U.S. patent application number 11/688019 was filed with the patent office on 2007-07-19 for compositions and methods for nucleic acid extraction from biological samples.
This patent application is currently assigned to SIGMA-ALDRICH CO.. Invention is credited to Derek K. Douglas, Carol Kreader, Scott A. Weber.
Application Number | 20070166751 11/688019 |
Document ID | / |
Family ID | 32507223 |
Filed Date | 2007-07-19 |
United States Patent
Application |
20070166751 |
Kind Code |
A1 |
Weber; Scott A. ; et
al. |
July 19, 2007 |
Compositions and Methods for Nucleic Acid Extraction from
Biological Samples
Abstract
Methods and compositions for extracting nucleic acids from a
biological sample are provided. The extraction compositions contain
a protease enzyme such as proteinase K at alkaline pH with little
or no surfactant present. Extraction can be efficiently performed
in 60 minutes or less at room temperature for certain mammalian
tissue samples and at elevated temperatures for certain plant
tissues.
Inventors: |
Weber; Scott A.; (St. Louis,
MO) ; Douglas; Derek K.; (St. Louis, MO) ;
Kreader; Carol; (St. Louis, MO) |
Correspondence
Address: |
SENNIGER POWERS
ONE METROPOLITAN SQUARE
16TH FLOOR
ST LOUIS
MO
63102
US
|
Assignee: |
SIGMA-ALDRICH CO.
3050 Spruce Street
St. Louis
MO
63103
|
Family ID: |
32507223 |
Appl. No.: |
11/688019 |
Filed: |
March 19, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10322103 |
Dec 17, 2002 |
7214484 |
|
|
11688019 |
Mar 19, 2007 |
|
|
|
Current U.S.
Class: |
435/6.12 ;
435/270 |
Current CPC
Class: |
C12N 15/1003
20130101 |
Class at
Publication: |
435/006 ;
435/270 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 1/08 20060101 C12N001/08 |
Claims
1. A method for extracting nucleic acids from a tissue sample
obtained from a mammal, the method comprising incubating the sample
at room temperature in an extraction composition which contains a
protease enzyme and a buffer component which buffers the
composition to a pH of 7.5 or greater for an incubation time of not
more than 30 minutes.
2. The method in accordance with claim 1 wherein the extraction
composition does not contain a surface active agent.
3. The method in accordance with claim 1 wherein the protease
enzyme is a subtilisin-like serine protease.
4. The method in accordance with claim 3 wherein the
subtilisin-like serine protease is proteinase K.
5. The method in accordance with claim 4 wherein the proteinase K
is present at a concentration of about 50 Units/ml or greater.
6. The method in accordance with claim 5 wherein the proteinase K
is present at a concentration of about 100 Units/ml or greater.
7. The method in accordance with claim 1 wherein the extraction
composition comprises not more than about 100 mM EDTA.
8. The method in accordance with claim 7, wherein the extraction
composition comprises about 10 mM EDTA.
9. The method in accordance with claim 1, wherein the buffer
component is Tris-HCl.
10. The method in accordance with claim 9, wherein the Tris-HCl is
present at a concentration of about 80 mM or greater.
11. The method in accordance with claim 1 wherein the extraction
composition is buffered to a pH of at least 8.0 or greater.
12. The method in accordance with claim 1 wherein the incubating is
for not more than about 20 minutes.
13. The method in accordance with claim 12 wherein the incubating
is for not more than about 10 minutes.
14. The method in accordance with claim 13 wherein the mammalian
tissue sample comprises a mouse tail tissue or a mouse ear tissue.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional and claims the benefit of
U.S. patent application Ser. No. 10/322,103, filed Dec. 17, 2002,
the content of which is hereby incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates generally to methods and compositions
for the extraction of nucleic acids from biological samples, and,
more particularly, to methods and compositions for rapid extraction
of nucleic acids from tissue samples using an alkaline solution
containing proteinase K. The extraction solution is suitable for
further processing of the extracted nucleic acid using PCR.
[0004] 2. Description of the Related Art
[0005] With the advent of modern molecular biology, the ability to
study nucleic acids in biological samples has allowed many
significant advances in biological and biochemical research. One
method that has provided such advances has been the polymerase
chain reaction (PCR) which allows the rapid amplification of target
nucleic acid from as little starting material as a single molecule
(for review see Baumforth et al, J. Clin. Pathol. Mol. Pathol.
52:1-10, 1999; Rapley et al, Medical Laboratory Sciences
49:119-128, 1992).
[0006] The application of PCR and other methods in molecular
biology require the extraction of nucleic acid from biological
samples and a number of approaches have been devised for performing
such extraction. Various approaches have included treatment with a
surface active agent such as sodium dodecyl sulfate and proteinase
K to lyse cells and release the nucleic acid along with extraction
using phenol and/or chloroform (see, for example, Sambrook et al.,
Molecular Cloning A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
[0007] In recent years, a number of approaches have been developed
for rapid extraction of nucleic acids from biological samples. The
methods not only provide an ease and convenience of tissue
processing, they allow the processing of a high volume of samples
(see for example, Steiner et al, Nucleic Acids Research
23:2569-2570, 1995). Nevertheless, efficient extraction of
biological samples has not always been achieved.
