U.S. patent application number 10/844602 was filed with the patent office on 2005-01-13 for compositions and methods for the release of nucleic acid molecules from solid matrices.
This patent application is currently assigned to Invitrogen Corporation. Invention is credited to Connolly, Michael A., Gebeyehu, Gulilat, Goldsborough, Mindy D., Xia, Jiu-Lin.
Application Number | 20050009063 10/844602 |
Document ID | / |
Family ID | 22964839 |
Filed Date | 2005-01-13 |
United States Patent
Application |
20050009063 |
Kind Code |
A1 |
Xia, Jiu-Lin ; et
al. |
January 13, 2005 |
Compositions and methods for the release of nucleic acid molecules
from solid matrices
Abstract
The present invention relates to compositions and methods for
releasing nucleic acid molecules from solid matrices. The invention
further relates to compositions and methods for purifying and
isolating nucleic acid molecules from biological materials such as
animal tissues and plant matter. The methods of the invention can
be readily adapted for rapid processing of multiple samples. Thus,
the invention further provides automated methods for the
purification of nucleic acid molecules from numerous samples. The
invention also relates to kits for removing nucleic acid molecules
from solid matrices.
Inventors: |
Xia, Jiu-Lin; (Germantown,
MD) ; Goldsborough, Mindy D.; (Gaithersburg, MD)
; Connolly, Michael A.; (Gaithersburg, MD) ;
Gebeyehu, Gulilat; (Potomac, MD) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Invitrogen Corporation
Carlsbad
CA
|
Family ID: |
22964839 |
Appl. No.: |
10/844602 |
Filed: |
May 13, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10844602 |
May 13, 2004 |
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10012443 |
Dec 12, 2001 |
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6803200 |
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60254583 |
Dec 12, 2000 |
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Current U.S.
Class: |
435/6.12 ;
536/25.4 |
Current CPC
Class: |
C12Q 2527/125 20130101;
C12Q 1/6806 20130101; C12N 15/1017 20130101; C12N 15/1006 20130101;
C12Q 1/6806 20130101 |
Class at
Publication: |
435/006 ;
536/025.4 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
1-58. (canceled).
59. A nucleic acid isolated by the method comprising: (a)
contacting the nucleic acid molecule with a solid matrix under
conditions which favor adherence, attachment, association, and/or
binding of the nucleic acid molecule to the solid matrix; and (b)
contacting the solid matrix containing the bound nucleic acid
molecule with a releasing reagent comprising one or more alkanol
amines, under conditions which favor release of the nucleic acid
molecule from the solid matrix.
60. (canceled).
61. A recombinant host cell comprising a nucleic acid produced by a
method comprising: (a) contacting the nucleic acid molecule with a
solid matrix under conditions which favor adherence, attachment,
association, and/or binding of the nucleic acid molecule to the
solid matrix; (b) contacting the solid matrix containing the bound
nucleic acid molecule with a releasing reagent comprising one or
more alkanol amines, under conditions which favor release of the
nucleic acid molecule from the solid matrix; (c) amplifying the
released nucleic acid molecules by PCR; and (d) transfecting a host
cell with the amplified nucleic acid molecule.
62. The host cell of claim 61, wherein the nucleic acid molecule of
(d) is contained by a vector.
63. A method of making a recombinant host cell comprising
introducing a nucleic acid into a host cell; wherein the nucleic
acid is produced by a method comprising: (a) contacting the nucleic
acid molecule with a solid matrix under conditions which favor
adherence, attachment, association, and/or binding of the nucleic
acid molecule to the solid matrix; (b) contacting the solid matrix
containing the bound nucleic acid molecule with a releasing reagent
comprising one or more alkanol amines, under conditions which favor
release of the nucleic acid molecule from the solid matrix; and (c)
amplifying the released the nucleic acid molecules.
64-78. (canceled).
79. A composition comprising nucleic acid molecules, a solid matrix
and a releasing reagent, wherein the releasing reagent comprises
one or more alkanol amines.
80. The composition of claim 79, wherein the solid matrix comprises
a weak base, a chelating agent, and an anionic surfactant or
anionic detergent.
81. The composition of claim 79, wherein the solid matrix further
comprises uric acid or a urate salt.
82. The composition of claim 79, wherein the solid matrix is a
cellulose based matrix or a micromesh synthetic plastic matrix.
83. The composition of claim 82, wherein the solid matrix is a
filter paper.
84. The composition of claim 82, wherein the solid matrix is
FTA.RTM. paper.
85. The composition of claim 79, wherein the releasing reagent
comprises more than one ethanolamine.
86. The composition of claim 79, wherein the releasing reagent
comprises an ethanolamine.
87. The composition of claim 86, wherein the ethanolamine is
mono-ethanol amine.
88. The composition of claim 86, wherein the ethanolamine is
di-ethanolamine.
89. The composition of claim 86, wherein the ethanolamine is
tri-ethanolamine.
90. The composition of claim 86, wherein the solution is an aqueous
solution.
91. The composition of claim 79, wherein the releasing reagent has
a pH from about 8.3 to about 13.
92. The composition of claim 91, wherein the releasing reagent has
a pH from about 10 to about 12.
93. The composition of claim 92, wherein the releasing reagent has
a pH of about 11.
94. The composition of claim 79, wherein the concentration of
alkanol amines is from about 0.01% to about 5% (vol./vol.).
95. The composition of claim 94, wherein the concentration of
alkanol amines is from about 0.01% to about 3% (vol./vol.).
96. The composition of claim 95, wherein the concentration of
alkanol amines is from about 0.01% to about 1% (vol./vol.).
97. The composition of claim 96, wherein the concentration of
alkanol amines is from about 0.1% to about 1% (vol./vol.).
98. The composition of claim 79, wherein the nucleic acid molecules
comprise DNA.
99. The composition of claim 79, wherein the nucleic acid molecules
comprise a vector.
100. The composition of claim 99, wherein the vector comprises a
plasmid or an artificial chromosome.
101. The composition of claim 79, wherein the nucleic acid
molecules are from a cell or a virus.
102. A kit for removing nucleic acid molecules from a solid matrix,
the kit comprising (1) a releasing reagent comprising one or more
alkanol amines and (2) one or more components selected from the
group consisting of: (a) a solid matrix; (b) an apparatus for
applying samples to a solid matrix; (c) an apparatus cutting a
solid matrix into sections which contain samples; and (d) a washing
solution.
103. The kit of claim 102, wherein the solid matrix comprises a
weak base, a chelating agent, and an anionic surfactant or anionic
detergent.
104. The kit of claim 102, wherein the solid matrix further
comprises uric acid or a urate salt.
105. The kit of claim 102, wherein the solid matrix is a cellulose
based matrix or a micromesh synthetic plastic matrix.
106. The kit of claim 105, wherein the solid matrix is a filter
paper.
107. The kit of claim 105, wherein the solid matrix is FTA.RTM.
paper.
108. The kit of claim 102, wherein the releasing reagent comprises
more than one ethanolamine.
109. The kit of claim 102, wherein the releasing reagent comprises
an ethanolamine.
110. The kit of claim 109, wherein the ethanolamine is
monoethanolamine.
111. The kit of claim 109, wherein the ethanolamine is
diethanolamine.
112. The kit of claim 109, wherein the ethanolamine is
triethanolamine.
113. The kit of claim 102, wherein the solution is an aqueous
solution.
114. The kit of claim 102, wherein the releasing reagent has a pH
from about 8.3 to about 13.
115. The kit of claim 114, wherein the releasing reagent has a pH
from about 10 to about 12.
116. The kit of claim 115, wherein the releasing reagent has a pH
of about 11.
117. The kit of claim 102, wherein the concentration of alkanol
amines is from about 0.01% to about 5% (vol./vol.).
118. The kit of claim 117, wherein the concentration of alkanol
amines is from about 0.01% to about 3% (vol./vol.).
119. The kit of claim 118, wherein the concentration of alkanol
amines is from about 0.01% to about 1% (vol./vol.).
120. The kit of claim 119, wherein the concentration of alkanol
amines is from about 0.1% to about 1% (vol./vol.).
121. The kit of claim 102, wherein the nucleic acid molecules
comprise a vector.
122. The kit of claim 121, wherein the vector comprises a plasmid
or an artificial chromosome.
123. The kit of claim 102, wherein the nucleic acid molecules are
from a cell or a virus.
124. The kit of claim 102, wherein the apparatus for applying
samples to a solid matrix comprises a micropipette.
125. The kit of claim 102, wherein the apparatus for applying
samples to a solid matrix is capable of applying multiple samples
to the solid matrix at one time.
126. The kit of claim 102, wherein the apparatus for applying
samples to a solid matrix results in the samples being crushed into
the surface of the solid matrix.
127. The kit of claim 102, wherein the apparatus cutting the solid
matrix into pieces which are circular, square, rectangular, or
irregular in shape.
128. The kit of claim 102, wherein the washing solution comprises
10 mM Tris HCl, 1 mM EDTA (pH 7.3).
129. The kit of claim 102, wherein the washing solution further
comprises a detergent.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to compositions and methods
for releasing nucleic acid molecules from solid matrices. The
invention further relates to compositions and methods for purifying
and isolating nucleic acid molecules from biological materials such
as animal tissues and plant matter.
BACKGROUND OF THE INVENTION
[0002] It is desirable in many instances to generate and analyze
nucleic acid molecules samples obtained from numerous individual
entities and/or organisms of populations. Often, such samples are
used to identify the genotypes of individuals which reside either
in the same or different geographic locations. Thus, the collection
and analysis of samples are often employed to determine the
genotypes of individual members of populations. Such analyses
generally result in the generation of data relating to both the
individuals from which the samples are obtained and the populations
as a whole.
[0003] The collection and analysis of samples which contain nucleic
acid molecules from populations of organisms are often performed to
obtain genotype data from viral, plant and animal populations. One
example of a situation where genotype analysis of large numbers of
individuals of members of populations is commonly performed is
where data; regarding the genotypes of plants in a geographic
location is sought. These data may be generated to determine the
spread rate of particular plant strains or to identify genetically
modified plants which either have been grown from seeds sold to
farmers or are the progeny of plants grown from such seeds.
[0004] A number of companies currently sell genetically modified
plants and seeds derived from these plants. In some cases, these
seeds are sold under the condition that the purchasers, generally
farmers, repurchase seeds from their suppliers instead of growing
plants from seeds which are obtained from plants grown from
purchased seeds. Further, a number of consumer groups, as well as
governmental organizations, have objected to the sale of
agricultural products prepared from genetically modified
plants.
[0005] In each instance described immediately above, genotype
analyses can be performed to identify genetically modified plants.
Such analyses often begin with the collection of large numbers of
plant samples obtained in rural settings. Thus, there is a need for
methods which allow for the collection and convenient storage of
large numbers of samples containing nucleic acid molecules derived
from plants which can then be used for genotype analyses.
