U.S. patent application number 10/346714 was filed with the patent office on 2003-12-25 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 McEwan, Paul, McKernan, Kevin, Morris, William.
Application Number | 20030235839 10/346714 |
Document ID | / |
Family ID | 26772764 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235839 |
Kind Code |
A1 |
McKernan, Kevin ; et
al. |
December 25, 2003 |
Solid phase technique for selectively isolating nucleic acids
Abstract
Described herein is a method in which genomic nucleic acid of a
cell can be separated from nucleic acid having a molecular weight
that is lower than the molecular weight of the genomic nucleic acid
(e.g., plasmid DNA) of the cell directly from a cell growth
culture. Also described herein, a method in which genomic nucleic
acid can be separated from nucleic acid having a molecular weight
that is lower than the molecular weight of the genomic nucleic acid
in a cell lysate without the need to prepare a cleared lysate.
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.: |
10/346714 |
Filed: |
January 16, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10346714 |
Jan 16, 2003 |
|
|
|
09311317 |
May 13, 1999 |
|
|
|
6534262 |
|
|
|
|
60085480 |
May 14, 1998 |
|
|
|
60121779 |
Feb 26, 1999 |
|
|
|
Current U.S.
Class: |
435/6.18 ;
435/6.1; 536/25.4 |
Current CPC
Class: |
C12N 15/1006 20130101;
C12Q 1/6806 20130101; C12Q 1/6806 20130101; C12N 15/1013 20130101;
C12Q 2527/137 20130101; C12Q 2523/308 20130101; C12Q 2563/143
20130101 |
Class at
Publication: |
435/6 ;
536/25.4 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. A method of separating genomic nucleic acid of a cell from
nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in the cell
comprising: a) combining i) solid phase carriers whose surfaces
have bound thereto a functional group which reversibly binds
nucleic acid, ii) a cell and iii) a reagent, wherein the reagent
causes lysis of the cell and precipitation of the nucleic acid of
the cell onto the solid phase carriers; thereby producing a
combination; b) maintaining the combination under conditions in
which lysis of the cell occurs and the nucleic acid of the cell
binds reversibly to the solid phase carriers, thereby producing
solid phase carriers having nucleic acid of the cell bound thereto;
c) separating the solid phase carriers from the combination; d)
contacting the solid phase carriers with an elution buffer that
causes elution of the nucleic acid having a lower molecular weight
than the genomic nucleic acid from the solid phase carriers,
thereby separating genomic nucleic acid of the cell from nucleic
acid having a molecular weight that is lower than the molecular
weight of the genomic nucleic acid in the cell.
2. The method of claim 1 wherein the nucleic acid having a lower
molecular weight is selected from the group consisting of: plasmid
DNA, episomal DNA, mitochondrial DNA, organelle DNA, and viral
DNA.
3. The method of claim 1 wherein the solid phase carriers magnetic
microparticles.
4. The method of claim 3 wherein the magnetic microparticles have a
coated surface wherein the coated surface is selected from the
group consisting of: a carboxyl group coated surface and amine
group coated surface.
5. The method of claim 1 wherein the reagent comprises an
alcohol.
6. The method of claim 5 wherein the alcohol is selected from the
group consisting of: ethanol, isopropanol and polyalkylene
glycol.
7. The method of claim 5 wherein the reagent further comprises a
salt.
8. The method of claim 7 wherein the salt is selected from the
group consisting of: NaCl, LiCl and MgCl.
9. The method of claim 1 wherein elution buffer comprises
water.
10. The method of claim 9 wherein the elution buffer further
comprises RNAse.
11. The method of claim 1 wherein the solid phase carriers are
separated from the combination using a method selected from the
group consisting of: applying a magnetic field, applying vacuum
filtration and applying centrifugation.
12. A method of separating genomic nucleic acid of a cell from
plasmid nucleic acid of the cell comprising: a) combining i)
magnetic microparticles whose surfaces have bound thereto a
functional group which reversibly binds nucleic acid, ii) a cell
and iii) a reagent, wherein the reagent causes lysis of the cell
and precipitation of nucleic acid of the cell onto the magnetic
microparticles; thereby producing a combination; b) maintaining the
combination under conditions in which lysis of the cell occurs and
the nucleic acid of the cell binds reversibly to the magnetic
microparticles, thereby producing magnetic microparticles having
nucleic acid of the cell bound thereto; c) separating the magnetic
microparticles having nucleic acid of the cell bound thereto from
the combination; d) contacting the magnetic microparticles with an
elution buffer that causes elution of the plasmid nucleic acid from
the magnetic microparticles, thereby separating genomic nucleic
acid of the cell from nucleic acid having a lower molecular weight
in the cell.
13. The method of claim 12 wherein the magnetic microparticles have
a coated surface wherein the coated surface is selected from the
group consisting of: a carboxyl group coated surface and amine
group coated surface.
14. The method of claim 12 wherein the reagent comprises an
alcohol.
15. The method of claim 14 wherein the alcohol is selected from the
group consisting of: ethanol, isopropanol and polyalkylene
glycol.
16. The method of claim 14 wherein the reagent further comprises a
salt.
17. The method of claim 16 wherein the salt is selected from the
group consisting of: NaCl, LiCl and MgCl.
18. The method of claim 12 wherein the elution buffer comprises
water.
19. The method of claim 18 wherein the elution buffer further
comprises RNAse.
20. The method of claim 12 wherein the magnetic microparticles are
separated from the combination using a method selected from the
group consisting of: applying a magnetic field, applying vacuum
filtration and applying centrifugation.