[0008] Both animal and plant tissues have been studied and
approaches for nucleic acid extraction have been developed for
both. For mammalian tissue extraction, some studies have reported
on the digestion of the tissue by incubation with proteinase K for
hours at elevated temperatures (see for example, Goldenberger et
al., PCR Methods and Applications 4: 368-370, 1995; Zimmermann et
al., Comparative Medicine 50:314-316, 2000) Such methods can form
the basis for kits, which are commercially available (see for
example GenElute.TM. Mammalian Genomic DNA Miniprep kit available
from Sigma-Aldrich, St. Louis Mo.).
[0009] Other studies have reported shorter incubation times, which
are more applicable for use in high throughput assays. For example,
U.S. Pat. No. 6,469,159 discloses an extraction method using a
buffer, a non-ionic surfactant and heating at alkaline pH. Heating
was to 70.degree. C. to 100.degree. C. for 5 minutes to 3 hours.
This reference, however, did not disclose extraction at
temperatures less than 70.degree. C., which could have been more
conveniently performed or the use of an extraction solution, which
did not contain a surfactant.
[0010] Drews et al. reported on a 15 minute procedure for
extraction of mouse tail sections at 55.degree. C. The procedure
used a Tris-HCl buffer at pH 8.0 and containing the ionic
surfactant, sodium dodecyl sulfate (SDS), and Proteinase K (Drews
et al., BioTechniques 17:866-867, 1994). Similarly, Chen et al.
reported on a 30 min procedure for extracting mouse ear-punch
tissues at 55.degree. C. using a Tris-HCl buffer at pH 8.0, SDS and
Proteinase K (Chen et al., BioTechniques 8:32-33, 1990). Although
SDS is known to inhibit Taq polymerase in PCR reactions (e.g. see
Gelfand, in PCR Technology, H. A. Erlich, Ed., Stockton Press,
N.Y., 1989 pp. 17-22), these groups provided no suggestion that
extraction could be carried out without surfactant. In addition,
there was no suggestion in these references that extraction of the
mouse tissues might have been carried out at room temperature,
which would have been more convenient.
[0011] Ren et al, however, reported on the extraction of mouse
ear-punch tissue using a detergent-free, proteinase K solution in
sterile water at room temperature for 30 min (Ren et al., Contemp.
Top. Lab. Anim. Sci. (US) 40:27-30, 2001). This group, however, did
not use a Tris-HCl buffer and particularly noted the absence of
strong bases and acids in their extraction solution.
[0012] Methods for rapid extraction of plant tissues have also been
reported. Thomson et al. reported on the extraction of DNA from
ground leaf, seeds and embryos using a Tris-HCl buffer at pH 9.5
and incubation at 95.degree. for 5-60 min and at 65.degree. for
10-60 min (Thomson et al, BioTechniques 19:394-400, 2002). McCarthy
et al. reported on the extraction of DNA from ground transgenic
wheat seeds at room temperature using an extraction buffer
containing urea, SDS and EDTA. A 1:1 phenol:chloroform mixture was
then added to the extraction buffer (BioTechniques 32:560-564,
2002). Steiner et al. reported on the extraction of lyophilized and
ground leaf tissue at 90.degree. for 20 min using an extraction
buffer containing Tris-HCl at pH 8, sodium lauryl sarkosyl and
polyvinylpolypyrrolidone. None of these groups reported on the use
of a protease enzyme such as Proteinase K in the extraction
buffer.
[0013] Guidet reported incorporating Proteinase K into the
extraction buffer to extract DNA from lyophilized and crushed leaf
samples (Guidet, Nucleic Acids Res. 21:4153-4154, 1994). The buffer
contained Tris-HCl at pH 8, EDTA, sodium lauryl sarkosyl and
Proteinase K and the extraction was at 50.degree. C. for 1 hour.
The Proteinase K, however, may not have significantly contributed
to the extraction since the EDTA, which is known to be a Ca.sup.2+
chelator, was present at a concentration of 450 mM. This is because
Proteinase K is Ca.sup.2+-dependent in that the enzyme is unstable
at the high temperatures used by Guidet as well as exhibiting a
decrease in enzyme activity in the absence of Ca.sup.2+ (Bajorath
et al., Nature 337:481-484, 1989; Muller et al, J. Biol. Chem.
269:23108-23111; Kolvenbach et al., Int. J. Pept. Protein Res
36:387-391, 1990). Moreover, the detergent, sodium lauryl sarkosyl
is present which may affect subsequent PCR amplification (Gelfand,
supra, 1989).
[0014] Thus, in view of the deficiencies of earlier methods and
compositions, there remains a continuing need for improved methods
and compositions for extracting nucleic acids from plant and animal
samples.