[0006] In other situations, genotype analyses are performed on
samples derived from animals (e.g., humans) to generate data
related, again, to either individuals or populations of which these
individuals are members. Further, genotype analyses performed on
samples derived from either animals or plants may be used to obtain
data relating to entities associated with these organisms. Examples
of such associated entities include viruses such as Human
Immunodeficiency Viruses (HIVs). In particular, genotype analyses
of HIV populations can be performed using nucleic acid molecules
obtained from human blood samples. Due to the rapid rate with which
HIVs alter their genomes, genotype analyses have been employed to
track the spread and regional predominance of various viral
strains.
[0007] The use of filter paper (e.g., Whatman 3MM filter paper)
provides an inexpensive method for the collection, shipment, and
storage of samples which contain nucleic acid molecules (e.g., RNA,
plasmids, viral vectors, and chromosomal DNA). This is especially
the case when samples are collected in remote areas where there is
no access to refrigeration.
[0008] One example, of a filter paper based medium used for the
collection, shipment, and storage of blood samples is FTA.RTM.
paper, which is composed of cellulose material impregnated with (i)
a monovalent weak base; (ii) a chelating agent; (iii) an anionic
detergent; and, optionally, (iv) uric acid or a urate salt.
FTA.RTM. paper can be used to store human genomic DNA, for example,
in the form of dried spots of whole blood, the cells of which lyse
after making contact with the paper. Stored at room temperature,
genomic DNA on FTA.RTM. paper is reported to be stable for at least
7.5 years. (Burgoyne et al., CONVENTIONAL DNA COLLECTION AND
PROCESSING: DISPOSABLE TOOTHBRUSHES AND FTA.RTM.0 PAPER AS A
NON-THREATING BUCCAL-CELL COLLECTION KIT COMPATIBLE WITH
AUTOMATABLE DNA PROCESSING, 8.sup.th International Symposium on
Human Identification, Sep. 17-20, 1997.) Thus, the placement of
nucleic acid samples on filter paper (e.g., FTA.RTM. paper) offers
a compact archival system compared to glass vials or plastic tubes
located in precious freezer space.
[0009] DNA from blood spots has been used in newborn screening
programs to identify genetic mutations implicated in several
diseases and to provide a means for identifying military personnel.
(See, e.g., Seltzer et al., Biochem. Med Metab. Biol. 46:105-109
(1991); Jinks et al., Hum. Genet. 81:363-366 (1989); Skogerboe et
al., Clin. Chem. 37:454-458 (1991); McEwen et al., Am. J. Hum.
Genet. 55:196-200 (1994).)
[0010] The storage of blood samples on dried filter paper has the
additional advantage of pathogen inactivation. More specifically,
HIV, as well as a number of other infectious agents, are believed
to lose viability upon drying. Further, nucleic acid molecules
obtained from these dried blood spots, as well as other dried
samples containing nucleic acid molecules, can also be used to
isolate and reverse transcribe messenger-RNA (mRNA).
[0011] The spotting of bacterial nucleic acids on filter paper can
also be used as part of a sample storage and retrieval system.
Recently, Rogers and Burgoyne characterized samples of several
bacterial strains of Staphylococcus and Escherichia coli stored on
FTA.RTM. paper by PCR-ribotyping. (Rogers et al., Anal. Biochem.
247:223 (1997).)
[0012] Before analysis of nucleic acids captured by filter papers,
washing steps generally need to be performed to remove stabilizing
chemicals, if present, and cellular inhibitors of enzymatic
reactions. Since-DNA, for the most part, remains with the paper
through these washing steps, manipulations to purify such nucleic
acids are simplified and amenable to automation.
[0013] Several methods have been developed for releasing nucleic
acids from materials such as FTA.RTM. paper. For example, Burgoyne
demonstrated that purified plasmid DNA, stored on paper encased in
polystyrene, can be recovered using a uric acid solution.
(Burgoyne, U.S. Pat. No. 5,496,562, the entire disclosure of which
is incorporated herein by reference.) Another method for nucleic
acid release employs a buffer containing a chelating agent in an
aqueous solution. (See, e.g., PCT Publications WO 99/39010, WO
99/38962, and WO 99/39009, each of which is incorporated herein by
reference.)
[0014] The invention provides methods for releasing DNA from solid
matrices which are relatively simple in comparison to methods
currently in use in the art. Further, the DNA released by methods
of the invention can be used directly in a number of processes
(e.g., genotyping analyses).
SUMMARY OF THE INVENTION
[0015] The present invention relates to compositions and methods
for the removal of nucleic acid molecules (e.g., DNA) from solid
matrices. In particular, the methods of the invention employ
releasing reagents to facilitate the release of nucleic acid
molecules. The invention further provides compositions relating to
these methods.
[0016] The present invention also relates to methods for purifying
and/or isolating nucleic acid molecules.
[0017] In one general aspect, the invention provides methods for
removing nucleic acid molecules from solid matrices comprising
contacting the solid matrices with releasing reagents which
comprise one or more alkanol amines.
[0018] In another general aspect, the invention provides methods of
purifying and/or isolating nucleic acid molecules comprising:
[0019] (a) contacting the nucleic acid molecules with solid
matrices under conditions which favor adherence, attachment,
association, and/or binding (covalently or non-covalently) of the
nucleic acid molecules to the solid matrices: and
[0020] (b) contacting the solid matrices containing the bound
nucleic acid molecules with releasing reagents comprising one or
more alkanol amines, under conditions which favor release of the
nucleic acid molecules from the solid matrices. In a related
aspect, the methods of the invention further comprise collecting
the releasing reagents containing nucleic acid molecules which have
been released from the solid matrices.
[0021] In specific embodiments, solid matrices used in methods of
the invention comprise compounds that prevent or inhibit
degradation of nucleic acid molecules, such as one or more weak
bases, one or more chelating agents, one or more anionic
surfactants, one or more anionic detergents, uric acid, and/or one
or more urate salts.
[0022] In other specific embodiments, solid matrices used in the
methods of the invention are cellulose based matrices or micromesh
synthetic plastic matrices. In specific embodiments of the
invention, the solid matrix is either a filter paper (e.g., Whatman
3MM paper) or an FTA.RTM. paper.
[0023] In yet other specific embodiments, the alkanol amine present
in releasing reagents used in methods of the invention comprises an
ethanolamine. In particular, the ethanolamine may be
mono-ethanolamine, di-ethanolamine, or tri-ethanolamine.
[0024] In related embodiments, releasing reagents used in methods
of the invention comprise more than one ethanolamine (e.g., two or
three ethanolamines).
[0025] In certain embodiments, releasing reagents used in methods
of the invention are aqueous solutions.
[0026] In specific embodiments, releasing reagents used in the
methods of the invention have a pH which falls within the range of
from about 8.3 to about 13 or from about 10 to about 12. In
specific embodiments of the invention, the releasing reagents have
a pH of about 11.
[0027] In other specific embodiments of the invention, the one or
more alkanol amines are present in the releasing reagents at
concentrations of from about 0.01% to about 5% (vol./vol.), from
about 0.01% to about 3% (vol./vol.), from about 0.01% to about 1%
(vol./vol.), or from about 0.1% to about 1% (vol./vol.).
[0028] In additional embodiments, solid matrices which contain
nucleic acid molecules are incubated with releasing reagents for a
time period ranging from about 1 to about 180 minutes, from about 1
to about 120 minutes, from about 10 to about 60 minutes, or from
about 10 to about 30 minutes.
[0029] In further embodiments, solid matrices which contain nucleic
acid molecules are incubated with releasing reagents at about
65.degree. C. to about 100.degree. C. or about 90.degree. C. to
about 100.degree. C.
[0030] In certain embodiments, the methods of the invention
comprise separating released nucleic acid molecules from the
releasing reagents.
[0031] In additional embodiments, nucleic acid molecules released
from solid matrices using methods of the invention comprise vectors
(e.g., plasmids, artificial chromosomes, etc.). Similarly, nucleic
acid molecules released from solid matrices using methods of the
invention may comprise nucleic acid molecules of cells or viruses
(e.g., cellular or viral genomic DNA, mitochondrial DNA,
chloroplast DNA, etc.).
[0032] In another aspect, the invention includes nucleic acid
molecules which are purified and/or isolated by methods of the
invention. In specific embodiments, the nucleic acid molecules
purified and/or isolated by methods of the invention may used in
molecular biological processes (e.g., may be amplified by PCR). In
a related aspect, the invention is further directed to methods of
making recombinant host cells comprising introducing nucleic acid
molecules produced by methods of the invention.
[0033] In an additional aspect, the invention provides methods for
separating RNA from DNA comprising:
[0034] (a) contacting solid matrices with samples which contain RNA
and DNA;
[0035] (b) contacting solid matrices of (a) with washing solutions
under conditions sufficient to remove the RNA while the DNA is
retained (for example, by washing the solid matrices for periods of
time ranging from 1 second to 90 minutes); and
[0036] (c) contacting the washed solid matrices with releasing
reagents comprising one or more alkanol amines, under conditions
which favor release of the DNA from the solid matrices.
[0037] In yet another aspect, the invention provides methods for
separating closed, circular nucleic acid molecules from linear
nucleic acid molecules comprising:
[0038] (a) contacting solid matrices with samples which contain
closed, circular nucleic acid molecules and linear nucleic acid
molecules;
[0039] (b) contacting solid matrices of (a) with washing solutions
under conditions sufficient to remove the closed, circular nucleic
acid molecules while the linear nucleic acid molecules are retained
(for example, by washing the solid matrices for periods of time
ranging from 1 second to 90 minutes): and
[0040] (c) contacting the washed solid matrices with releasing
reagents comprising one or more alkanol amines, under conditions
which favor release of the linear nucleic acid molecules from the
solid matrices.
[0041] In another aspect, the invention further provides methods
for separating nucleic acid molecules on the basis of size
comprising:
[0042] (a) contacting solid matrices with samples which contain
nucleic acid molecules of different sizes;
[0043] (b) contacting solid matrices of (a) with washing solutions
under conditions sufficient to remove smaller nucleic acid
molecules while larger nucleic acid molecules are retained (for
example, by washing the solid matrices for periods of time ranging
from 1 second to 90 minutes); and
[0044] (c) contacting the washed solid matrices with releasing
reagents comprising one or more alkanol amines, under conditions
which favor release of the larger nucleic acid molecules from the
solid matrices.
[0045] In specific embodiments, washing solutions used in methods
of the invention comprise 10 mM Tris-HCI, 1 mM EDTA (pH 7.3),
water, or FTA.RTM. Purification Reagent (Invitrogen Corp., Life
Technologies Division, Cat. No. 10876-019). In related specific
embodiments, these washing solutions further comprise one or more
detergents.
[0046] In further specific embodiments, solid matrices are washed
for a time period selected from the group consisting of about 1
second, about 3 seconds, about 5 seconds, about 20 seconds, about
30 seconds, about 45 seconds, about 1 minute, about 5 minutes,
about 10 minutes, and about 30 minutes.