21. A method of separating genomic nucleic acid of a cell from
nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in the cell
comprising: a) combining i) solid phase carriers whose surfaces
have bound thereto a functional group which reversibly binds
nucleic acid, ii) a cell lysate and iii) a reagent, wherein the
reagent causes precipitation of the nucleic acid of the cell lysate
onto the solid phase carriers; thereby producing a combination; b)
maintaining the combination under conditions in which the nucleic
acid of the cell lysate binds reversibly to the solid phase
carriers, thereby producing solid phase carriers having nucleic
acid of the cell bound thereto; c) separating the solid phase
carriers from the combination; d) contacting the solid phase
carriers with an elution buffer that causes elution of the nucleic
acid having a lower molecular weight than the genomic nucleic acid
from the solid phase carriers, thereby separating genomic nucleic
acid of the cell from nucleic acid having a molecular weight that
is lower than the molecular weight of the genomic nucleic acid in
the cell.
22. The method of claim 21 wherein the nucleic acid having a lower
molecular weight is selected from the group consisting of: plasmid
DNA, episomal DNA, mitochondrial DNA, organelle DNA and viral
DNA.
23. The method of claim 21 wherein the solid phase carriers are
magnetic microparticle.
24. The method of claim 23 wherein the magnetic microparticles have
a coated surface wherein the coated surface is selected from the
group consisting of: a carboxyl group coated surface and amine
group coated surface.
25. The method of claim 21 wherein the reagent comprises an
alcohol.
26. The method of claim 25 wherein the alcohol is selected from the
group consisting of: ethanol, isopropanol and polyalkylene
glycol.
27. The method of claim 25 wherein the reagent further comprises a
salt.
28. The method of claim 27 wherein the salt is selected from the
group consisting of: NaCl, LiCl and MgCl.
29. The method of claim 21 wherein elution buffer comprises
water.
30. The method of claim 29 wherein the elution buffer further
comprises RNAse.
31. The method of claim 21 wherein the solid phase carriers are
separated from the combination using a method selected from the
group consisting of: applying a magnetic field, applying vacuum
filtration and applying centrifugation.
32. The method of claim 21 wherein the cell is selected from the
group consisting of: a bacterial cell, a viral cell and a mammalian
cell.
33. The method of claim 21 wherein the mammalian cell is a whole
blood cell.
Description
BACKGROUND OF THE INVENTION
[0001] 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.
[0002] 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
[0003] As described herein, Applicants provide a method in which
genomic nucleic acid of a cell can be separated from nucleic acid
having a molecular weight that is lower than the molecular weight
of the genomic nucleic acid (e.g., plasmid DNA) of the cell,
directly from a cell growth culture. In addition, Applicants
provide a method in which genomic nucleic acid can be separated
from nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in a cell lysate,
without the need to prepare a cleared lysate.
[0004] Traditional alkaline lysis requires the following steps:
concentrating or pelleting cells diluted in growth media;
centrifuging or vortexing; lysing cells with alkaline detergent;
shaking and/or agitating lysate; adding neutralization buffer;
filtering and/or manipulating sample to remove the flocculent mass;
adding a solid phase carrier; and adding binding buffer. Additional
purification steps are generally required to remove the detergents
and salts as follows: addition of solid phase carriers and addition
of a binding buffer.
[0005] An advantage of the invention is that it allows for a
simplified procedure for separating genomic nucleic acid of a cell
from nucleic acids having a molecular weight lower than the
molecular weight of the genomic nucleic acid of the cell. By
providing solid phase carriers and a reagent that causes lysis of
cells and precipitation of the nucleic acid of the cells onto the
solid phase carriers (a nucleic acid precipitation agent), one or
more steps can be removed from the standard purification process of
nucleic acid from intact or whole cells. Furthermore, by providing
solid phase carriers and a reagent that causes precipitation of the
nucleic acid of the cell onto the solid phase carriers, one or more
steps can be removed from the standard purification process of
nucleic acid from cell lysates. The order in which the solid phase
carriers and the reagent are combined with a cell or cell lysate is
not critical. The solid phase carriers and the reagent that causes
precipitation of the nucleic acid of the cell onto the solid phase
carriers and/or lysis of the cells, can be combined with a cell or
cell lysate sequentially (e.g., as separate components) or
simultaneously (e.g., as a single component). When the solid phase
carriers and the reagent are combined into a single component, the
methods described herein allow for the addition of a single reagent
to a cell or cell culture, followed by an incubation, a separation
of a solid phase carrier and a selective elution to achieve
separation of a cell's genomic nucleic acid from the cell's nucleic
acid which has a molecular weight that is lower than the molecular
weight of the genomic nucleic acid. No pH adjustments are required
by the methods of the invention. The reduced number of steps
provided by the reagents and methods described herein simplifies
the automation of the nucleic acid purification process of cells or
cell lysates.
[0006] Accordingly, the present invention relates to a method of
separating genomic nucleic acid of a cell from nucleic acid having
a molecular weight that is lower than the molecular weight of the
genomic nucleic acid in the cell (e.g., plasmid DNA, episomal DNA,
mitochondrial DNA, organelle DNA, viral DNA) comprising combining
i) solid phase carriers (e.g., magnetic microparticles) whose
surfaces have bound thereto a functional group (e.g., carboxyl
group, amine group) which reversibly binds nucleic acid, ii) a cell
and iii) a reagent, wherein the reagent causes lysis of the cell
and precipitation of the nucleic acid of the cell onto the solid
phase carriers, thereby producing a combination. The combination is
maintained under conditions in which lysis of the cell occurs and
the nucleic acid of the cell binds reversibly to the solid phase
carriers, thereby producing solid phase carriers having nucleic
acid of the cell bound thereto. The solid phase carriers are
separated from the combination and contacted with an elution buffer
(e.g., water) that causes elution (selective elution) of the
nucleic acid having a lower molecular weight than the genomic
nucleic acid from the solid phase carriers. The genomic nucleic
acid remains bound to the solid phase carrier, thereby resulting in
the separation of genomic nucleic acid of the cell from nucleic
acid having a molecular weight that is lower than the molecular
weight of the genomic nucleic acid in the cell.