BRIEF SUMMARY OF THE INVENTION
[0015] Accordingly, the inventors herein have succeeded in
discovering that an extraction composition which comprises a
protease enzyme and which contains a buffering component to buffer
the composition to an alkaline pH of 7.5 or greater, can be used to
efficiently extract nucleic acids from animal or plant samples. One
protease enzyme suitable for use in the present invention is
Proteinase K. By extraction of nucleic acids from a sample it is
meant that the release of nucleic acid from tissue components is
effected so that subsequent procedures such as PCR can be
performed. In the subsequent PCR procedures, the threshold cycle
value (C.sub.t) is detectable over background. The C.sub.t value
will depend upon instrumentation and detection system used and can
be readily ascertained by the skilled artisan. Typical C.sub.t
values are less than or equal to about 20, less than or equal to
about 35 or less than or equal to about 50 or less than or equal to
any value therebetween. Nucleic acids are, thus, extracted in such
a manner that the nucleic acids can be subsequently amplified by
PCR. It is possible that PCR inhibitors which would prevent
amplification could be present in the extraction solution, whether
obtained as part of the extraction process from the biological
sample or from some other source. The presence of PCR inhibitors in
the extraction solution would result in little or no amplification
of nucleic acids and this would be deemed to constitute absence of
effective extraction from the sample. Because surfactants can
sometimes interfere with subsequent PCR amplification, the
extraction composition, in certain embodiments, preferably, does
not contain surfactants. The term "surfactant" as used herein is
intended mean a surface active substance, which is used herein
interchangeably with the term "surfactant". Surfactants include
anionic surfactants, cationic surfactants, non-ionic surfactants
and zwitterionic surfactants. Although surfactants are not included
in the extraction compositions of certain embodiments, surfactants
may, nevertheless, be included in the extraction compositions of
certain other embodiments.
[0016] In addition, because an absence of Ca.sup.2+ can, in some
instances, decrease protease activity either directly or indirectly
(Id., Muller et al., supra, 1994; Bajorath et al, supra, 1989), the
extraction composition, preferably, does not contain a high
concentration of a Ca.sup.2+-chelator such as EDTA. Preferably, if
present at all, the Ca.sup.2+-chelator is at a concentration of not
more than about 100 mM, not more than about 50 mM, not more than
about 25 mM or not more than about 10 mM in the extraction
composition.
[0017] Samples can be from any of a wide variety of biological
sources including the non-limiting examples of species of animals
such as, mammals including humans, fish, birds, insects such as
drosophila, nematodes such as C. elegans and the like; plants
including moss, ferns, trees, bushes, flowering plants and the
like; fungi including mushrooms, molds, yeast and the like;
protista including amea, paramecium, algae, seaweed, diatoms and
the like; or monera including species of bacteria or cyanobacteria.
Samples of tissues are considered to be distinct from samples of
isolated cells such as, for example, blood cells, cells from tissue
culture preparations or cells isolated by any process from a tissue
or biopsy sample. The term tissue as used herein includes plant
seeds and seed tissues. Generally, it is more difficult to extract
nucleic acids from tissues than to extract nucleic acids from cells
although this is not always the case.
[0018] Samples such as tissues or cells from a human or other
mammal can be extracted at room temperature upon incubation for not
more than about 30 minutes, and in certain aspects of the
invention, not more than 20 minutes, and in other aspects, not more
than about 10 minutes or not more than about 5 minutes. A preferred
incubation time is about 10 minutes. Reference to room temperature
or ambient temperature which may be used interchangeably herein, is
intended to mean a laboratory temperature in which humans can
perform tissue extractions, in absence of applied heat to the
extraction mixture. Room temperature is typically about 20.degree.
C. or less, about 22.degree. C. or less, up to about 25.degree. C.
or less, or, in some instances, up to even higher temperatures as
would be readily understood by the skilled artisan.
[0019] Samples from plant tissues, such as portions of leaf or seed
can be extracted at room temperature or at an increased temperature
above room temperature. Extraction temperatures for plant tissues
are sufficiently high to facilitate extraction, but not so high as
to cause denaturation of the protease enzyme. In certain
embodiments, extraction of plant tissue is carried out at an
increased temperature of at least about 37.degree. C., at least
about 45.degree. C., at least about 50.degree. C., at least about
55.degree. C., or at least about 60.degree. C. or higher, depending
upon the heat stability of the particular protease enzyme.
[0020] Thus in certain aspects, the present invention is directed
to a method for extracting nucleic acid from a biological sample.
The method comprises incubating the sample in an extraction
composition, which is buffered to an alkaline pH of about 7.5 or
greater. The extraction composition comprises a protease enzyme and
does not contain a surface active agent. Incubation time for
extraction of nucleic acids from samples are, preferably for
sufficient time to release the nucleic acid, but not more than
about 60 minutes. Incubation time, i.e. extraction time can be, in
certain instances, about 5 minutes, about 10 minutes, about 20
minutes, about 30 minutes or about 45 minutes.
[0021] In another aspect, the present invention provides a method
for extracting nucleic acid from a biological sample obtained from
a mammal. The method comprises incubating the sample at room
temperature in an extraction composition, which is buffered to a pH
of 7.5 or greater for an incubation time of not more than 30
minutes.
[0022] In still another aspect of the present invention, there is
provided a method for extracting nucleic acid from a biological
sample obtained from a plant. The method comprises incubating the
sample at room temperature or at an increased temperature in a
range of temperatures of from a minimum of at least about
37.degree. C. or at least about 45.degree. C. up to a maximum of
not more than about 60.degree. C. in an extraction composition. The
extraction composition is buffered to a pH of 7.5 or greater.
Incubation is for a period of time of not more than 30 minutes.