[0047] In additional specific embodiments, when nucleic acid
molecules are separated from each other on the basis of size,
nucleic acid molecules having an average size of from about 1
kilobase to about 50 kilobases are separated from nucleic acid
molecules having an average size of from about 100 kilobases to
about 1,000 kilobases; nucleic acid molecules having an average
size of from about 50 kilobase to about 100 kilobases are separated
from nucleic acid molecules having an average size of from about
250 kilobases to about 500kilobases; or nucleic acid molecules
having an average size of from about 50kilobase to about 100
kilobases are separated from nucleic acid molecules having an
average size of from about 1,000 kilobases to about 4,000
kilobases.
[0048] The invention further provides compositions comprising
nucleic acid molecules, solid matrices, and releasing reagents,
wherein the releasing reagents comprises one or more alkanol
amines.
[0049] The present invention also relates to kits for carrying out
methods of the invention, as well as for preparing compositions of
the invention. Thus, in one general aspect, the invention provides
kits for removing nucleic acid molecules from solid matrices, the
kits comprising (1) one or more releasing reagents of the invention
and (2) one or more components selected from the group consisting
of:
[0050] (a) at least one solid matrix;
[0051] (b) at least one apparatus for applying samples to solid
matrices;
[0052] (c) at least one apparatus for cutting solid matrices into
sections which contain samples; and
[0053] (d) at least one washing solution.
[0054] In specific embodiments, the apparatus for applying samples
to solid matrices comprises a pipette (e.g., Pipeteman.TM. Models
P-2, P-10, P-20, P-100, P-200, etc., Rainin Instrument Company,
Inc. Rainin Road, Box 4026, Woburn, Ma. 01888). In a related
embodiment, the apparatus for applying samples to the solid matrix
(e.g., a hammer, device which employs a piston, etc.) results in
the samples being crushed into the surface of the solid matrix. An
apparatus of this type will be especially useful when the sample
comprises material obtained from a plant. In another related
specific embodiments, the sample application apparatus is capable
of applying multiple samples to solid matrices at one time.
[0055] In additional embodiments, the apparatus for cutting solid
matrices into sections produces pieces of the matrices which are of
varying shapes (e.g., circular, square, rectangular, irregular,
etc.).
[0056] In further additional specific embodiments, washing
solutions of kits of the invention comprise 10 mM Tris-HCI, 1 mM
EDTA (pH 7.3), water, or FTA.RTM. Purification Reagent (Invitrogen
Corp., Life Technologies Division, Cat. No. 10876-019). In related
specific embodiments, washing solutions of kits of the invention
further comprise one or more detergents.
[0057] Other embodiments of the invention will be apparent to one
of ordinary skill in light of what is known in the art, the
following drawings and description of the invention, and the
claims.
BRIEF DESCRIPTION OF THE FIGURES
[0058] FIG. 1 depicts the results of a DNA quantitation assay
performed using an ACES.TM. 2.0.sup.+ Human DNA Quantitation System
(Invitrogen Corp., Life Technologies Division, Cat. No. 10294-015).
The nylon membrane shown in FIG. 1 was prepared according to the
manufacturers instructions using probe supplied with the System.
The results demonstrate the ability of different reagents to
release DNA from 2 mm blood punches of FTA.RTM. paper upon heating
for 10-20 minutes at 100.degree. C. All reagents tested released
50-100 ng of DNA per 2 mm punch after heating for 20 minutes.
Heating time had very little effect on the
3-(cyclohexylamino)-1-propane sulfonic acid (CAPS) buffer with
approximately the same amount of DNA being released at all heating
times.
[0059] FIG. 2 depicts ACES quantitation assay results, performed as
described for FIG. 1, showing concentration and pH effects on the
ability of ethanolamine to release DNA from FTA.RTM. paper. DNA
concentrations on the left hand side of the figure are 0.2, 0.4. 1,
2, 4, 10, 20 and 40 ng. Punches were incubated in 50 .mu.l of
ethanolamine solution at 100.degree. C. for 10 minutes. Ten .mu.l
of the released DNA was used for quantitation. Between 0.025% and
0.2%, the concentration of the ethanolamine (all at pH 11) does not
have much effect on the release of DNA from the FTA.RTM. paper.
However, a change in the pH does effect the efficiency of DNA
release. At pH 8.3, 0.5 ng of DNA is released per punch. At pH 9.7
and pH 13, 2 ng of DNA is released per punch. At pH 11, 10 ng of
DNA is released per punch. Therefore, as discussed below in Example
2, the maximum yield, in terms of amount of released DNA, was
obtained at a pH of 11.
[0060] FIG. 3 depicts ACES quantitation assay results, performed as
described for FIG. 1, showing the effect of ethanolamine
concentration on the release of DNA from FTA.RTM. paper. 10 .mu.l
of released DNA was used in this assay. The pH of all ethanolamine
solutions was 11. As the concentration of ethanolamine is increased
from 0.00025% to 0.025% more DNA is released from the punch. The
most DNA, approximately 50 ng, is released at a concentration of
1%. At 0.2% approximately 10 ng of DNA is released. For testing
purposes, ethanolamine concentrations of 0.2% and 1% were used.
[0061] FIG. 4 depicts an agarose gel showing PCR performed on
nucleic acid molecules released from FTA.RTM. paper using different
reagents. Punches were heated for 10 minutes, 15 minutes, or 20
minutes in 0.2% ethanolamine, pH 11. PCR was performed as follows.
Each 15 .mu.l PCR reaction was performed using 1 .mu.l of released
DNA. The PCR reaction buffer contained 20 mM Tris-HCl (pH 8.4), 50
mM KCl, 1.5 mM MgCl.sub.2, 200 .mu.M dNTPs, 5 .mu.l primer, 0.04
units of Platinum.RTM. Taq DNA polymerase (Invitrogen Corp., Life
Technologies Division, 9800 Medical Center Drive, Rockville, Md.
20850 USA, Catalog No. 10966-018), and a primer set which amplifies
D1S197 microsatellite marker DNA. Thermocycling was performed using
the following temperature shifts: 80.degree. C. for 10 minutes, and
94.degree. C. for 1 minute; ten cycles of 94.degree. C. for 30
seconds, 55.degree. C. for 30 seconds, and 72.degree. C. for 1
minute; twenty cycles of 89.degree. C. for 30seconds, 55.degree. C.
for 30 seconds, and 72.degree. C. for 1 minute; and final extension
of 72.degree. C. for 10 minutes. PCR reaction products were stored
at 4.degree. C. until analyzed. Five .mu.l of the PCR reaction was
loaded onto an agarose gel. In addition, 5 .mu.l of the PCR
reaction diluted 1:10 was also loaded onto the gel in order to
determine if there was a difference in the amount of product
generated for each reagent tested. From this agarose gel, it was
determined that the 0.2% ethanolamine performed as well as Gentra
Elution Buffer 2. Both of these reagents had visible PCR products
when diluted 1:10 (right of short dividing line on the gel
photograph).
[0062] FIG. 5 depicts an agarose gel which shows the effect of
releasing reagent ethanolamine concentration (%) and pH on the
formation of PCR products. PCR was performed as described in FIG. 4
using 1 .mu.l of released DNA in a 15 .mu.l PCR reaction. 5 .mu.l
of the PCR reaction was loaded onto an agarose gel for
electrophoresis. These results indicate that there is a slight
increase in the amount of amplification seen when the concentration
of ethanolamine is increased. The effect of pH is also clearly seen
here. PCR products are generated at a pH range of 8.3 to 11. At pH
6.2 and 13 no PCR product is generated. These data support the
previous data generated from the ACES quantitation kit regarding
ethanolamine concentration and pH (see FIG. 3).
[0063] FIG. 6 depicts an agarose gel which shows the effect of
releasing reagent pH on the formation of PCR products. PCR was
performed as described in FIG. 4 using 1 .mu.l of released DNA in a
15 .mu.l PCR reaction. 5 .mu.l of the PCR reaction was loaded onto
an agarose gel for electrophoresis. No PCR product was generated at
pH 6.2 and 13. The amount of PCR product produced increases with
the pH. These data support the data shown above in FIG. 2, which
indicate that greater quantities of DNA are released as the pH of
the releasing reagent is increased from 8.3 to 11. From this it can
be concluded that the most effective pH for releasing DNA from the
FTA.RTM. paper is pH 11.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0064] The following definitions are provided to clarify the
subject matter of the present invention.
[0065] Solid Matrix: As used herein, the phrase "solid matrix"
refers to any solid support to which nucleic acid molecules adhere,
bind (covalently or non-covalently), attach, and/or associate with
including but not limited to cellulose based materials (e.g.,
cellulose based filter papers) and micromesh synthetic plastic
matrices. One example of such a material is FTA.RTM. paper. (Fitzco
Inc., 5600 Pioneer Creek Drive, Maple Plain. Mn. 55359 USA;
Invitrogen Corp., Life Technologies Division, 9800 Medical Center
Drive, Rockville, Md. 20850 USA, Catalog No. 10786-036.) Other
examples include Schleicher and Schuell grade 903, 704E, 402, 404,
and 577 filter papers (Schleicher and Schuell, 10 Optical Avenue,
Keene, N.H. 03431 USA); Whatman BFC180. No. 1, No. 40, No. 42, No.
50, and 3MM filter papers (Whatman International, LTD, Maidstone,
Kent, UK); nitrocellulose; and cellulose acetate.
[0066] In many embodiments of the invention, the solid matrix used
will be a cellulose based filter paper or other type of papers
(e.g., laminar conglomerates obtained by pulping fibers, such as
plant fibers). Solid matrices of these types are especially useful
because they are relatively inexpensive and normally work well with
methods of the invention.
[0067] Solid matrixes suitable for use with the invention include
those which inactivate pathogenic agents (e.g., Herpes Simplex,
Cytomegalovirus, Hepatitis B, Hepatitis C, etc.). This feature is
especially advantageous when the sample being applied to the solid
matrix is obtained from a human (e.g., saliva, buccal swab, whole
blood, etc.).
[0068] Solid matrixes may also be impregnated with agents which
induce lysis of cells. Examples of such agents include anionic
detergents (e.g., sodium dodecyl sulfate, sodium deoxycholate),
cation detergents (e.g., cetyl trimethylammonium bromide (CTAB),
cetyl pyridinium chloride (CPC), myristyltrimethyl ammonium
chloride (MTAB), dioctadecyldimethyl ammonium chloride (DODMAC)),
non-ionic detergents (e.g., TWEEN 80, TRITON X-100), enzymes,
salts, chaotropic agents (e.g., guanidine hydrochloride), and the
like.
[0069] In specific embodiment, solid matrixes may comprise glass
(e.g., controlled pore glass beads) and plastics (e.g.,
polystyrene, polyvinylchloride, polypropylene, polyethylene,
polyvinylidenedifluoride, nylon, etc.). Solid matrixes suitable for
use with the invention may be in any form or configuration
including beads, filters, membranes, sheets, columns and the
like.