[0007] The present invention also relates to a method of separating
genomic nucleic acid of a cell from plasmid nucleic acid of the
cell comprising combining i) magnetic microparticles whose surfaces
have bound thereto a functional group which reversibly binds
nucleic acid, ii) a cell and iii) a reagent (e.g., alcohol such as
ethanol, isopropanol, polyallylene glycol), wherein the reagent
causes lysis of the cell and precipitation of nucleic acid of the
cell onto the magnetic microparticles, thereby producing a
combination. The combination is maintained under conditions in
which lysis of the cell occurs and the nucleic acid of the cell
binds reversibly to the magnetic microparticles, thereby producing
magnetic microparticles having nucleic acid of the cell bound
thereto. The magnetic microparticles having nucleic acid of the
cell bound thereto are separated from the combination and contacted
with an elution buffer that causes elution of the plasmid nucleic
acid from the magnetic microparticles. The genomic nucleic acid
remains bound to the solid phase carrier, thereby resulting in the
separation of genomic nucleic acid of the cell from plasmid nucleic
acid of the cell.
[0008] The present invention also relates to a method of separating
genomic nucleic acid of a cell from nucleic acid having a molecular
weight that is lower than the molecular weight of the genomic
nucleic acid in the cell comprising combining i) solid phase
carriers whose surfaces have bound thereto a functional group which
reversibly binds nucleic acid, ii) a cell lysate and iii) a
reagent, wherein the reagent causes precipitation of the nucleic
acid of the cell lysate onto the solid phase carriers, thereby
producing a combination. The combination is maintained under
conditions in which the nucleic acid of the cell lysate binds
reversibly to the solid phase carriers, thereby producing solid
phase carriers having nucleic acid of the cell bound thereto. The
solid phase carriers are separated from the combination and
contacted with an elution buffer that causes elution of the nucleic
acid having a lower molecular weight than the genomic nucleic acid
from the solid phase carriers. The genomic nucleic acid remains
bound to the solid phase carrier, thereby separating genomic
nucleic acid of the cell from nucleic acid having a molecular
weight that is lower than the molecular weight of the genomic
nucleic acid in the cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 is an illustration of the protocol for separation of
genomic nucleic acid of a cell from nucleic acid having a lower
molecular weight than the genomic nucleic acid in the cell.
[0010] FIG. 2 is a 96-well Agarose gel which shows separation of E.
coli genomic nucleic acid of a cell from a plasmid in the cell.
[0011] FIG. 3 is a histogram of the Phred 20 (red) and Phred 30
(black) bases generated by the reads; the Y axis is number of
reads, the X axis if Phred 20 binds in 50 bp increments.
[0012] FIG. 4 shows gDNA duplicates prepared from 50 ul horse
blood.
[0013] FIG. 5 shows the PicoGreen Analysis of 8 samples prepared
gDNA from horse blood.
[0014] FIG. 6 shows a gradient PCR of prepared gDNA from horse
blood (using Y3B19 markers with an expected amplicon size of 225
bp).
DETAILED DESCRIPTION OF THE INVENTION
[0015] As described herein, the present invention provides methods
in which a cell's genomic nucleic acid can be separated from the
cell's nucleic acid which has a molecular weight that is lower than
the molecular weight of the genomic nucleic acid (e.g., plasmid
DNA) using a minimal number of steps. The separation can be
performed on a cell culture directly without the need to pellet the
cells. In addition, the methods described herein can be performed
directly on a cell lysate without the need to clear the lysate of
genomic nucleic acid using traditional methods (e.g.,
centrifugation, chemical treatment).
[0016] The present invention provides methods and reagents for
isolating nucleic acids. The reagents described herein can be used
to separate genomic nucleic acid of a cell (one or more) from
nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid of the cell, by
combining the cell with solid phase carriers and a reagent which
causes lysis of the cell and precipitation of nucleic acid of the
cell onto the solid phase carriers. Alternatively, the reagents
described herein can be used to separate genomic nucleic acid of a
cell lysate from nucleic acid having a molecular weight that is
lower than the molecular weight of the genomic nucleic acid of the
cell lysate, by combining the cell lysate with solid phase carriers
and a reagent which causes precipitation of nucleic acid of the
cell lysate onto the solid phase carriers. The nucleic acid having
a molecular weight that is lower than the molecular weight of the
genomic nucleic acid of the cell is then selectively eluted from
the solid phase carriers.
[0017] As described herein, the method comprises binding the
nucleic acid of a cell or cell lysate nonspecifically and
reversibly to solid phase carriers (e.g., magnetic microparticles)
having a functional group coated surface (e.g., carboxyl coated
surface). The microparticles are then separated from the
supernatant, for example, by applying a magnetic field to draw down
the magnetic microparticles. The remaining solution, (e.g.,
supernatant) can then be removed, leaving the microparticles with
the bound nucleic acid. Once separated from the supernatant, the
microparticles can be contacted with an elution buffer that
selectively elutes the nucleic acid having a molecular weight that
is lower than the molecular weight of genomic nucleic acid of the
cell. As a result, an elution buffer containing unbound nucleic
acid (the cell's nucleic acid which has a lower molecular weight
than the molecular weight of the cell's genomic nucleic acid) and
magnetic microparticles to which genomic nucleic acid of the cell
is still bound are produced. An elution buffer is a solution in
which the concentration of a nucleic acid precipitating reagent is
below the range required for binding of nucleic acid having a
molecular weight that is lower than the molecular weight of genomic
nucleic acid onto magnetic microparticles. In one embodiment, the
eluent is water. In addition, sucrose (20%) and formamide (100%)
solutions can be used to elute the nucleic acid. Elution of the
nucleic acid from the microparticles occurs in thirty seconds or
less when an elution buffer of low ionic strength, for example,
water, is used. Once the bound nucleic acid has been eluted, the
magnetic microparticles are separated from the elution buffer that
contains the eluted nucleic acid. Preferably, the magnetic
microparticles are separated from the elution buffer by magnetic
means. Other methods known to those skilled in the art can be used
to separate the magnetic microparticles from the supernatant. For
example, filtration or centrifugation can be used.