[0023] The present invention also includes compositions and kits
for extracting nucleic acid from a biological sample. The
compositions and kits are comprised of an extraction composition,
which comprises a protease enzyme and a buffering agent, which
maintains the pH of the composition at 7.5 or greater. In certain
instances a Ca.sup.2+-chelator such as EDTA can be present and, if
present, it is at a concentration of not more than 100 mM. The
extraction composition of the kits can be a single composition or
the individual components of the extraction composition can be
maintained separately for mixing prior to use. Thus the components
of the nucleic acid extraction composition in the kits of the
present invention, can be packaged separately or in one or more
mixtures of any combination the components. Each of the separate
components or one or more mixtures are in a different container and
a single container can contain the overall kit. Preferably, the
extraction compositions of the kits and compositions of the present
invention do not include a surface active agent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 illustrates in duplicate samples, agarose gel
electrophoresis of PCR amplification products from extracted
tissues as follows: Row I shows the products of duplicate samples
of mouse ear punch tissues using the extraction method herein with
incubation at room temperature for 10 minutes (lanes 1 and 2), 20
minutes (lanes 3 and 4) or for 30 minutes (lanes 5 and 6) or
incubation at 55.degree. C. is for 10 minutes (lanes 7 and 8), 20
minutes (lanes 9 and 10) and 30 minutes (lanes 11 and 12). Row II
shows amplified DNA extracted from mouse ear punches illustrating
the effect of the absence (lanes 1 and 2) of heating to 95.degree.
C. for 3 minutes following a 10 minute, room-temperature
extraction; the lack of deleterious effect of omitting addition of
the Neutralization Solution B (lanes 3 and 4) and the comparative
effect of amplification of DNA obtained from mouse tails (lanes
5-12). Row III shows the effect of extraction of mouse tail at a pH
adjusted to pH 10.5 (lanes 1 and 2), adjusted to pH 9.5 (lanes 3
and 4), adjusted to pH 8.5 (lanes 5 and 6), adjusted to pH 7.5
(lanes 7 and 8) and negative and positive controls (lanes 9 and
10). Row IV shows the lack of effect of varying the KCl
concentration on extraction efficiency at 200 mM KCl (lanes 1 and
2), at 100 mM KCl (lanes 5 and 6), at 50 mM KCl (lanes 9 and 10) or
at 0 mM KCl (lanes 13 and 14) with negative controls shown in lanes
3, 7, 11 and 15 and positive controls shown in lanes 4, 8, 12 and
16. Row V shows the PCR amplification products from extraction of
mouse ear punch tissue using the methods of Ren et al. (Ren et al.,
Contemp. Top. Lab. Anim. Sci. (US) 40:27-30, 2001) following
incubation at room temperature for 10 minutes (lanes 1 and 2), 20
minutes (lanes 3 and 4) or 30 minutes (lanes 5 and 6) and following
incubation at 55.degree. C. for 10 minutes (lanes 7 and 8), 20
minutes (lanes 9 and 10) or 30 minutes (lanes 11 and 12). Row VI
shows the PCR amplification products from extraction of mouse ear
punch tissue using a modification of the method of Ren et al. in
which Neutralization Solution B was added at the end of extraction
(lanes 1 and 2) and amplification by the method of Chen et al.
(Chen et al., BioTechniques 8:32-33, 1990) following incubation at
55.degree. C. for 30 minutes (lanes 3 and 4) with negative and
positive control (lanes 5 and 6).
[0025] FIG. 2 illustrates in duplicate samples, agarose gel
electrophoresis of PCR amplification products obtained from
extraction of seeds from soybean as extracted (lanes 1-4) and as
diluted by ten-fold (lanes 5-8) and from extraction of seeds from
sorghum as extracted (lanes 10-13) and as diluted by ten-fold
(lanes 14-17) in the presence (lanes 1, 2, 5, 6, 10, 11, 14, and
15) and absence of proteinase K (lanes 3, 4, 7, 8, 12, 13, 16, and
17).
[0026] FIG. 3 illustrates in duplicate samples, agarose gel
electrophoresis of PCR amplification products obtained from
extraction of seeds from Arabidopsis as extracted (lanes 1-4) and
as diluted by ten-fold (lanes 5-8) in the presence (lanes 1, 2, 5,
and 6) and absence (lanes 3, 4, 7, and 8) of proteinase K.
[0027] FIG. 4 illustrates in duplicate samples, agarose gel
electrophoresis of PCR amplification products obtained from
extraction of seeds from soybean, sorghum, canola, wheat, and
arabidopsis as extracted (Row I, lanes 1, 2, 5, 6, 7, 8, 9, 10, 13,
14, 15, 16, 17, 18, 21, 22, 23, and 24; Row II, lanes 1, 2, 5, 6,
7, 8, 9, 10, 13, 14, 15, and 16) and as diluted by ten-fold (Row I,
lanes 3, 4, 11, 12, 19 and 20; Row II, lanes 3, 4, 11, and 12)
using the extraction method herein (Row I, lanes 1-4, 9-12 and
17-20; Row II, lanes 1-4 and 9-12) the extraction method described
by Guidet (Guidet, Nucleic Acids Res. 21:4153-4154, 1994) with the
PCR mix from the method herein (Row I, lanes 5, 6, 13, 14, 21, and
22; Row II, lanes 5, 6, 13, and 14) and the extraction method
described by Guidet with the PCR setup as described by Guidet (Row
I, lanes 7, 8, 15, 16, 23, and 24; Row II, lanes 7, 8, 15, and
16)
[0028] FIG. 5 illustrates in single samples, agarose gel
electrophoresis of PCR amplification products obtained from
extraction of mouse tails in which various components were absent
or present showing the effect of absence (lanes 1-4 and 9-12) and
presence (lanes 5-8 and 13-16) of proteinase K, the effects of
water (lanes 1 and 5), EDTA (lanes 2 and 6), Tris-HCl (lanes 3 and
7), Tris-HCl and EDTA (lanes 4 and 8), KCl (lanes 9 and 13), KCl
and EDTA (lanes 10 and 14), KCl and Tris-HCl (lanes 11 and 15), and
Extraction Solution (E7526)(lanes 12 and 16).