[0070] Alkanol Amines: As used herein, the phrase "alkanol amines"
refers to C.sub.2-C.sub.50 compounds which contain at least one
amino group and at least one alcohol group. Examples of such
compounds include N.N-dimethylethanolamine, N-methyldiethanolamine,
3-aminopropyldiethanola- mine, diisopropano lamine,
N-methylethanolamine, 2-(dibutylam ino)ethanol,
2-(diisopropylamino)ethanol, 2-(isopropylamino)ethanol,
2-(propylamino)ethanol, 2-(tert-butylamino)ethanol,
2-benzylaminoethanol, 2-butylaminoethanol, N-phenylethanolamine,
mono-ethanolamine, di-ethanolamine, and tri-ethanolamine, as well
as mixtures of various alkanol amines. Alkanol amines, such as
those listed above, are available from commercial suppliers such as
Sigma-Aldrich Corporation, 3050 Spruce Street, St. Louis, Mo. 63103
USA. In certain embodiments, alkanol amines will not include
3-(cyclohexylamino)-2-hydroxypropanesulfonic acid (CAPSO),
proteins, and/or molecules having a molecular weight over 100, 150,
200, 250, 300, 350, 400, 450, or 500.
[0071] Releasing Reagents: As used herein, the phrase "releasing
reagents" refers to reagents of the invention which induce the
release of nucleic acid molecules from solid matrices. Releasing
reagents of the invention comprise one or more alkanol amines.
Additional characteristics of releasing reagents are described
below.
[0072] Nucleic Acid Molecules: As used herein, the phrase "nucleic
acid molecules," refers to molecules composed chains of alternating
units of phosphoric acid and deoxyribose, linked to purine and
pyrimidine bases, such as DNA (e.g., RNA, cDNA, mitochondrial DNA,
chloroplast DNA, genomic.DNA, double minutes, artificial
chromosomes, extrachromosomal elements, synthetic DNA, etc.).
Representative examples of nucleic acid molecules include vectors
(e.g., plasmids, yeast artificial chromosomes, mammalian artificial
chromosomes, bacterial artificial chromosomes, and the like) and
genomic nucleic acid molecules of prokaryotic organisms, eukaryotic
organisms, and viruses (e.g., Epstein Barr virus, bovine
papillomavirus 1, duck Hepatitis B viruses, Mycoplasma virus P1,
etc.).
[0073] Purified: As used herein, the term "purified," when used in
reference to a biological molecule (e.g., a nucleic acid) means
that the molecule has been separated from some surrounding
molecules and/or materials. "Purified" is thus a relative term
which is based on a change in conditions in terms of molecules
and/or materials in close proximity to the molecules being
purified. Thus, genomic nucleic acid molecules, for example, which
adhere to, attach to, bind to (covalently or non-covalently),
and/or associate with solid matrices after cell lysis are
considered to be purified when at least some cellular debris,
proteins and/or carbohydrates are removed by washing. These same
genomic nucleic acid molecules are purified again, when they are
released from solid matrices using methods of the invention. The
term purified is not intended to mean that the all of matter
intended to be removed is removed from the molecules being
purified. Thus, some amount of contaminants may be present along
with the purified molecules.
[0074] For practical applications, the concentration of materials
such as water, salts, and buffer are not considered when
determining whether a biological molecule has been purified. Thus,
as an example, nucleic acid molecules which have been separated
from other biological molecules using column chromatography but
have been diluted with an aqueous buffer in the process are still
considered to have been purified by the chromatographic separation
process.
[0075] Isolated: As used herein, the term "isolated," when used in
reference to a biological molecule (e.g., a nucleic acid) means
that the molecule has been separated from substantially all of the
molecules and/or materials present which surround the molecule when
the molecule was associated with a biological system (e.g., inside
a cell). As when determining whether a biological molecule has been
purified, the concentration of materials such as water, salts, and
buffer are not considered when determining whether a biological
molecule has been "isolated."
[0076] Average Size: As used herein, the phrase "average size"
means that at least 85% of the molecules in the population are of a
size which is about +/-10% of a recited value. For example, if the
recited value is 100 kilobases and 90% of the members of a
population of nucleic acid molecules fall within the range of 90 to
110 kilobases, then the population has an average size of 100
kilobases. Of course, the same would be true if 98% of the members
of a population of nucleic acid molecules were to fall within the
range of 90 to 110 kilobases.
[0077] One or More: As used herein, the phrase "one or more" means
one or more than one. As one skilled in the art would recognize,
the meaning of more than one will vary with the particular context.
For example, when reference is made to the use of one or more
ethanolamine, one skilled in the art would recognize that this
means, due to the limited number of ethanolamines, one, two, or
three ethanolamines, or mixtures of these ethanolamines. However,
when one or more nucleic acid molecules are referred to, one
skilled in the art would recognize that, due to the number of
different nucleic acid molecules which can be present in a
population of such molecules, the phrase "one or more" means, one,
two, three, four, five, ten, twenty, thirty, fifty, one hundred,
one thousand, or one million. Thus, depending on the particular
situation, the phrase "one or more" means, for example, one, two,
three, four, five, ten, fifteen, twenty, thirty, fifty, one
hundred, two hundred, one thousand, two thousand, five thousand,
ten thousand, one hundred thousand, one million, five million, ten
million, fifty million, one hundred million, one billion, etc.
[0078] Vector. As used herein, the term "vector" refers to nucleic
acid molecules which are capable of replicating autonomously in a
host cell. Such vectors may also be characterized by having a small
number of endonuclease restriction sites at which these molecules
may be cut without loss of an essential biological function and
into which nucleic acid molecules may be spliced to bring about its
replication and cloning. Examples of vectors include plasmids,
autonomously replicating sequences (ARS), centromeres, cosmids,
fosmids, phagemids, bacterial artificial chromosomes (BACs), yeast
artificial chromosomes (YACs), mammalian artificial chromosomes
(MACs), and the like. Vectors can further provide primer sites for
PCR, transcriptional and/or translational initiation and/or
regulation sites, recombinational signals, repl icons, etc.
Further, vectors can further contain one or more selectable markers
(e.g., nucleic acid molecules which confer kanamycin resistance,
tetracycline resistance, amplicillin resistance, etc.) suitable for
use in the identification of cells transformed or transfected with
these vectors.
[0079] In specific embodiments of the invention, the term "vector"
does not include nucleic acid molecules which are less than about
50, about 75, about 100, about 125, about 150, about 175, about
200, or about 250 kilobases. In other specific embodiments of the
invention, the term "vector" does not include plasmids.
[0080] In accordance with the invention, any vector may be used. In
particular, vectors known in the art and those commercially
available (and variants or derivatives thereof) may be used in
accordance with the invention. Such vectors may be obtained from,
for example, Vector Laboratories Inc., Invitrogen Corp., Promega,
Novagen. NEB, Clontech, Boehringer Mannheim, Pharmacia, EpiCenter,
OriGenes Technologies Inc., Stratagene, Perkin Elmer, Pharningen.,
and Research Genetics. Such vectors may then for example be used
for cloning or subcloning nucleic acid molecules of interest.
General classes of vectors of particular interest include
prokaryotic and/or eukaryotic cloning vectors, expression vectors,
fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttle
vectors for use in different hosts, mutagenesis vectors,
transcription vectors, vectors for receiving large inserts and the
like.
[0081] Other vectors of interest include viral origin vectors (M13
vectors, bacteriophage .lambda. vectors, baculovirus vectors,
adenovirus vectors, and retrovirus vectors), high, low and
adjustable copy number vectors, vectors which have compatible
replicons for use in combination in a single host (pACYC 184 and
pBR322), and eukaryotic episomal replication vectors (pCDM8).
[0082] Sample. As used herein, the term "sample" refers to a
material which contains nucleic acid molecules and is applied to a
solid matrix. Examples of samples include bacterial cells,
bacterial cell homogentates, fungal cells, protist cells, viral
plaques obtained from plates, viral material (e.g., DNA or RNA)
isolated by cesium chloride centrifugation, human buccal swabs,
human blood, purified human blood cells, blood cell homogenates,
and plant materials such as leaves, roots, stems, and fruits. Thus,
the term "sample" encompasses any material which contains nucleic
acid molecules and is in a form which can be applied to a solid
matrix.
[0083] As described below, solid materials may be directly applied
to solid matrices or a liquid solution or suspension of these
materials may be prepared prior to application.
[0084] Storage. As used herein, the term "storage" refers to
maintaining the solid matrices, to which samples have been applied,
for periods of time. Solid matrices may be stored, for example, at
a constant humidity, at about 20.degree. C. to 30.degree. C. for
five years. Lower storage temperatures may range from about
0.degree. C. to 20.degree. C., -20.degree. C. to 0C., and
-80.degree. C. to -20.degree. C.
[0085] Other terms used in the fields of recombinant DNA technology
and molecular and cell biology as used herein will be generally
understood by one of ordinary skill in the applicable arts.
II Compositions and Methods of the Invention
[0086] As noted above, solid matrices which nucleic acid molecules
adhere to, attach to, bind to (covalently or non-covalently),
and/or associate with have been used in the art for various
applications, including storage. Further, nucleic acid molecules
bound to solid matrices can be used in biochemical reactions (e.g
PCR).
[0087] The present invention relates to novel compositions and
methods for the removal of nucleic acid molecules from solid
matrices. In particular, the methods of the invention relate to the
contacting of solid matrices to which nucleic acid molecules are
bound with solutions comprising one or more (e.g., one, two, three,
etc.) alkanol amines.
[0088] In one relatively specific aspect, the invention relates to
the following processes and reagents for releasing nucleic acid
molecules from solid matrices. A sample which contains nucleic acid
molecules in aqueous solution is spotted on a solid matrix (e.g.,
Whatman 3MM paper, FTA.RTM. paper, etc.). The solid matrix
containing the sample is then allowed to dry. The dried solid
matrix is then placed in a releasing reagent comprising one or more
alkanol amines (e.g. ethanolamine) and boiled for 30 minutes. The
resulting solution is then cooled to room temperature and the solid
matrix is removed. The solution is then diluted with 50 volumes of
a PCR buffer and nucleic acid molecules are amplified by PCR using
standard protocols.
[0089] Ethanolamine is one of a group of alkanol amines which are
often used as buffering agents. Due to strong hydrogen bonding and
hydrophobic interactions, ethanolamine is normally a viscous fluid
at room temperature. Further, ethanolamine has strong affinity for
--OH and --H+ groups. It also forms complexes with some metal ions
in solution. All of these combined properties make this compound a
superior reagent for releasing nucleic acids from solid
matrices.
[0090] Ethanolamine has considerable buffer capacity at pHs around
pH 10. At pH 10, most solid matrices are negatively charged due to
the presence of --COOH and --Si--OH groups. While not wishing to be
bound by theory, the negative charge of solid matrices at certain
basic pHs is believed to result in the release of nucleic acid
molecules from the charged surface.
[0091] Further, ethanolamine is believed to strongly interact with
nucleic acid molecules through hydrogen bonding. This interaction
is believe to facilitate the extraction of nucleic acid molecules
from solid matrices. In addition, ethanolamine may also share metal
ions with nucleic acid molecules since both of these compounds can
form soluble metal ion complexes. This is believed to result in the
formation of soluble complexes of ethanolamine and nucleic acid
molecules.