[0018] The isolation of high quality nucleic acid preparations from
starting solutions of diverse composition and complexity is a
fundamental technique in molecular biology. As a result of the
reagents and methods described herein, rapid and readily
automatable methods of separating genomic nucleic acid from nucleic
acid having a lower molecular weight than genomic nucleic acid are
now available. Nucleic acids isolated by the disclosed methods can
be used for molecular biology applications requiring high quality
nucleic acids, such as the preparation of DNA sequencing templates,
microinjection, transfection or transformation of mammalian cells,
in vitro synthesis of RNAi hairpins, reverse transcription cloning,
cDNA library construction, PCR amplification, and 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.
[0019] The reagents and methods described herein can be used
together with a variety of nucleic acid purification techniques,
including those described in U.S. Pat. Nos. 5,705,628 and 5,898,071
as well as WO 99/58664, the contents of which are herein
incorporated by reference.
[0020] In one embodiment, the present invention relates to a method
of separating genomic nucleic acid of a cell from nucleic acid
having a molecular weight that is lower than the molecular weight
of the genomic nucleic acid (e.g., plasmid DNA) in the cell,
comprising combining i) solid phase carriers whose surfaces have
bound thereto a functional group which reversibly binds nucleic
acid, ii) a cell and iii) a reagent, wherein the reagent causes
lysis of the cell and precipitation of the nucleic acid of the cell
onto the solid phase carriers, thereby producing a combination. The
combination is maintained under conditions in which lysis of the
cell occurs and the nucleic acid of the cell binds reversibly to
the solid phase carriers, thereby producing solid phase carriers
having nucleic acid of the cell bound thereto. The solid phase
carriers are separated from the combination and contacted with an
elution buffer that causes elution of the nucleic acid having a
molecular weight that is lower than the molecular weight of the
genomic nucleic acid from the solid phase carriers, but does not
cause elution of the genomic nucleic acid from the solid phase
carriers, thereby separating genomic nucleic acid of the cell from
nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in the cell.
[0021] In another embodiment, the present invention relates to a
method of separating genomic nucleic acid of a cell from nucleic
acid having a molecular weight that is lower than the molecular
weight of the genomic nucleic acid in the cell, comprising
combining i) solid phase carriers whose surfaces have bound thereto
a functional group which reversibly binds nucleic acid, ii) a cell
lysate and iii) a reagent, wherein the reagent causes precipitation
of the nucleic acid of the cell lysate onto the solid phase
carriers, thereby producing a combination. The combination is
maintained under conditions in which the nucleic acid of the cell
lysate binds reversibly to the solid phase carriers, thereby
producing solid phase carriers having nucleic acid of the cell
bound thereto. The solid phase carriers are removed from the
combination and contacted with an elution buffer that causes
elution of the nucleic acid having a molecular weight that is lower
than the genomic nucleic acid, but does not cause elution of the
genomic nucleic acid from the solid phase carriers, from the solid
phase carriers, thereby separating genomic nucleic acid of the cell
from nucleic acid having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in the cell.
[0022] The present invention further relates to a method of
separating genomic nucleic acid of a cell from nucleic acid having
a molecular weight that is lower than the molecular weight of the
genomic nucleic acid in the cell, wherein the nucleic acid having a
lower molecular weight is suitable for use in either manual or a
high-throughput automated sequencing methods.
[0023] In one embodiment, the invention is a readily automatable
method of isolating a plasmid DNA template for nucleotide
sequencing. The use of the reagents described herein affords an
alternative, and readily automatable, means of nucleic acid
separation.
[0024] As used herein the term "separating" 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.
[0025] 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, and relaxed circular forms),
RNA (e.g., single-stranded and double-stranded), RNA/DNA hybrids
and polyamide nucleic acids (PNAs).
[0026] "Genomic nucleic acid" refers to the genomic or chromosomal
nucleic acid present in a cell. Typically, the molecular weight of
genomic or chromosomal nucleic acid is from about 500 kilobases
(kb) (e.g., mycoplasma) to about 500 gigabases (Gb). In particular
embodiments, the molecular weight of genomic or chromosomal nucleic
acid ranges from about 1000 kb to about 250 Gb (e.g., onion); from
about 10,000 kb to about 5 Gb; from about 100,000 kb to about 1 Gb;
and from about 500,000 kb to 1,000,000 kb.