[0029] FIG. 6 illustrates in duplicate samples, agarose gel
electrophoresis of PCR amplification products obtained from
extraction of mouse tails in which various components were absent
or present showing the effect of Tris-HCl and proteinase K (lanes 1
and 2); EDTA, Tris-HCl and proteinase K (lanes 3 and 4), KCl,
Tris-HCl and proteinase K (lanes 8 and 9) and Extraction Solution
(lanes 10 and 11) with lanes 5 and 12 and lanes 6 and 13 showing
negative and positive controls, respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0030] In accordance with the present invention, it has been
discovered that nucleic acids can be efficiently extracted from
biological samples using an extraction composition which contains a
protease enzyme and which is buffered to an alkaline pH of 7.5 or
greater.
[0031] The protease enzyme serves to produce at least a partial
tissue break down such that nucleic acids are released. As such,
any substance, which serves the same function, can be used in the
extraction composition. The terms proteases, proteinases and
peptidases are used interchangeably herein and refer to the group
of enzymes that catalyze the hydrolysis of covalent peptidic bonds.
Protease enzymes are well known in the art and serine proteases are
one group of proteases. Six clans of serine proteases have been
identified, the two largest of which are the chymotrypsin-like and
the subtilisin-like clans (Rawlings et al. Methods Enzymol
244:19-61,1994; Barrett et al, Arch. Biochem. Biophys. 318:247-250,
1995). A large number of subtilases are known (Siezen et al,
Protein Science 6:501-523, 1997; Siezen et al., Protein Engineering
4:719-737, 1991). Some of the subtilases which have been
extensively studied include those obtained from various species of
Bacillus including subtilisin DY, subtilisin Carlsberg, subtilisin
BPN' (also called nagarse), mesentericopeptidase as well as
proteinase K which is obtained from Tritirachium album Limber and
thermitase which is obtained from Thermoactinomyces vulgaris. In
certain embodiments of the present invention, proteinase K is
preferred as a protease enzyme. Other protease enzymes, however,
can also be used in certain embodiments, such as, for example,
nagarse (for characterization of nagarse see Masaharu et al, J.
Clin. Lab. Anal. 14:97-100, 2000). The protease enzyme can thus be
any of a number of proteases that produce at least a partial break
down of the biological sample such that nucleic acids are released
within a short period of, for example about 60 minutes or less.
[0032] Proteinase K is commercially available as a lyophilized
powder or in aqueous solutions or suspension (Sigma-Aldrich, St.
Louis, Mo.). Activity is generally referenced in Units, which are
defined as the activity necessary to release folin-positive amino
acids and peptides corresponding to 1 .mu.M of tyrosine per minute
using hemoglobin as substrate in 30 mM Tris-HCl, pH 7.5 at
37.degree. C. The typical activity of proteinase K preparations is
about 30 Units/mg. The concentrations of proteinase K in the
extraction composition is preferably at least about 25 Units/ml or
greater, at least about 50 Units/ml or greater or at least about
100 Units/ml or greater. Maximal concentrations can be up to about
200 Units/ml, up to about 400 Units/ml, or up to about 800
Units/ml, the upper limit of concentration being effected by
solubility of the enzyme as well as the ability to sufficiently
denature the enzyme at the end of extraction so that there will be
no decrease in DNA polymerase activity during subsequent PCR
amplification of the extracted nucleic acid.
[0033] The extraction composition of the present invention is
typically an aqueous solution, however, in certain embodiments, the
extraction composition can be in the form of an aqueous dispersion,
suspension, emulsion or the like. The aqueous portion of the
extraction composition is at an alkaline pH of about 7.5 or
greater. Preferably, the extraction composition pH is at least
about 8.0 or greater, at least about 8.5 or greater, at least about
9.0 or greater and in a range of from about 7.5 to about 10, in a
range of from about 8.0 to about 9.0, or in a range of from about
8.0 to about 8.5.
[0034] Any of a number of buffering agents can be used in the
extraction composition the selection and use of which can be
readily performed by the skilled artisan (see for example Beynon
and Esterby, Buffer Solutions: The basics, BIOS Scientific
Publishers, Oxford, 1996). Preferably, the buffer is prepared from
a substance have a pKa value from one unit less than to one unit
greater than the desired pH. Thus, for example, a pH 8.0 buffer can
be prepared using a substance having a pKa from about 7.0 to 9.0.