[0092] As discussed below, any nucleic acid molecules may be stored
and later recovered using the methods and compositions of the
invention. Further, nucleic acid molecules may be separated from
other materials by their ability to adhere to, attach to, bind to
(covalently or non-covalently), and/or associate with and then
released from solid matrices. In particular, the methods and
compositions of the invention relate to simple and efficient
processes in which nucleic acid molecules (e.g., chromosomal DNA,
vectors, viral nucleic acid molecules, etc.) are contacted with a
solid matrix (e.g., FTA.RTM. paper or derivatives, variants or
modifications thereof) and released using a releasing reagent. In
addition, as discussed in more detail below, released nucleic acid
molecules may also be separated from other cellular materials which
are also released by the releasing reagent. Thus, the invention
further provides methods for purifying and/or isolating nucleic
acid molecules.
[0093] Solid matrices suitable for use with the invention include
those which comprise an absorbent cellulose-based matrix (e.g.,
cellulose based paper), or a micromesh of synthetic plastic
material, such as those described in U.S. Pat. No. 5,496,562, which
is incorporated by reference herein in its entirety. Further, the
solid matrix may be a composition comprising a weak base, a
chelating agent, an anionic surfactant or anionic detergent, and,
optionally, uric acid or a urate salt. FTA.RTM. paper and
derivatives, variants and modifications thereof are included among
such solid matrices.
[0094] In general, when samples are intended for only short term
storage (e.g., time periods of less than one month or so), the
solid matrix will normally not contain agents which stabilize
nucleic acid molecules (e.g., agents such as chelating agents,
detergents, and uric acid or urate salts). Further, when the
samples are intended for only long term storage (e.g., time periods
of greater than one month or so), the solid matrix will often
contain agents which stabilize nucleic acid molecules.
[0095] Solid matrices suitable for use with the invention may be of
any number of shapes or forms. For example, the solid matrices may
be in the form of a flat sheet or packing in a tube. It will
normally be advantageous to use solid matrices in a flat sheet form
when large number of separate samples are used. Flat sheet and
tubular packing solid matrix forms may be used for nucleic acid
purification and isolation protocols employing pressurized,
centrifugal, or gravity based sample and reagent flows.
[0096] Compositions and methods of the invention may be used with
(1) purified nucleic acid molecules or (2) crude preparations which
contain nucleic acid molecules. Thus, the invention provides
methods for purifying and/or isolating nucleic acid molecules from
samples (e.g., human blood, plant cell homogenates, plant cell
homogenates, leaves, seeds, bacterial cells, viruses, viral
plaques, etc.).
[0097] Samples suitable for use with the invention may be obtained
from numerous sources. Sources from which samples suitable for use
with the present invention may be obtained include buccal swabs,
plant cell extracts, plant tissues, seeds, animal fluids, animal
tissues, organs, bacterial cultures, fungal cultures, protozoan
cultures, and viral plaques.
[0098] As noted above, one common sample applied to solid matrices
is human blood: Much of the DNA present in human blood is
mitochondrial and nuclear DNA of white blood cells. The application
of human blood to FTA.RTM. paper, for example, results in the lysis
of blood cells, adhesion of nucleic acid molecules to the paper,
and the inactivation of many pathogenic agents.
[0099] In some instances, the solid matrix will be one which
protects against degradation of nucleic acid molecules bound
thereto and inactivates pathogens. A considerable number of agents
which can be used to perform these functions are known in the art
and include detergents, chelating agents, antibiotics, and enzymes
(e.g., proteinases, lipases, and nucleases).
[0100] Agents used with the solid matrix will generally be selected
or used in such a concentration so that they do not substantially
decrease the quality of the nucleic acid molecules which are to be
later released from the matrix. For example, when one seeks to
obtain DNA from a solid matrix, the matrix could be treated with
agents that will selectively degrade RNA. Examples of such agents
include RNAses and strong bases. As is known in the art, RNA, but
not DNA, undergoes hydrolysis under alkaline conditions. Alkaline
conditions are also known to inhibit the growth of many
microorganisms. Thus, the pH of solid matrices can be adjusted to
both degrade RNA and inhibit the growth of microorganisms.
[0101] Prior to release of nucleic acid molecules using releasing
reagents of the invention, solid matrices to which nucleic acid
molecules have bound may be treated to remove solvents, detergents,
proteins, other nucleic acid molecules (e.g., RNA when DNA is
sought), salts, etc. One method currently used in the art for
removing proteins from, for example, FTA.RTM. papers is phenol
extraction. (See, e.g., Burgoyne, U.S. Pat. No. 5,496,562.)
Further, water soluble compounds (e.g., detergents, salts, etc.)
can be removed by treating matrices with aqueous solutions (e.g.,
deionized water). Similarly, volatile agents may be removed by
exposure of matrices to air or vacuum.
[0102] Treatment of the solid matrices after sample application but
before nucleic acid release will vary with the particular
application. For example, in instances where the sample contains
considerable quantities of contaminants such as proteins and lipids
it may be advantageous to treat solid matrices with agents that
either remove or degrade these materials. Further, as noted above,
proteins can be removed from solid matrices by phenol extraction,
optionally with a metal ion chelator such as EDTA to stabilize the
phenol. (See Perlman. U.S. Pat. No. 5,098,603.) Similarly, lipids
and other hydrophobic molecules can be removed by extraction with
organic solvents or detergent (e.g., anionic detergents, cationic
detergents, and non-ionic detergents) washes.
[0103] Solid matrices may also be treated to remove agents which
are present prior to sample addition. For example, FTA.RTM. paper
contains detergent which facilitates the lysing of cells contained
in samples. Under some circumstances, it may be advantageous to
remove the detergent prior to nucleic acid release. One method of
removing this detergent is by washing the FTA.RTM. paper with
water. Depending on the particular situation, other suitable
washing solutions, in addition to water, include 10 mM Tris, 1 mM
EDTA (pH 7.3 or 8.0) and FTA.RTM. Purification Reagent (Invitrogen
Corp., Life Technologies Division, Cat. No. 10876-019).
[0104] The methods of the invention can also be used to separate
nucleic acid molecules based on physical properties such as size
and/or type (e.g., RNA v. DNA, plasmid v. chromosomal DNA). For
example, large nucleic acid molecules associate more tightly with
solid matrices than small nucleic acid molecules. Further, as the
size of nucleic acid molecules increases, these molecules are
believed to associate more tightly with solid matrices. Thus,
smaller nucleic acid molecules (e.g., plasmids) are more easily
removed from solid matrices than larger nucleic acid molecules
(e.g., chromosomal DNA). Further, closed, circular nucleic acid
molecules, such as plasmids, often release from solid matrices
during washing steps. This is so because linear nucleic acid
molecules are believed to associate more tightly with solid
matrices than closed, circular nucleic acid molecules. In addition,
DNA is believed to associate with solid matrices more tightly than
RNA.
[0105] In view of the above, the invention further provides methods
for separating nucleic acid molecules from other nucleic acid
molecules based on any one of a series of physical properties. For
example, the invention provides, in one aspect, methods for
separating DNA from RNA comprising contacting a solid matrix with a
sample which contains both DNA and RNA, followed by contacting the
solid matrix with a washing solution. Nucleic acid molecules (e.g.,
DNA) which remain bound to the solid matrix may then be released by
contacting the washed solid matrix with a releasing reagent of the
invention.
[0106] As one skilled in the art would recognize, when plasmid DNA,
for example, is sought, it will often be advantageous to treat the
solid matrix containing the sample with a releasing reagent either
without intervening washing or after a very brief washing (e.g.,
washing for about 3 seconds). Further, when eukaryotic genomic DNA
(e.g. genomic DNA from plant or animal cells), for example, is
sought, it will often be advantageous to wash the solid matrix
containing the sample with one or more washing solutions or steps
prior to contacting the matrix with a releasing reagent.
[0107] In one aspect, the invention, provides methods for
separating linear nucleic acid molecules (e.g., sheared chromosomal
DNA) from closed, circular nucleic acid molecules (e.g., plasmids).
Thus, the invention further provides methods for separating linear
nucleic acid molecules from closed, circular nucleic acid molecules
comprising contacting a solid matrix with a sample which contains
both linear nucleic acid molecules and closed, circular nucleic
acid molecules, followed by contacting the solid matrix with a
washing solution. Nucleic acid molecules which remain bound to the
solid matrix may then be released by contacting the washed solid
matrix with a releasing reagent of the invention.
[0108] Further, because larger nucleic acid molecules associate
more tightly with solid matrices than smaller nucleic acid
molecules, the invention further provides methods for separating
nucleic acid molecules which differ in size. Thus, the invention
further provides methods for separating smaller nucleic acid
molecules from larger nucleic acid molecules comprising contacting
a solid matrix with a sample which contains both smaller nucleic
acid molecules and larger nucleic acid molecules, followed by
contacting the solid matrix with a washing solution. Nucleic acid
molecules which remain bound to the solid matrix may then be
released by contacting the washed solid matrix with a releasing
reagent of the invention.
[0109] Washing conditions, for example, may be adjusted to
facilitate the release of smaller nucleic acid molecules of
specific sizes while the larger nucleic acid molecules remain
adhered to, attached to, bound to (covalently or non-covalently),
and/or associated with matrices. One example of a washing condition
which can be adjusted is the length of time for which washing
occurs. For example, solid matrices may be washed in a washing
solution for about 1 second, about 5 seconds, about 10 seconds,
about 20 seconds, about 30 seconds, about 45 seconds, about 1
minute, about 2 minutes, about 5 minutes, about 10 minutes, about
15 minutes, about 20 minutes, about 30 minutes, about 45minutes,
about 60 minutes, about 75 minutes, or about 90 minutes. As one
skilled in the art would recognize, the effect that various washing
conditions have on the release of nucleic acid molecules of various
size can be readily assayed using, for example, gel
electrophoresis. Thus, one skilled in the art can readily adjust
washing conditions such that nucleic acid molecules of specific
sizes are removed from solid matrices by washing and nucleic acid
molecules of specific sizes remain associated with these matrices.
Further, as noted above, nucleic acid molecules which remain
associated with these matrices can later be removed from the
matrices using releasing reagents of the invention.
[0110] The "larger nucleic acid molecules" referred to above may be
of an average size of about 50 kilobases or larger (e.g., about 50,
about 100, about 150, about 200, about 250, about 300, about 350,
about 400, about 450, about 500, about 600, about 700, about 800,
about 900, about 1,000, about 1,500, about 2,000, about 2,500,
about 3,000, or about 4,000 kilobases). Further, the "smaller
nucleic acid molecules" referred to above may have an average size
of less than about 50 kilobases (e.g., about 1, about 5, about 10,
about 20, or about 40 kilobases).
[0111] Similarly, the "larger nucleic acid molecules" referred to
above may be of an average size of about 150 kilobases or larger
(e.g., about 150, about 200, about 250, about 300, about 350, about
400, about 450, about 500, about 600, about 700, about 800, about
900, about 1,000, about 1,500, about 2,000, about 2,500, about
3,000, or about 4,000 kilobases) and the "smaller nucleic acid
molecules" referred to above may have an average size of less than
about 150 kilobases (e.g., about 1, about 5, about 10, about 20,
about 40, about 80, about 100, about 120, or about 140
kilobases).