[0027] "Nucleic acid having a molecular weight that is lower than
the molecular weight of the genomic nucleic acid in the cell"
refers to nucleic acid other than genomic or chromosomal nucleic
acid that is present in a cell and can be endogenous or exogenous
nucleic acid. Nucleic acid having a molecular weight that is lower
than the molecular weight of the genomic nucleic acid in the cell
typically has a molecular weight between about 1 kb to about
250,000 kb (e.g., BAC). In particular embodiments, nucleic acid
having a molecular weight that is lower than the molecular weight
of the genomic nucleic acid in the cell ranges from about 5 kb to
about 100,000 kb; from about 100 kb to about 10,000 kb; and from
about 1000 kb to about 5000 kb. As used herein "endogenous nucleic
acid" (host cell nucleic acid) refer to nucleic acid that are
present in the cell as the cell is obtained. "Exogenous nucleic
acid" (foreign nucleic acid; recombinant nucleic acid) refer to
nucleic acid that is not present in the cell as obtained (e.g.
transfected cell, transduced cell). Exogenous nucleic acid may be
present in a cell as a result of being introduced into the cell or
being introduced into an ancestor of the cell. The exogenous
nucleic acid may be introduced directly or indirectly into the cell
or an ancestor thereof by means known to one of ordinary skill in
the art (e.g., transformation or transfection). Examples of
exogenous nucleic acid introduced into a cell include bacterial
artificial chromosome (BAC), yeast artificial chromosomes (YAC),
plasmids, cosmids, P1 vector and nucleic acid introduced due to an
amplification process (e.g., polymerase chain reaction (PCR)). 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. Examples of vectors used to introduce
nucleic acid into a cell include pUC, pOT, pBluescript, pGEM, pTZ,
pBR322, pSC101, pACYC, SuperCos and pWE15. Alternatively, the
exogenous nucleic acid may be introduced into the cell from a phage
into which the nucleic acid has been packaged (e.g., cosmid, P1).
Additional examples of "nucleic acid having a molecular weight that
is lower than the molecular weight of the genomic nucleic acid in
the cell" include, but are not limited to, episomal nucleic acid,
mitochondrial nucleic acid, organelle nucleic acid, RNA, siRNA,
plastids, microchromosomes, organelle nucleic acid, primers, viral
nucleic acid, bacterial nucleic acid and nucleic acid from other
pathogens.
[0028] A "solid phase carrier" is an entity that is essentially
insoluble under conditions upon which a nucleic acid can be
precipitated. Suitable solid phase carriers for use in the methods
of the present invention 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,
microparticles, fibers, beads and supports which have an affinity
for nucleic acid 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. In one
embodiment, the solid phase carrier is paramagnetic, e.g., a
paramagnetic microparticle (magnetically responsive). In another
embodiment, the solid phase carrier includes a functional group
coated surface. For example, the solid phase carrier can be an
amine-coated paramagnetic microparticle, a carboxyl-coated
paramagnetic microparticle, or an encapsulated carboxyl
group-coated paramagnetic microparticle.
[0029] 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/encapsulation 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 micro spheres),
Agencourt Biosciences and Seradyn.
[0030] 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 1 00.mu. mean diameter. A preferred size is
about 1.0.mu. mean diameter. Suitable magnetic microparticles are
commercially available from PerSeptive Diagnostics and are referred
to as BioMag COOH.
[0031] 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 precipitated nucleic acid (e.g., 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. Carboxylic
acid-coated magnetic particles are commercially available from
PerSeptive Diagnostics (BioMag COOH). 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.
[0032] Appropriate starting material include cells (intact or whole
cells), cell lysates (cells in growth or culture media) and lysates
prepared from such cells. Appropriate starting material 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. Thus, any type of cell having genomic
nucleic acid and nucleic acid having a molecular weight lower than
the molecular weight of the genomic nucleic acid can be used.
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) which is targeted for isolation from host cell
chromosomal DNA and other host cell biomolecules. Alternatively,
the starting material can be lysates prepared from such cells.
[0033] As used herein a "host cell" is any cell into which
exogenous nucleic acid can be introduced, thereby producing a host
cell which contains exogenous nucleic acid, in addition to host
cell nucleic acid. As used herein the terms "host cell nucleic
acid" and "endogenous nucleic acid" refer to nucleic acid species
(e.g., genomic or chromosomal nucleic acid) that are present in a
host cell as the cell is obtained. As used herein, the term
"exogenous" refers to nucleic acid other than host cell nucleic
acid (e.g., plasmid); exogenous nucleic acid 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 which is exogenous to a particular host cell
is a nucleic acid species which is non-endogenous (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.
[0034] As described herein, an advantage of the present invention
is that a cell's genomic nucleic acid can be separated from the
cell's nucleic acid which has a molecular weight that is lower than
the molecular weight of the genomic nucleic acid using a minimal
number of steps. As described herein, the separation can be
performed on a cell culture directly without the need to pellet the
cells. In addition, the method can be performed on a cell lysate,
wherein the methods of the present invention make it unnecessary to
clear the lysate in order to separate genomic nucleic acid of a
cell from nucleic acid having a molecular weight that is lower than
the molecular weight of the genomic nucleic acid.
[0035] As used herein, a "lysate" is a solution containing cells
which contain genomic nucleic acid and nucleic acid having a lower
molecular weight than genomic nucleic acid and whose cell 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), isotonic shock, or
physical disruption (e.g., homogenization).
[0036] 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.
[0037] According to the methods of the present invention, 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.
[0038] "Non-specific nucleic acid binding" refers to binding of
different nucleic acid molecules with approximately the same
affinity to magnetic microparticles, despite differences in the
nucleic acid sequence or size of the different nucleic acid
molecules. 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.
[0039] 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.
[0040] Appropriate alcohol (e.g., ethanol, isopropanol)
concentrations (final concentrations) for use in the methods of the
present invention are from about 40% to about 60%; from about 45%
to about 55%; and from about 50% to about 54%.
[0041] 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 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%.
[0042] As described above, in the embodiment in which the starting
material is a cell the reagent is 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). Optionally, 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.
[0043] The reagent used in the methods described above is useful
for isolating a nucleic acid from a cell. This reagent contains a
nucleic acid precipitating agent and a solid phase carrier, and can
also be formulated to cause lysis of a cell(s). The nucleic acid
precipitating agent is of sufficient concentration to precipitate
the nucleic acid of the cell. The solid phase carrier in this
reagent contains a surface that binds nucleic acid of the cell. The
components of the reagent can be contained in a single reagent or
as separate components. The components can be combined
simultaneously or sequentially with cells. The order in which the
elements of the combination are combined is not 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.