Such buffer substances include, for example, AMPD
(2-Amino-2-methyl-1,3-propanediol), bicarbonate (Sodium hydrogen
carbonate), Bicine (N,N-Bis-(2-hydroxyethyl)-glycine),
Bis-Tris-Propane
(1,3-Bis-[tris-(hydroxymethyl)-methylamino]-propane),),
DIPSO(N,N-Bis-(2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic
acid), glycylglycine HEPES
(4-(2-Hydroxyethyl)-piperazine-1-ethanesulfonic aicd), HEPPS
(4-(2-Hydroxyethyl)-piperazine-1-propanesulfonic acid), HEPPSO
(4-(2-Hydroxyethyl)-piperazine-1 (2-hydroxy)-propanesulfonic acid),
phosphate, POPSO (piperazine-1,4-bis-(2-hydroxypropanesulfonic
acid)), TAPS(N-[Tris-(hydroxymethyl)-methyl]-3-aminopropanesulfonic
acid),
TAPSO(N-[Tris-(hydroxymethyl)-methyl]-3-amino-2-hydroxypropanesulf-
onic acid) TES
(N-[Tris-(hydroxymethyl)-methyl]-2-aminoethanesulfonic acid),
Tricine (N-[Tris-(hydroxymethyl)-methyl]-glycine), Tris
(Tris-(hydroxymethyl)-aminomethane) and triethanolamine. Preferred
as a buffer component is Tris-HCl, which is typically prepared by
dissolving Tris base in water and adding HCl to achieve the desired
pH. Buffers from other substances as those identified above, can be
readily prepared by the skilled artisan by methods well known in
the art.
[0035] As noted above, surfactants are preferably, not present in
the extraction compositions of certain embodiments. Both ionic and
non-ionic surfactants are absent from such embodiments. By a
surfactant not being present of the composition lacking a
surfactant or the surfactant being absent from the extraction
composition, it is meant that any concentration of the surfactant
is negligible as would be readily understood by the skilled
artisan. Such negligible amounts present are considered to be less
than about 0.5% (w/w), preferably, less than about 0.2% (w/w),
preferably, less than about 0.1% (w/w).
[0036] Ca.sup.2+ chelators are also, preferably, not present or at
low concentrations in the extraction compositions of certain
embodiments of the present invention. Thus, the Ca.sup.2+ chelator,
EDTA, if present in the extraction composition, is at a
concentration of about 100 mM or less, preferably at a
concentration of about 50 mM or less, preferably at a concentration
of about 20 mM or less, preferably at a concentration of about 10
mM or less.
[0037] The extraction composition can be in two or more
compositions which are mixed together to form the extraction
composition. In one embodiment, the components can be in two
solutions, an extraction solution and a sample preparation
solution. The extraction solution can contain a buffer component,
for example Tris-HCl. The Tris-HCl can be at a concentration of
from about 10 mM to about 1000 mM, preferably, from about 20 mM to
about 300 mM, preferably, from about 50 mM to about 200 mM and,
preferably, about 100 mM. Along with the Tris-HCl, the extraction
solution can contain a salt such as, for example KCl at a
concentration of from about 0.1 to about 0.4 mM, preferably 0.2 to
about 0.3 mM, and preferably about 0.25 mM. EDTA can also be
present in the solution, for example at a concentration of from
about 1 to about 40 mM, preferably, from about 5 to about 20 mM and
preferably about 10 mM.
[0038] A sample preparation solution can contain the protease
enzyme, such as, for example, proteinase K at a concentration of
from about 100 to about 1000 Units/ml, preferably from about 200 to
about 800 Units/ml, and preferably about 600 Units/ml. The second
solution can also contain a buffer component such as, for example
Tris-HCl, a water soluble calcium compound such as, for example
calcium acetate or calcium chloride, and stabilizers and/or
solubilizers for the proteinase K, such as, for example, glycerol.
The extraction solution and sample preparation solution are
contacted with the biological sample and the mixture incubated.
[0039] Incubation is, preferably, for about 60 minutes or less,
preferably, about 45 minutes, preferably, about 30 minutes,
preferably about 20 minutes, preferably about 10 minutes,
preferably about 5 minutes or less. Typically, incubation is for
about 10 minutes.
[0040] The temperature for incubation will depend upon the
biological sample being extracted. For example, for mammalian
tissues, extraction is preferably, at room temperature, preferably
no more than about 25.degree. C., preferably no more than about
22.degree. C. and, in particular, about 20.degree. C., about
22.degree. C., or about 25.degree. C. For other biological samples,
for example, plant tissues, extraction temperatures can be from
about 37.degree. C. to about 60.degree. C., preferably from about
50.degree. C. to about 55.degree. C. The incubation temperature is
maintained below the temperature at which the protease enzyme
becomes denatured, for example below about 65.degree. C. for
proteinase K.
[0041] Nucleic acids can be extracted from any of a wide variety of
biological samples using the extraction composition of the present
invention. As discussed above, the samples can be obtained from
animals such as, mammals including humans, fish, birds, insects
such as drosophila, nematodes such as C. elegans and the like;
plants including moss, ferns, trees, bushes, flowering plants and
the like; fungi including mushrooms, molds, yeast and the like;
protista including amoeba, paramecium, algae, seaweed, diatoms and
the like; or monera including species of bacteria or cyanobacteria.
Biological samples from plants can include leaf samples, seed
samples or samples from other plant tissues. Samples from animals
can include tissue and cell samples from mammals and, in
particular, from humans. Mammalian tissue samples can be obtained
by any method known in the art such as for example from mouse ear
punch or from mouse tail portion. The samples can be prepared such
as by lyophilization or drying by other methods. In certain
embodiments such as, for example, for the extraction of certain
plant tissues, samples may be minced or ground to facilitate the
extraction process.
