal of the template from the
reaction. For example the following procedure has been successfully
used to detemplate Sanger sequencing reactions thereby producing a
sample enriched for extension products: [0103] Transfer 20 ul
aliquots of either fluorescently labeled dye terminator or dye
primer sequencing products into the wells of a shallow well
microtiter plate. [0104] Add 10 ul of prewashed carboxyl coated
magnetic microparticles to the sequencing products. [0105] Add 40
ul of Binding Buffer which has been empirically determined to be
suitable for the selective precipitation of the DNA template
targeted for isolation, but not extension products present in the
same mixture. For example, a Binding Buffer comprising 11% PEG
8000, 1.1 M NaCl can be used. [0106] Mix by shaking and incubated
for a time sufficient to promote the precipitation and facilitated
adsorption of the extension products to the microparticles. [0107]
Remove the paramagnetic microparticles by exposing the assay plate
to a magnetic field of at least 1000 Gauss. [0108] Wash the
particle-bound extension products three times with 250 ul volumes
of a wash solution comprising 70% EtOH 10 mM EDTA. [0109] Air dry
the washed microparticles. [0110] Elute the purified sequencing
products by contacting the washed with a small volume (e.g., 10 ul)
of loading buffer (70% formamide, 1 mM EDTA). Results
Electrophoresis: Sanger sequencing reaction products obtained using
this procedure were placed on an agarose gel for electrophoretic
analysis. Template and extension products are visible prior to
cleanup, while only extension products are visible after.
Readlengths assessed in phred 20s (99.0%) accuracy)=550 bp
utilizing a ABI 377, 48 cm WTR, 10 hour gel. Phred 15 10 bp window
from the end of trace falls at 716 bp.
EXAMPLE 5
[0110] Use of Alcohol Precipitation and Paramagnetic Microparticles
for the Isolation of High Purity Extension Products Suitable for
Capillary Electrophoresis
[0111] The purity of extension products prepared for capillary
sequencing can be even further improved by subjecting extension
products selected according to the method of Example 3 to an
alcohol-induced precipitation reaction performed under conditions
suitable to promote the adsorption of the extension products
targeted for isolation to paramagnetic microparticles. The ability
to adsorb the extension products to a solid phase carrier
facilitates an investigator's ability to wash the extension
products free of any reagents and/or nucleotides present in the
sequencing reaction. The presence of these components of the
sequencing reaction are known to adversely effect the quality of
the sequencing data obtained. For example the following protocol
has been used to improve the purity of Sanger sequencing products,
and concomitantly improve the quality of the resulting sequencing
data: [0112] Transfer 20 ul aliquots of either fluorescently
labeled dye terminator or dye primer sequencing products into the
wells of a shallow well microtiter plate. [0113] Add 10 ul of
prewashed carboxyl coated magnetic microparticles to the sequencing
products. [0114] Add 80 ul of Alcohol Precipitation Solution. For
example, a 95% Ethanol or Isopropanol [0115] Solution can be used.
[0116] Mix by shaking and incubated for a time sufficient to
promote the precipitation and facilitated adsorption of the
extension products to the microparticles. [0117] Remove the
paramagnetic microparticles by exposing the assay plate to a
magnetic field of at least 1000 Gauss. [0118] Air dry the nucleic
acid coated microparticles. [0119] Elute the purified extension
products with 10 ul 70% Formamide or 12% pyrrolidinone. Results
Capillary Electrophoresis: Sequencing products obtained using this
procedure have a reduced representation of excess primer which aids
in lane tracking and signal processing.
[0120] 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
spirit and scope of the invention as defined by the appended
claims.
* * * * *