[0044] Optionally, salt may be added to the reagent to cause
precipitation of the nucleic acid of the cell 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 0.1M to about
0.5M; from about 0.15M to about 0.4M; and from about 2M to about
4M.
[0045] 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). Additional components may
be added to the reagent. In one embodiment, RNAse is added to the
nucleic acid precipitating agent.
[0046] According to the methods of the invention, the isolation of
the nucleic acid molecules of the cell 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. Once separated from the mixture, the nucleic acid having a
lower molecular than the molecular weight of the genomic nucleic
acid of the cell which is adsorbed to the solid phase carrier can
be recovered by contacting the solid phase carrier with a suitable
elution buffer. As a result, 1) a solution comprising the nucleic
acid molecules having a molecular weight that is lower than the
molecular weight of the genomic nucleic acid in the elution buffer
and 2) solid phase carriers to which is bound genomic nucleic acid
of the cell, are produced.
[0047] A suitable "elution buffer" for use in the methods of the
present invention is a buffer that selectively elutes a cell's
nucleic acid which has a molecular weight that is lower than the
molecular weight of the cell's genomic nucleic acid. In one
embodiment, a suitable elution buffer for use in the present
invention can be water or any aqueous solution in which the nucleic
acid precipitating agent (e.g., isopropanol and/or PEG)
concentration is below the concentration required for binding of a
cell's nucleic acid which has a molecular weight that is lower than
the molecular weight of the cell's genomic nucleic acid to the
solid phase carrier, 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 a cell's
nucleic acid which has a molecular weight that is lower than the
molecular weight of the cell's genomic nucleic acid 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 DNA has been eluted, the solid phase carrier, to which is
bound the cell's genomic nucleic acid, is separated from the
elution buffer.
[0048] Optionally, impurities (e.g., host cell components,
proteins, metabolites, chemicals or cellular debrts) 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 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 nucleic acid 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. The magnetic
microparticles with bound nucleic acid can also be washed with more
than one wash buffer solution. The 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 nucleic acid. 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 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); 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.
[0049] 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 having a molecular weight that is
lower than the molecular weight of genomic nucleic acid 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 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.
[0050] In another embodiment, the isolated nucleic acid of one or a
plurality of samples is subjected to further analysis (e.g.,
sequence analysis). 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,
in vitro siRNA, RNAi hairpins, preparation of DNA probes and
primers and detemplating protocols.
EXEMPLIFICATION
Example 1
[0051] One Embodiment of a Protocol for Separation of Nucleic Acid
Having a Lower Molecular Weight than Genomic Nucleic Acid in a
Cell
[0052] The following protocol is illustrated in FIG. 1.
[0053] Growth of Bacterial Cultures
[0054] 1. Pipette 200 .mu.L of 2.times.YT bacterial growth media
containing the appropriate antibiotic into each well of a 300 .mu.l
Costar 96 well round bottom plate (Cat.#3750).
[0055] 2. Innoculate each well with a single plasmid containing E.
coli bacterial colony (DH10B, DH5alpha: Invitrogen). Growth
cultures can be innoculated directly from agar lawns or from
glyceral stocks.
[0056] 3. Cover the plate with a porous seal and shake vigorously
(e.g., 300 rpm) at 37.degree. C. for 16 hours.
[0057] Purification Procedure
[0058] 1. Add 60 .mu.L of paramagnetic lysis buffer (0.4N NaOH, 2%
SDS, 0.0016% solids Agencourt COOH magnetic microparticles)
solution to each well of the source plate and shake or tip mix.
[0059] 2. Add 60 .mu.L of Wash A solution (100% isopropanol) to the
samples. Perfonn 15 tip mixes once the Wash A solution is added.
Tip mixing conditions may vary depending on the tup orifice.
[0060] 3. Place source plate onto a magnetic SPRIplate96-R(TM)
(cat.# Agencourt Biosciences) for 5 minutes to separate beads from
solution.
[0061] 4. Aspirate and discard the cleared solution from the
destination plate while it is situated on a SPRIplate96-R (TM)
(RING Shaped magnets).
[0062] 5. Dispense 200 .mu.L of Wash B solution (70% ethanol) into
each well of the Destination Plate as a wash solution.
[0063] 6. Remove ethanol wash solution and repeat step four
times.
[0064] 7. Dry the destination plate at 37.degree. C. for 30
minutes.
[0065] 8. Add 40 .mu.L of elution buffer (ddH20+1.75 ng/.mu.l RNAse
A) to each well of the plate and shake. Reagent grade water is the
recommended elution buffer, Vortex or shake the plate after adding
elution buffer or wait 1 0 minutes for the elution to occur.
[0066] Sequencing Procedure:
[0067] For 1/24th.times.BigDye reaction add:
[0068] 60 .mu.l of purified DNA
[0069] 1 .mu.l BigDye Cocktail
[0070] BigDye Cocktail
[0071] 5 parts BigDye Terminator
[0072] 1 part 200 .mu.m M13-21 primer
[0073] 4 parts 15.times.BigDye Buffer
[0074] Cycle Sequence
[0075] 95.degree. C. for 2 minutes
[0076] 50 cycles of (54.degree. C. for 15 seconds, 60.degree. C.
for 2.5 minutes, 95.degree. C. for 5 seconds)
[0077] 4.degree. C. until cleanup
[0078] Purify sequencing reactions using either standard EtOH
precipitation of Agencourt CleanSeq kit (cat. #000121 and
000222).