[0112] Additionally, the "larger nucleic acid molecules" referred
to above may be of an average size of about 250 kilobases or larger
(e.g., about 250, about 300, about 350, about 400, about 450, about
500, about 600, about 700, about 800, about 900, about 1,000, about
1,500, about 2,000, about 2,500, about 3,000, or about 4,000
kilobases) and the "smaller nucleic acid molecules" referred to
above may have an average size of less than about 250 kilobases
(e.g., about 1, about 5, about 10, about 20, about 40, about 80,
about 100, about 120, about 140, about 160, about 180, about 200,
about 220, or about 240 kilobases).
[0113] In addition, the "larger nucleic acid molecules" referred to
above may be of an average size of about 500 kilobases or larger
(e.g., about 500, about 600, about 700, about 800, about 900, about
1,000, about 1,500, about 2,000, about 2,500, about 3,000, or about
4,000 kilobases) and the "smaller nucleic acid molecules" referred
to above may have an average size of less than about 500 kilobases
(e.g., about 1, about 5, about 10, about 20, about 40, about 80,
about 100, about 120, about 140, about 160, about 180, about 200,
about 220, about 240, about 250, about 300, about 350, about 400,
or about 450 kilobases).
[0114] Furthermore, the "larger nucleic acid molecules" referred to
above may be of an average size of about 1,000 kilobases or larger
(e.g., about 1,000, about 1,500, about 2,000, about 2,500, about
3,000, or about 4,000 kilobases) and the "smaller nucleic acid
molecules" referred to above may have an average size of less than
about 1,000 kilobases (e.g., about 1, about 5, about 10, about 20,
about 40, about 80, about 100, about 120, about 140, about 160,
about 180, about 200, about 220, about 240, about 250, about 300,
about 350, about 400, about 450 kilobases, about 500, about 550,
about 650 kilobases, about 750, about 850, or about 950
kilobases).
[0115] Further, when releasing reagents used in methods of the
invention will not solubilize substantial quantities of cellular
contaminants or nucleic acid molecules which one seeks to remove
from the solid matrix, then no intervening treatment (e.g.,
washing) of the solid matrix will often be necessary prior to
releasing agent treatment. One example of such a situation is when
the sample is obtained from seed material. Seeds contain a
considerable amount of carbohydrates. The releasing reagents of the
invention do not result in the release of substantial amounts of
seed carbohydrates from solid matrices. Thus, when the sample
comprises seed materials, in many instances, the sample may be
directly applied to the solid matrix and then treated with a
releasing agent without intervening treatment. As a result, nucleic
acid molecules are released from the solid matrix and most of the
seed carbohydrates remain associated with the matrix.
[0116] The releasing reagents of the invention may be aqueous or
non-aqueous. As one skilled in the art would recognize, different
alkanol amines suitable for use in releasing reagents demonstrate
different solubility properties in both aqueous and non-aqueous
solutions. Further, the choice of solvent system will vary with the
particular application, the sample, and the solid matrix. Examples
of non-aqueous solvents which can be used in releasing reagents of
the invention include ethanol, methanol, propanol, butanol,
acetonitrile, dimethyl sulfoxide (DMSO), and N,N-dimethylformamide
(DMF).
[0117] In one specific embodiment of methods of the invention, 2 mm
FTA.RTM. paper punches, to which nucleic acid molecules are bound,
are soaked for 20 minutes at 90-100.degree. C. in a releasing
reagent which contains, is an aqueous solution, 1%
mono-ethanolamine at pH 11.0. In this embodiment, described in more
detail below in Example 2, substantial quantities of nucleic acid
molecules are released from the FTA.RTM. punches. As one skilled in
the art would recognize, these methods may be modified to suit the
particular application. Thus, in one general aspect, the invention
relates to methods for removing nucleic acid molecules from solid
matrices which comprise contacting these matrices with solutions
comprising an alkanol amine under specified conditions for a
specified period of time.
[0118] In specific embodiments, the alkanol amine (e.g.,
ethanolamine) concentration in the releasing reagent is in the
range of from about 0.01% to about 5.0% (vol./vol.), from about
0.01% to about 4.0% (vol./vol.), from about 0.01% to about 3.0%
(vol./vol.), from about 0.01% to about 2.0% (vol./vol.), from about
0.01% to about 1.0% (vol./vol.), from about 0.01% to about 0.9%
(vol./vol.), from about 0.01% to about 0.8% (vol./vol.), from about
0.01% to about 0.5% (vol./vol.), from about 0.1% to about 5.0%
(vol./vol.), from about 0.1% to about 4.0% (vol./vol.), from about
0.1% to about 3.0% (vol./vol.), from 30 about 0.1% to about 2.0%
(vol./vol.), from about 0.1% to about 1.0% (vol./vol.), from about
0.1% to about 0.9% (vol./vol.), from about 0.1% to about 0.8%
(vol./vol.), from about 0.1% to about 0.5% (vol./vol.), from about
0.2% to about 5.0% (vol./vol.), from about 0.2% to about 4.0%
(vol./vol.), from about 0.2% to about 3.0% (vol./vol.), from about
0.2% to about 2.0% (vol./vol.), from about 0.2% to about 1.0%
(vol./vol.), from about 0.2% to about 0.9% (vol./vol.), from about
0.2% to about 0.8% (vol./vol.), from about 0.2% to about 0.5%
(vol./vol.), from about 0.4% to about 5.0% (vol./vol.), from about
0.4% to about 4.0% (vol./vol.), from about 0.4% to about 3.0%
(vol./vol.), from about 0.4% to about 2.0% (vol./vol.), from about
0.4% to about 1.0% (vol./vol.), from about 0.4% to about 0.9%
(vol./vol.), from about 0.4% to about 0.8% (vol./vol.), from about
0.4% to about 0.7% (vol./vol.), from about 0.8% to about 5.0%
(vol./vol.), from about 0.8% to about 4.0% (vol./vol.), from about
0.8% to about 3.0% (vol./vol.), from about 0.8% to about 2.0%
(vol./vol.), or from about 0.8% to about 1.0% (vol./vol.). The
invention further includes releasing reagents which contain about
0.01% (vol./vol.), about 0.1% (vol./vol.), about 0.2% (vol./vol.),
about 0.4% (vol./vol.), about 0.7% (vol./vol.), about 0.8%
(vol./vol.), about 0.9% (vol./vol.), about 1.0% (vol./vol.), about
1.1% (vol./vol.), about 1.2% (vol./vol.), about 1.4% (vol./vol.),
about 1.6% (vol./vol.), about 1.8% (vol./vol.), about 2.0%
(vol./vol.), about 2.5% (vol./vol.), about 3.0% (vol./vol.), about
3.5% (vol./vol.), about 4.0% (vol./vol.), about 4.5% (vol./vol.),
or about 5.0% (vol./vol.).
[0119] The alkanol amine concentration used will vary with a number
of factors, including the particular alkanol amine, the solubility
of non-nucleic acid compounds present in the sample, the pH of the
releasing reagent, the incubation conditions (e.g., length of
incubation period and incubation temperature), and the solid matrix
used. One of ordinary skill in the art would know how to identify
alkanol amine concentrations suitable for particular applications.
Similarly, one of ordinary skill in the art would also know how to
select alkanol amines suitable for particular applications.
[0120] The pH of releasing reagents of the invention can vary
within, for example, the following ranges: from about 8.0 to about
14.0, from about 8.5 to about 14, from about 9.0 to about 14.0,
from about 9.5 to about 14.0, from about 10.0 to about 14.0, from
about 10.5 to about 14.0, from about 11 to about 14.0, from about
7.5 to about 13.5, from about 7.5 to about 13.0, from about 7.5 to
about 12.5, from about 7.5 to about 12.0, from about 7.5 to about
11.5, from about 7.5 to about 11.0, from about 7.5 to about 10.5,
from about 7.5 to about 10.0, from about 7.5 to about 9.5, from
about 8.0 to about 12.0, from about 8.0 to about 11.5, from about
8.0 to about 11.0, from about 8.5 to about 14.0, from about 8.5 to
about 13.0, from about 8.5 to about 12.5, from about 8.5 to about
12.0, from about 8.5 to about 11.5, from about 8.5 to about 11.0,
from about 9.0 to about 12.0, from about 9.0 to about 11.5, from
about 9.0 to about 11.0, and from about 9.0 to about 10.5. The
invention further includes releasing reagents which have a pH of
about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, about 10.5,
about 11.0, about 11.5, about 12.0, about 12.5, about 13.0, about
13.5, or about 14.0.
[0121] Alkanol amines suitable for use with the invention will
often have the capacity to act as a buffer. Thus, in some instances
it will be possible to dilute the alkanol amines used in the
practice of the invention with a solvent (e.g., water) and directly
adjust the pH using either an acid or a base. In other instances,
it may be necessary, or desirable, to include a buffering agent
(e.g., Tris, 2-[(Tris(hydroxymethyl)methyl)amino]-1-ethanesulfonic
acid (TES), methylamine, CAPS,4-(Cyclohexylamino)-1-butanesulfonic
acid (CABS),2-(N-Cyclohexylamino)ethanesulfonic acid (CHES),
Tricine, or Bicine) in the releasing reagent. Buffers suitable for
use in releasing reagents of the invention can be obtained from
commercial suppliers such as Sigma-Aldrich Corporation, 3050 Spruce
Street, St. Louis, Mo. 63103 USA.
[0122] The methods of the invention can be performed by incubation
of the solid matrix with a releasing reagent at a number of
temperatures and for varying incubation times. For example,
incubations may be performed at temperatures ranging from about
65.degree. C. to about 100.degree. C., about 70.degree. C. to about
100.degree. C., about 80.degree. C. to about 100.degree. C., about
90.degree. C. to about 100.degree. C., about 65.degree. C. to about
90.degree. C. about 70.degree. C. to about 90.degree. C., about
80.degree. C. to about 90.degree. C., about 65.degree. C. to about
80.degree. C., or about 70.degree. C. to about 80.degree. C.
Further, incubations may be performed at temperature such as about
65.degree. C., about 70.degree. C., about 75.degree. C., about
80.degree. C., about 85.degree. C., about 90.degree. C., about
95.degree. C., or about 100.degree. C.
[0123] As noted above, the solid matrix from which nucleic acid
release is desired can be incubated with the releasing reagent for
various periods of time. As one skilled in the art would recognize,
longer incubation periods will result in the release of nucleic
acid molecules until an equilibrium is reached between the
concentration of nucleic acid molecules in the releasing reagent
and that bound to the solid matrix.
[0124] Solid matrices may be incubated with the releasing reagent
for varying periods of time such as for 1 to 60 minutes, 5 to 50
minutes, 10 to 40 minutes, 20 to 30 minutes. 20 to 40 minutes, 30
to 50 minutes, 40 to 60 minutes, or 40 to 90 minutes. Further,
incubations may be performed for about 1, 5, 10, 15, 20, 25, 30,
35, 40, 45, 50. 55, 60, 65, 70, 75, 80, 85, 90, 120, 180, or240
minutes. Such incubation periods will often be useful when nucleic
acid molecules are released from a solid matrix which is in sheet
form.