[0042] One particular method can involve pipetting the extraction
solution and the sample preparation solution into a microcentrifuge
tube or well of a multiwell plate. For mammalian tissue extraction
about 100 .mu.l of extraction solution and about 25 .mu.l of sample
preparation solution are added and then mixed. A mammalian tissue
sample of about 2 to 10 mg of tissue is placed in the solution. The
sample is then incubated at room temperature for 10 minutes. After
the incubation period, the sample and solution are heated to
95.degree. C. for about 3 minutes. This stops the extraction
process and denatures the proteinase K, which will allow for
subsequent PCR amplification of the extracted DNA.
[0043] Optionally, a neutralization solution, containing albumin
can then be added to the mixture. The extracted DNA can then be
used in further methods as are known in the art such as, for
example, in PCR, sequencing and the like.
[0044] Kits of the present invention can contain components of the
extraction composition packaged separately or in one or more
mixtures of any combination to components. Thus, a kit can contain,
for example, an extraction solution and a sample preparation
solution each packaged in a separate container. Optionally, other
components can be included in the kit such as, for example, a
neutralization solution. In addition, the kits can also contain
components for performing PCR amplifications such as are
commercially available in kit form (Sigma-Aldrich, St. Louis,
Mo.).
[0045] Illustrative examples are described below. Unless otherwise
indicated, all materials used in the examples were obtained from
Sigma-Aldrich Corporation, St. Louis, Mo.
EXAMPLE 1
[0046] This example illustrates the extraction of DNA from
mammalian tissue samples, Drosophila samples, C. elegans samples
and from plant seed samples.
[0047] The extraction method uses an extraction solution, a tissue
preparation solution and a neutralization solution. The Extraction
Solution (referenced herein as E7526) contains 0.25 M KCl, 0.01 M
EDTA and 0.1 M Tris-HCl, at a pH of 9.5. The Sample Preparation
Solution contains Proteainase K from Tritirachium Album Libium in
an aqueous solution (.about.800 Units/mL) containing 40% glycerol,
1 mM calcium acetate and 10 mM Tris-HCl at pH 7.5. The
Neutralization Solution B contains 3% bovine albumin and 10 ppm
Kathon.
[0048] The following procedures are carried out for extraction of
DNA from mammalian, drosophila or C. elegans samples at room
temperature and from seed samples at 55.degree. C.
[0049] A. DNA Extraction from Mouse Tails, Animal Tissues, Hair,
Saliva, Drosophila or C. Elegans.
[0050] 100 .mu.l of Extraction Solution is placed into a
microcentrifuge tube or well of a multiwell plate. 25 .mu.l of
Sample Preparation Solution are added to the tube or well and the
two solutions are mixed by pipetting up and down. If several
extractions are to be performed, sufficient volumes of Extraction
and Sample Preparation Solutions may be pre-mixed in a ratio of 4:1
up to 2 hours before use.
[0051] Sample preparation and addition to the prepared solution is
as follows.
[0052] (i) For Fresh or Frozen Mouse Tails: Scissors and forceps
are rinsed in 70% ethanol prior to use and between different
samples. A 0.5 to 1 cm piece of mouse tail tip is placed into the
solution, cut end down. Thorough mixing is achieved by vortexing or
pipetting. Care is taken to ensure that the mouse tail is in
solution. For fresh mouse tails, extractions are within 30 minutes
of the tail being snipped.
[0053] (ii) For Animal tissues: Scissors or scalpel and forceps are
rinsed in 70% ethanol prior to use and between different samples. A
2 to 10 mg piece of tissue is placed into the solution. Thorough
mixing is achieved by vortexing or pipetting. Care is taken to
ensure that the tissue is in the solution.
[0054] (iii) For Hair Shafts: Scissors and forceps are rinsed in
70% ethanol prior to use and between different samples. Excess hair
shaft is trimmed off leaving the root and the sample is placed into
solution, root end down.
[0055] (iv) For Saliva: 10 .mu.l of saliva are pipetted into the
solution. Thorough mixing is achieved by vortexing or
pipetting.
[0056] (v) For Saliva Dried on Card: 50 .mu.l of saliva are
pipetted onto collection card and card is allowed to dry. Punch is
rinsed in 70% ethanol prior to use and between different samples.
Disk (preferably 1/8 inch) is punched out of card from area with
dried saliva sample. Disk is placed into the solution. Tube or
plate is tapped on hard surface to insure disk is in solution for
incubation period.
[0057] (vi) For Drosophila: 1 to 10 anesthetized flies are crushed
into the solution. Thorough mixing is achieved by vortexing or
pipetting. Care is taken to ensure that the fly material is in the
solution.
[0058] (vii) For C. elegans: 1 to 10 worms are added to the
solution. Thorough mixing is achieved by vortexing or pipetting.
Care is taken to ensure that the C. elegans material is in the
solution.
[0059] Following addition of the sample to solution the sample is
incubated at room temperature for 10 minutes to extract the DNA.
For less than 10 Drosophila or C. Elegans, release of DNA may be
enhanced by performing this incubation at 55.degree. C.
[0060] Following the incubation for extraction, the sample is
heated to 95.degree. C. for 3 minutes to denature the proteinase
K.
[0061] 100 .mu.l of Neutralization Solution B is then added to the
sample and mixing is achieved by vortexing.
[0062] The neutralized tissue extract is used immediately in PCR or
stored at 4.degree. C. for PCR at a later time. Extracts may be
stored at 4.degree. C. for up to 6 months; however, it is desirable
to remove the undigested tissue before long term storage.
[0063] B. DNA Extraction for Buccal Swabs.
[0064] First, buccal cells are collected on swab and allowed to
dry. Drying time is approximately 10 to 15 minutes.