[0079] Elute Precipitation of CleanSeq product in 30 .mu.l of ddH2O
and place on an ABI 3700 or 3730 for sequencing detection.
[0080] Over 11,000 reads sequences using POP5 polymer on the ABI
3700 produced 90% pass rates (passing read must average phred 20
from bp 100-300), and 625 Phred20 bases.
Example 2
[0081] Isolation of Exogenous Nucleic Acid Having a Lower Molecular
Weight than Genomic Nucleic Acid in Bacterial Cells
[0082] Methods and Materials
[0083] Cloning and purification:
[0084] Chimpanzee genomic DNA was sheared, end repaired with T4
polymerase and Klenow (NEB), and cloned in pOT bacterial vector.
DH10B cells (Invitrogen) were electroporated and plated on 25 ug/ml
chloramphenicol agar and grown overnight. Colonies were picked with
a Gentix Qpix into 200 ul of 2.times.YT, 50 ug/ml Chloramphenicol
broth and grown for 16 hours. The clones were purified in the
growth plate on a Beckman FX robotic platform.
[0085] Purification Process
[0086] Comparing method described herein (OneStep Prep) to the
method described in U.S. Pat. No. 6,534,262 (McPrep).
[0087] 24 samples were purified using two different purification
methods; McPrep and the single step purification method described
herein. Controlled samples were loaded on an agarose gel to compare
recovery (FIG. 2).
[0088] 60ul of paramagnetic lysis buffer (0.4N NaOH, 2% SDS, 160
mg/liter Seradyn COOH paramagnetic beads) in addition to 60 ul of
100% isopropanol (or 100 ul 40% PEG) was added to the cell culture
and tip mixed 15 times. Samples were separated for 6 minutes on an
Agencourt Magnet Plate (1000 gauss). Supernatant was removed and
the separated beads were rinsed 5 times with 70% ethanol. After
elution with 1. 75ng/ul RNAse A/ddH2O (Sigma), samples were run on
a 96 E-Gel(Invitrogen) to estimate relative DNA recovery (FIG. 2).
Pico Green Analysis (Molecular Probes) was also run on the samples
and average DNA recovery was 20 ng/ul. Average conductivity of the
samples were recorded to estimate any salt contamination from the
growth broth by pooling all 40 ul of eluent from 10 wells.
Conductivity was less than 20 us/cm.
[0089] Sequencing Reaction Setup:
[0090] Samples where eluted in 40 ul of 1.75ng/ul RNAse/ddH2O and
robotically agitated for 20cycles of 0.5 cm at 2 Hz. 3 ul of DNA
was aspirated and placed into 0.5 ul (1/3.times.BigDye) reagent
plus 3 pmole of -21 primer. Reactions were overlayed with mineral
oil and cycle sequenced in a 384 well plate. Samples were cycle
sequenced according to the manufacturers recommendations.
Sequencing reactions were purified with Agencourt's CleanSeq kit
and eluted in 15 ul of ddH2O and heat sealed prior to loading on
the ABI 3700.
[0091] Detection:
[0092] DNA sequencing Data generated using ABI3700 sequencing
platform and 1/48th.times.BigDye V3.1 Sequencing Chemistry and POP5
polymer. Electrophoresis was performed at 5800V with a 30 second
injection.
[0093] Results:
[0094] 12672 sequencing reads were generated from 6336 purified
samples (Table 1). The high Pass Rates, high Signal Intensity and
high Average Phred 20 base pairs (Ewing et al., Genome Research,
8:175-185 (1998)) obtained are exceptional for 48 fold diluted
BigDye sequencing chemistry. A histogram of the read performance is
plotted in FIG. 3.
[0095] FIG. 2 shows a 96 well Agarose gel with 13 columns by 8
rows. The 13.sup.th column is 200 ng pGEM 3.2 kb vector (Promega).
Positive electrode is at the bottom of the picture. Prep samples
are eluted in 40 ul of various elution buffers and 10 ul loaded on
the gel. One can see a 3-5 kb plasmid on the gels with no sign of
E. coli genomic DNA remaining in the Agarose wells. RNA can be seen
in wells that were not eluted in 1.75 ng/ul of RNAse (row F).
[0096] One can see more plasmid DNA is recovered using the single
step method versus using the McPrep method (Row A vs Row B &
Row C vs Row D). This is expected as the McPrep method requires
E.coli genomic DNA to be bound to one set of beads and the plasmid
enriched supernatant to be removed to another plate for binding to
a second mixture. This supernatant removal step makes it difficult
to aspirate 100% of the plasmid supernatant without disturbing the
genomic DNA magnetic beads, so a loss of DNA is expected.
1 Row A: 10 ul/40 ul McPrep Row B: 10 ul/40 ul OneStep Prep Row C:
10 ul/40 ul McPrep Row D: 10 ul/40 ul OneStep Prep Row E & F:
10 u//40 ul OneStep with and without RNAse Row C: 10 ul/40 ul
McPrep without RNAse
[0097] 3 ul eluted plasmid DNA was used for BigDye sequencing.
Eletropherogram read quality was determined by running Phred (Ewing
et al, Genome Research, 8:175-185 (1998)). Phred 30 bases have an
error rate of 1 in 1000, Phred 40 bases have an error rate 1 in
10,000. Counting the number of phred 20 bases that each read
produces is a standard sequencing quality metric (Table).
[0098] Pass Rate is defined as the number of reads meeting the PASS
criteria/Total Reads
[0099] P30=Average Number of Phred 30 per 384 well plate
[0100] P20=Average Number of Phred 20 per 384 well plate
[0101] CP20=Average Number of Contiguous Phred 20s per 384 well
plate
[0102] P15=Average Number of Phred 15 per 384 well plate
[0103] Qual=Average Phred scores throughout the whole read:
Averaged over 384 well plate
[0104] PASS criteria--A read must average Phred20 quality in a 200
bp window from bp 100 to bp 300.