[0125] Of course, the length of the incubation period used will
vary with the particular conditions. Examples of conditions where
relatively long incubation periods (e.g., eight hours, one day, two
days, three days, five days, seven days) may be desirable include
situations where the concentration of nucleic acid molecules on the
solid matrix is low, the concentration of the alkanol amine is low
(e.g., 0.1% ethanolamine), and/or the incubation temperature is low
(e.g., 70.degree. C. or lower).
[0126] Further, the length of the incubation period, as well as
other incubation conditions, can be varied so that a particular
amount or percentage of the nucleic acid molecules in the sample is
released into the releasing reagent. For example, incubation
conditions can be adjusted such that 0.5%, 5%, 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, or 90% of the nucleic acid molecules present on
the solid matrix are released into the releasing reagent. One of
ordinary skill in the art would know how to determine incubation
conditions suitable for particular applications.
[0127] Tubular/column forms of solid matrices suitable for use in
methods of the invention may have, for example, the following
attributes. The solid matrix can be placed in a tube where it runs
from one end to the other end, with room at one end (i.e., the top)
for sample addition. In such embodiments, the solid matrix will
generally extend from side wall to side wall within the tube. Thus,
the majority of fluids which passes through the tube will have to
pass through the solid matrix. In such situations, the solid matrix
and tube can form a structure similar to that of columns used for
chromatography. Thus, these solid matrix/tube formats are referred
to as "solid matrix columns." As implied above, the solid matrix
may form a solid or loose packing in these column forms.
[0128] As with the flat sheet embodiments of the invention, samples
applied to solid matrix columns may be washed using solutions
described above. Further, release of nucleic acid molecules from
solid matrix columns may be induced by the passage of releasing
reagents through columns. In one specific aspect of the invention,
the matrix in the solid matrix column is washed at room temperature
to remove contaminants. The bottom of the column is then plugged to
prevent fluid flow, the column is heated to 95.degree. C., and the
releasing reagent (also heated to 90.degree. C.) is then applied to
the column. The bottom of the column is unplugged for sufficient
time to allow the releasing reagent to fill the column and then
replugged. After a 30 minute incubation at 95.degree. C., the
column in unplugged again and the releasing reagent with nucleic
acid molecules is drained from the column. In one more specific
aspect, the solid matrix columns are spin columns which can be
placed in a centrifuge and the releasing reagent is removed from
the columns by centrifugation.
[0129] As one skilled in the art would recognize, incubation
conditions can be varied similar to the incubation conditions as
described above. Further, the particular incubation conditions used
will again vary with the sample, the nucleic acid molecules which
are released from the solid matrix, and the solid matrix itself One
of ordinary skill in the art would know how to determine incubation
conditions suitable for particular applications.
[0130] Nucleic acid molecules released from solid matrices may be
used in or manipulated by one or more standard molecular biology
techniques, such as nucleic acid synthesis, restriction
endonuclease digestion, hybridization reactions, ligation to other
nucleic acid molecules, sequencing, amplification (e.g., PCR),
transformation, and transfection. Generally, the released nucleic
acid molecules will be used after dilution with another solution
(e.g., Tris-EDTA) to render the released/solubilized nucleic acid
molecules suitable for the intended use (e.g., PCR).
[0131] The compositions and methods of the invention can be
designed so that the nucleic acid molecules are released from solid
matrices into a solution having a relatively low salt content.
Further, as implied above, the salt concentration of the releasing
reagent containing the nucleic acid molecules can be decreased by
dilution. In situations where the salt content is higher than
desired, the nucleic acid molecules can be desalted prior to use
(e.g., the nucleic acid molecules can be separated from the alkanol
amine) using standard techniques.
[0132] The invention further provides recombinant host cells which
comprise nucleic acid molecules prepared by methods of the
invention, as well as methods for preparing recombinant host cells
comprising these nucleic acid molecules. Representative examples of
appropriate hosts include bacterial cells (e.g., Escherichia coli,
Salmonella typhimurium), fungal cells (e.g., Saccharomyces
cerevisiae, Schizosaccharomyces pombe, Neurospora crassa), insect
cells (e.g., Drosophila S2 cells, Spodoptera Sf9 cells), animal
cells (e.g., CHO cells. COS cells, Bowes melanoma cells), and plant
cells. Appropriate media and conditions for culturing the
above-described host cells are known in the art.
[0133] Nucleic acid molecules prepared by methods of the invention
may be introduced into host cells by methods described in standard
laboratory manuals (see, e.g., Sambrook, J. et al., eds., MOLECULAR
CLONING, A LABORATORY MANUAL, 2 nd. edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1989), Chapter 9;
Ausubel, F. et al., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John H. Wiley & Sons, Inc. (1997), Chapter 16), including
methods such as electroporation, DEAE-dextran mediated
transfection, transfection, microinjection, cationic lipid-mediated
transfection, electroporation, transduction, scrape loading,
ballistic introduction, and infection. Methods for the introduction
of nucleic acid molecules into host cells are discussed, for
example, in Felgner et al., U.S. Pat. No. 5,580,859.
[0134] Nucleic acid molecules prepared by methods of the invention
may also contain genetic elements which allow for chromosomal
integration of vector sequences. Such elements are useful for the
stable maintenance of heterologous sequences and include sequences
which confer both site-specific and site-independent integration.
Site-specific integration (e.g., homologous integration) and
site-independent integration, sometimes referred to as "random
integration" can be used to introduce heterologous sequences of
interest into host cell chromosomes. Descriptions and methods for
inserting genetic material into eukaryotic chromosomes, for
example, are available from numerous sources including Sambrook, J.
et al., eds. (MOLECULAR CLONING, A LABORATORY MANUAL, 2 nd.
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)).
III. Automated Sample Processing
[0135] The invention provides compositions, methods and reagents
suitable for a number of applications, in addition to the
purification and/or isolation of nucleic acid molecules from
individual samples. One example of such an application is the
purification and/or isolation of nucleic acid molecules derived
from collections of samples which represent individual members of
populations.
[0136] In one embodiment, samples comprising viral plaques (e.g.,
plaques of T4 phage) on plates are transferred to a solid matrix.
Depending on the particular application, the solid matrix may then
be either stored for later use or treated with a releasing reagent
to release viral nucleic acid molecules. In many instances, the
solid matrix will be cut into pieces which contain different
nucleic acid molecules. This segmentation of the solid matrix may
be random or may result in the production of individual pieces
which contain, for example, nucleic acid molecules corresponding to
particular viral plaques.
[0137] Similarly, samples from a variety of non-viral sources can
be transferred to solid matrices and used to prepare individual
nucleic acid molecules. For example, samples (e.g., bacterial
cultures) which contain different nucleic acid molecules can be
prepared in the individual wells of a 96 well plate (e g., a 96
well MicroPlate). Portions of these samples can then be transferred
to solid matrices using a DNA Card Registration Tool (Cat. No. VP
382CS) and Slot Pin Spotter (Cat. No. VP 408S5), available from
V&P Scientific, Inc., 9823 Pacific Heights Boulevard, Suite T,
San Diego, Calif. 92121 USA. Using this device, 5 .mu.l from each
of the 96 sample wells can be transferred at one time to an
FTA.RTM. CloneSaver 96 Card, available from Invitrogen Corp., Life
Technologies Division, 9800 Medical Center Drive, Rockville, Md.
20850 USA. Optionally, a dye (e.g., bromophenol blue) may be added
to each of the sample wells to mark the sample location after
application to the FTA.RTM. CloneSaver 96 Card.
[0138] Once samples have been applied to a solid matrix, such as an
FTA.RTM. CloneSaver 96 Card, the solid matrix can be cut into
pieces to separate the portions of the matrix to which individual
samples have been applied. In situations where the sample has been
applied in a specific pattern (e.g., where a DNA Card Registration
Tool, a Slot Pin Spotter, and an FTA.RTM. CloneSaver 96 Card have
been used as described above), a machine can be readily designed to
cut the solid matrix in such a manner so as to separate individual
samples. Using FTA.RTM. loneSaver 96 Cards as an example, a machine
can be designed which punches out portions of the card
corresponding to some or all of the 96 positions which contain
samples. These individual card pieces can then be placed in the
individual wells of a 96 well plate and treated with various
reagents to remove contaminants and release nucleic acid molecules.
For example, reagents (e.g., washing solutions) may be added to
each of the wells and then removed by aspiration. Further, the
releasing reagent may be added to each of the wells and then
removed by aspiration and transferred to another vessel. As one
alternative, the solid matrices (e.g., the card pieces) can be
removed from the wells after the incubation period, resulting in
the nucleic acid molecules remaining behind. As another
alternative, the solid matrices can be left in the wells with the
released nucleic acid molecules. The most appropriate course of
action will vary with the particular application(s) in which the
nucleic acid molecules are intended to be used.
[0139] One specific application of automated methods of the
invention is in the high-throughput amplification of nucleic acid
molecules by PCR, followed by sequencing of amplification products.
Systems can be designed so that each sample applied to solid
matrices contains cells or plaques having different nucleic acid
molecules.
[0140] Further, nucleic acid samples from which nucleic acid
molecules can be released using compositions and methods of the
invention can be conveniently prepared, stored, and sold, or
otherwise transferred to individuals or entities, for use in
methods of the invention.
[0141] In other specific embodiments of the invention, robotic
systems perform essentially all of the steps required for
determining genotypes using samples. In such applications, a sample
card designed to hold 96 samples and having a reinforced backing
may be used. This reinforced backing is advantageous because it
increases the durability of the card and thus makes the card easier
to handle by robotic systems. A liquid handling system is used to
deposit fluid containing the samples (e.g., 5 .mu.l of each) at the
appropriate locations on the sample card (e.g., an FTA.RTM.
loneSaver 96 Card). After the samples have dried, either the same
or another machine cuts out portions of the card (e.g., 2 mm
circles are punched out of the card) which contain the individual
samples. These portions of the card are then placed by the machine
into the wells of a 96 well plate. Reagents are added and removed
from the wells using a liquid handler. Thermocycling, for example,
can also be performed on the plates. Further, portions of fluid
from the wells containing solubilized nucleic acid molecules can be
removed and placed in other containers for use in restriction
fragment length polymorphism (RFLP) or Southern blot analyses.
[0142] Processes of the invention are especially useful for
generating genotype data using large numbers of samples. For
example, genotype analyses of a large number of individuals can be
performed using human blood in processes of the invention, followed
by PCR using flourescent primers and by sequencing using automated
sequencing machines.
[0143] Processes of the invention are also useful for genotype
analysis of large numbers of samples obtained from plants,
microorganisms and non-human animals. Using plants as an example,
large numbers of plant samples can be prepared and screened from
genetic properties associated with one or more genotypes. This will
be especially useful for identifying particular genotypes of plants
grown in specific geographic regions. For example, material from
soybeans (e.g., leaves or seeds) may be collected from fields in a
particular region. The plant material may be either applied
directly to the solid matrix or may be dispersed into a solution
prior to sample addition.
[0144] When plant material, or any other solid material, is applied
directly to a solid matrix, it will generally be advantageous to
apply the material to the matrix by the use of compression force.
In other words, the solid material will often be crushed against
the surface of the solid matrix.
[0145] A device may be employed for applying solid materials to
solid matrices (e.g., a hammer). In one embodiment, this device
contains a chamber, where the solid material containing the nucleic
acid molecules is initially placed. An piston, or piston-like
object, is located at one end of this chamber and the solid matrix
is located at the other end. Thus, the movement of the piston, or
piston-like object, towards the solid matrix results in the solid
material being forced against the surface of the solid matrix.
Nucleic acid molecules from the solid material are deposited on the
solid matrix during this process. The device optionally has a solid
portion which supports the back portion of the solid matrix and
prevents the solid material from being forced through the matrix.
This solid portion is not necessary when the solid matrix is rigid
enough to withstand the pressure exerted by compression of the
piston or piston-like object with damage to its structural
integrity.
[0146] The above device especially is useful for the collection of
plant material samples over a particular geographic region. The
device will generally be relatively small, portable, and capable of
being operated using manually induced pressure. Further, the device
eliminates the need to prepare sample solutions for application to
solid matrices. The device also provides uniform sample application
on the surface of the solid matrix. Thus, in another aspect, the
invention provides a device for the application of samples to solid
matrices, wherein the device has (1) a chamber which houses solid
materials containing nucleic acid molecules (ie., the sample), (2)
a piston, or piston-like object, at one end of the chamber, and (3)
an attachment site for holding solid matrices at the other end of
the chamber.
[0147] Plants which can be used in the methods of the invention
include, but are not limited to, soybeans, corn, Rey, wheat,
sorghum, rice, green peppers, red peppers, peas, pine trees, blue
spruce, maple trees, grass (e.g. Kentucky Blue Grass), and cotton.
Portions of plants which can be used in methods of the invention
include stems, roots, seed, leaves, petals, and sepals.
[0148] The genotype analysis methods of the invention are useful
for monitoring the spread of plant strains and genetic markers
across geographic regions. These method are also useful for
detecting genetically modified organisms (G.M.O.s). One example of
a G.M.O, is a plant which has been grown from seeds derived from
another G.M.O. Thus, the invention provides methods for identifying
G.M.O.s comprising obtaining samples from plants, applying these
samples to solid matrices, releasing nucleic acid molecules
associated with the solid matrices, and analyzing the nucleic acid
molecules to determine a genotype related to one genetic trait of
the plant from which the sample was obtained. In related
embodiments, genotypes related to multiple traits (e.g., two,
three, four or five) may be determined.
[0149] When screening plant materials to identify/detect G.M.O.s,
as well as specific plant strains, molecular marker detection
methods will generally be used. For example, PCR can be performed
to amplify nucleic acid molecules which represent single copy
genes. These amplified nucleic acid molecules may then be analyzed
using methods such as sequencing, RFLP analysis, or hybridization
analysis to determine whether a particular plant is a G.M.O.
IV. Kits
[0150] Other embodiments of the invention, include kits for
releasing nucleic acid molecules from solid matrices and kits for
the purification and/or isolation of nucleic acid molecules. Kits
serve to expedite the performance of for example, methods of the
invention by providing multiple components and reagents packaged
together. Further, reagents of these kits can be supplied in
pre-measured units so as to increase precision and reliability of
the methods.
[0151] Kits of the invention for removing nucleic acid molecules
from solid matrices will generally comprising a carton such as a
box, one or more containers such as boxes, tubes, ampules, jars,
bags, plates and the like, and one or more releasing reagents and
one or more individual components selected from the group
consisting of:
[0152] (a) at least one (e.g, one, two, three, four, five, ten,
twenty, fifty, one hundred) solid matrix;
[0153] (b) at least one apparatus for applying samples to solid
matrices;
[0154] (c) at least one apparatus for cutting solid matrices into
sections which contain samples; and
[0155] (d) at least one washing solution.
[0156] Apparatuses for applying samples to solid matrices include
essentially any device which can be used for sample application.
One example of such a device is a DNA Card Registration Tool and
Slot Pin Spotter which, as noted above, can be used to apply 96
samples at one time to a solid matrix. Other examples include
microcapillary tubes and pipettes (e.g., micropipettes such as a
Pipeteman.TM.), as well as other devices which can be used to
transfer and/or deliver fluids. Yet other examples include the
device described both below in Example 1 and above for applying
solid material to solid matrices.
[0157] Apparatuses for cutting solid matrices into sections which
contain samples include devices for cutting materials such as
papers and cards. Examples of such devices include scissors,
razors, knives, and the HARRIS MICRO PUNCH.TM. Apparatus. Devices
such as HARRIS MICRO PUNCH.TM. Apparatuses are particularly useful
because the can be used to cut solid matrices into circular pieces
of relatively uniform size (e.g., 1.2 mm, 2 mm, etc.).
[0158] Washing solutions supplied with kits of the invention will
vary with the particular application that the kit is intended for.
As an example, when the kit is designed for the purification and/or
isolation of high molecular w eight nucleic acid molecules from
samples which contain high concentrations of fats and lipids (e.g.,
adipose tissues), one or more washing solutions contained with the
kit will generally contain a detergent (e.g., TRITON X-100).
[0159] Further, when multiple washing solutions are contained in
the kit, the solutions used for washing steps earlier in the
washing process may contain agents which are removed by later
washing steps. For example, when two washing steps are employed,
the washing solution used in the first washing step may contain a
detergent. The washing solution used in the second washing step,
however, may not contain a detergent and may, in part, be used to
remove the detergent added to the solid matrix by the first washing
solution.
[0160] Examples of washing solutions which can be used with various
embodiments of the invention include 10 mM Tris, 1 mM EDTA (pH
7.3); water; and solutions which contain detergents.
[0161] Sample containers and containers solid matrices may also be
included in kits of the invention. One example a container suitable
for use in a number of embodiments of the invention is a FTA.RTM.
96-well MicroPlate (Invitrogen Corp, Life Technologies Division,
Cat. No. 10786-028). Plates such as these can be used, for
examples, to house both the samples prior to application to solid
matrices and pieces of solid matrices which contain samples.
[0162] Columns may also be included in kits of the invention (e.g.,
columns suitable for centrifugation in, for example, micro
centrifuges). These columns may be used, for example, to desalt
samples or to separate larger nucleic acid molecules from smaller
molecules such as nucleotides, proteins, RNA, etc. Such columns are
known in the art and may contain for example, material designed for
molecular weight separations such as Sephadex.RTM. G-50.
[0163] It will be understood by one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are readily apparent
from the description of the invention contained herein in view of
information known to the ordinarily skilled artisan, and may be
made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
[0164] Pine needles from individual trees (Pinus pinus) are
collected at various locations in the Shenandoah National Park.
Needles from each tree are placed over the surface of an FTA.RTM.
CloneSaver 96 Card and crushed into the Card using a hammer. The
needles are placed in the Card such that each position on the card
contains sample material from only one tree. As each sample is
applied to the card, a log is kept so that the sample can be linked
to a particular tree. Once samples have been applied to all of the
sample positions on the card, the card is stored at ambient
temperature until it is transported to a lab for analysis.
[0165] Once in the lab, the samples are "punched" from the FTA.RTM.
card using a HARRIS MICRO PUNCH.TM. Apparatus with mat. The
resulting 2 mm punches are then placed in wells of a 96 well plate
and washed with Tris-EDTA (pH 7.3). 50 .mu.l of ethanolamine,
adjusted to pH 11.0 with KOH, is then added to each of the wells.
After incubation at 90.degree. C. for 30 minutes, the fluid in each
well containing the released nucleic acid molecules is removed by
aspiration, transferred to 0.5 ml microcentrifuge tubes, and frozen
for later use in PCR reactions.
Example 2
[0166] Methods
[0167] Twenty-five .mu.l of fresh blood was spotted onto individual
locations of FTA.RTM. CloneSaver 96 Cards using a Model P-200
Pipeteman.TM. (Rainin Instrument Company, Inc. Rainin Road, Box
4026, Woburn, Mass., 01888). These Cards were stored at room
temperature until used.
[0168] The releasing reagents were composed of ethanolamine diluted
in water to a concentration of between 0.01% and 1%. The pH range
was between 8.3 and 13. The pH was adjusted by the addition of
aqueous KOH. The best results were obtained at a pH of 11 at a
concentration of 1%. The ethanolamine reagents were compared to 15
mM and 30 mM CABS [4-(cyclohexylamino)-1-butane sulfonic acid]
buffer, CAPS [3-(cyclohexylamino)-1-propane sulfonic acid] buffer,
Gentra Elution Buffer 2 (used with the GENERATION.TM. CAPTURE
COLUMN.TM. Kit, Gentra Systems Inc., 13355 10 th Avenue N., Suite
120, Minneapolis, Minn. 55441, USA), and AmpDirect (Shimadzu, Inc.,
1, Nishinokyo Kuwabaracho, Nakagyou-ku, Kyoto 604-8511, Japan).
[0169] After sample application, FTA.RTM. punches were washed with
FTA.RTM. Purification Reagent (Invitrogen Corp., Life Technologies
Division, Cat. No. 10876) and TE buffer according to manufacturer's
recommendations.
[0170] The releasing reagents were added at 50 .mu.l/2 mm punch.
The punches were then heated to 90-100.degree. C. for 1-30 minutes,
with the best results being obtained between 10 and 20 minutes
based on PCR analysis. This conclusion was based on the results
obtained in FIGS. 1 and 3. FIG. 1 shows that as much as 100 pg/ml
is released after 10 minutes. At 20 minutes approximately 1 ng/ml
is released using the releasing reagent. FIG. 4 shows that the PCR
results obtained using the released DNA is similar after heating
for 10, 15 or 20 minutes. However, when the PCR product is diluted
1:10 differences in the amount of product is noticeable. The Gentra
Elution Buffer 2 and the ethanolamine releasing reagent perform
better that the other reagents tested. PCR products are clearly
visible for the 15 and 20 minute heating times when the PCR
reaction is diluted 1:10 prior to loading on the gel.
[0171] The concentration of the released DNA was determined using
the ACES.TM. 2.0.sup.+ Human DNA Quantitation System. In this
system 10 .mu.l of released DNA is used for quantitation. Following
quantitation the performance of each released DNA was tested for
its ability to produce amplified products when used in genotyping
PCR assays. This was tested by setting up a standard genotyping PCR
assay and then adding 1 .mu.l of the released DNA to be tested.
Each PCR assay was in a total volume of 15 .mu.l. The PCR products
were analyzed by electrophoresis on a 1.5% agarose gel.
Electrophoresis was performed at 100 volts for 1 hour. The gels
were then stained with ethidium bromide and a photograph was
taken.
[0172] Having now fully described the present invention in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be obvious to one of ordinary skill in
the art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
[0173] All publications, patents and patent applications mentioned
in this specification are indicative of the level of skill of those
skilled in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
* * * * *