[0105] SigA=Average Relative Fluorescent Units in the A channel for
the 96 reads.
[0106] SigG=Average Relative Fluorescent Units in the G channel for
the 96 reads
[0107] SigC=Average Relative Fluorescent Units in the C channel for
the 96 reads
[0108] SigT=Average Relative Fluorescent Units in the T channel for
the 96 reads
2 Seq Pass Sig Sig Sig Sig Barcode Pass Total % P30 P20 CP20 P15
Qual Length A G C T 000048032741 (KF) 351 384 91.41 576 669 540 706
47 763 188 134 177 187 000028713241 (KN) 360 384 93.75 528 626 482
670 46 743 149 118 161 158 000028713341 (KP) 345 384 89.84 534 625
489 663 46 736 51 33 46 50 000028713641 (HK) 345 384 89.84 538 620
509 660 44 744 93 64 85 103 000028714941 (GQ) 353 384 91.93 552 639
515 677 43 741 202 151 191 208 000028715041 (KF) 336 384 87.50 555
654 520 694 46 754 197 125 168 186 000048032841 (KF) 350 384 91.15
568 659 527 696 46 754 165 114 146 162 000028712941 (KN) 357 384
92.97 588 681 560 717 47 778 188 156 173 197 000028713041 (KH) 369
384 96.09 585 670 545 705 47 768 94 56 63 82 000028713141 (KJ) 341
384 88.80 538 627 521 663 47 726 113 79 110 148 000028713441 (KP)
341 384 88.80 569 665 534 703 46 760 115 75 98 114 000028713541
(KN) 339 384 88.28 528 632 487 677 46 755 268 217 295 292
000028713741 (GZ) 347 384 90.36 549 639 515 677 44 741 165 117 143
146 000028713941 (HK) 349 384 90.89 548 638 522 676 45 740 177 121
148 177 000028714141 (KN) 352 384 91.67 498 598 447 643 45 727 108
83 113 114 000028714241 (KK) 347 384 90.36 548 646 516 685 46 747
120 83 99 127 000028714441 (GY) 350 384 91.15 529 621 503 660 46
727 138 98 124 135 000028714541 (GQ) 343 384 89.32 525 620 483 663
43 738 194 118 182 184 000028714641 (KF) 311 384 80.99 540 638 481
678 45 747 146 89 119 141 000028714741 (XB) 351 384 91.41 488 612
371 661 38 726 170 120 128 178 000028714841 (KF) 333 384 86.72 569
666 527 706 47 759 205 122 161 191 000028715141 (HH) 363 384 94.53
536 618 507 652 46 712 191 129 157 201 000028715541 (GQ) 358 384
93.23 532 619 505 660 43 731 110 86 102 110 000028715641 (KR) 362
384 94.27 530 612 495 645 46 710 51 45 65 65 000028712841 (HG) 361
384 94.01 537 634 504 675 44 741 360 220 335 339 000028714041 (KF)
349 384 90.89 542 637 499 677 45 746 77 49 61 75 000028715241 (GW)
359 384 93.49 497 580 462 618 43 699 132 67 102 117 000028715441
(HH) 345 384 89.84 487 570 450 610 43 696 104 70 81 109
000028715741 (KS) 354 384 92.19 464 546 393 585 45 675 39 29 39 38
000028713841 (HA) 358 384 93.23 534 623 507 660 46 721 126 96 144
167 000028712741 (GR) 326 384 84.90 434 520 401 561 41 674 76 53 64
73 000028714341 (GR) 328 384 85.42 481 570 446 613 42 704 99 70 84
96 000028715341 (GR) 334 384 86.98 478 563 455 603 42 698 114 69 84
99 11467 12672 90.49 531 623 492 662 45 733 143 99 129 145
[0109] FIG. 3 is a Histogram of the Phred 20 (red) ad Phred3O
(black) bases generated by the reads. Y Axis is number of Reads, X
axis is phred20 bins in 50 bp increments.
Example 3
[0110] Isolation of Nucleic Acid Having a Lower Molecular Weight
than Genomic Nucleic Acid in Horse Whole Blood Cells
Materials and Methods
[0111] Source
[0112] Horse whole blood was obtained in 1:1 ratio with Alsevers
anti-coagulant (2.05% dextrose, 0.5% sodium citrate, 0.055% citric
acid, 0.42% sodium chloride).
[0113] Purification Process
[0114] 60 ul paramagnetic lysis buffer (0.4NaOH, 2% SDS, 160
mg/liter Seradyn COOH paramagnetic beads) in addition to 80 ul of
100% isopropanol is added to the cell culture and tip mixed 15
times. Samples were separated for 15 minutes on an Agencourt Magnet
Plate (1000 gauss). Supernatant was removed and the separated beads
were rinsed 5 times with 70% ethanol. After elution with ddH.sub.2O
(Sigma), samples were run on a 96 E-Gel (Invitrogen) to estimate
relative DNA recovery (FIG. 4). Pico Green Analysis (Molecular
Probes) was also run on the samples and average DNA recovery is 0.5
ng/ul (FIG. 5). DNA quality was verified in its applicability to
the Polymerase Chain Reaction (PCR), a common downstream
application (FIG. 6).
[0115] FIG. 4 shows gDNA duplicates prepped from 50 ul horse blood.
FIG. 5 shows the PicoGreen Analysis of 8 samples prepped gDNA. FIG.
6 shows a gradient PCR of prepped gDNA above (using Y3B19 markers
with an expected amplicon size of 225 bp).
[0116] 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.
* * * * *