[0065] 200 .mu.l of Extraction Solution is pipetted into a
microcentrifuge tube. 25 .mu.l of Sample Preparation Solution is
added to the tube and pipetted up and down to mix. If several
extractions are to be performed, sufficient volumes of Extraction
and Sample Preparation Solution may be pre-mixed in a ratio of 8:1
up to 2 hours before use.
[0066] The dried buccal swab is placed into the solution and
incubated at room temperature for 1 minute.
[0067] The swab is twirled in solution 10 times and then excess
solution is removed from the swab into the tube by twirling the
swab firmly against the side of the tube. The swab is then
discarded. The tube is then closed and vortexed briefly.
[0068] The sample is then incubated at room temperature for 10
minutes to extract DNA.
[0069] Following the incubation for extraction, the sample is
heated to 95.degree. C. for 3 minutes to denature the proteinase
K.
[0070] 200 .mu.l of Neutralization Solution B is then added to the
sample and mixing is achieved by vortexing.
[0071] The neutralized tissue extract is used immediately in PCR or
stored at 4.degree. C. for PCR at a later time. Extracts may be
stored at 4.degree. C. for up to 6 months without notable loss in
most cases.
[0072] C. DNA Extraction from Plant Seeds:
[0073] First, seed is ground by hand using plastic pestle or by use
of a beadmill (2000 Geno/Grinder, Spex Certiprep, Inc., Metuchen,
N.J.) in a volume of water of from 100 to 800 .mu.l depending upon
the volume of seed used. If grinding is to be done manually with a
plastic pestle, the seed is soaked in water at 55.degree. C. for 1
hour before grinding.
[0074] 45 .mu.l of Extraction Solution and 5 .mu.l of Sample
Preparation Solution are mixed together in a microcentrifuge tube
or well of a multiwell plate. 5 .mu.l of ground seed is placed into
the mixture.
[0075] The mixture is then heated at 55.degree. C. for 10 minutes
to extract DNA.
[0076] The Extract is then heated at 95.degree. C. for 3 minutes to
denature the proteinase K.
[0077] 50 .mu.l of Neutralization Solution B is then added to the
extract and the solutions are then mixed.
[0078] The neutralized seed extract is used immediately in PCR or
stored at 4.degree. C. for PCR at a later time. Extracts may be
stored at 4.degree. C. for up to 6 months without notable loss in
most cases.
[0079] PCR can be directly performed on sample extracts obtained
from the procedures above using Extract-N-Amp PCR Ready Mix or
REDExtract-N-Amp PCR Ready Mix (Sigma-Aldrich, St. Louis, Mo.).
EXAMPLE 2
[0080] This example illustrates the extraction of DNA by the
present method using tissues from mouse ear punches and tail snips
and compares extraction with earlier methods (Chen et al., 1990,
supra; Ren et al., 2001, supra).
[0081] All materials were obtained from Sigma-Aldrich (St. Louis,
Mo.) unless otherwise noted. PCR primers were obtained from
SigmaGenosys (The Woodlands, Tex.).
[0082] Tissue samples obtained were mouse ear punches (1/8 inch
disc) and mouse tails (0.5 cm tail tip and next 0.5 cm section up
from that tip). The tail pieces were either fresh or frozen from a
previous tissue collection which had been stored at -20.degree. C.
Each extraction was setup by placing 1 mouse ear punch or mouse
tail into a 1.5 ml microcentrifuge tube. Extraction using the
present method was performed as described in Example 1. Comparative
extractions were performed using the methods of Chen et al. (1990,
supra) and Ren et al. (2001, supra).
[0083] Two tubes were prepared for the extractions of each
condition. In the set of tubes using the instant method, 100 .mu.l
of Extraction Solution and 25 .mu.l Sample Preparation Solution
were pipetted and mixed together in each tube, which already
contained either an mouse ear punch or mouse tail. For samples
using the procedures of earlier methods, the methods outlined by
Ren et al. in Table 1 of that reference were used (see Ren et al,
2001, supra). Briefly, for the Chen et al. method a solution of 20
.mu.l of 50 mM Tris-HCl at pH 8.0, 20 mM NaCl, 1 mM EDTA, 1% SDS
and 1 .mu.g/.mu.l proteinase K was used and for the Ren et al.
method, 5 .mu.l of proteinase K solution (3 .mu.g/.mu.l) in sterile
water was used. The Ren et al. mixture was incubated at room
temperature or 55.degree. C. for 10, 20 & 30 minutes to extract
the DNA. The Chen et al. method was performed as described earlier
(Chen et al., 1990, supra).
[0084] Following incubation to extract the DNA, 100 .mu.l of
Neutralization Solution B was added. The extractions were then
vortexed to ensure thorough mixing of reagents.
[0085] PCR Amplifications: For standard PCR in 8 tube strips, 10
.mu.l of REDExtract-N-amp PCR.TM. Ready mix (Sigma-Aldrich, St.
Louis, Mo.) for Extract-N-Amp extracts or 12.5 .mu.l of JumpStart
REDTaq ReadyMix PCR Reaction Mix (Sigma-Aldrich, St. Louis, Mo.)
for Ren et al. and Chen et al. methods, both forward and reverse
primers at 0.4 .mu.M., and aliquots of the extracts, 4 .mu.l of the
extract using the present method or 1 .mu.l of the extract using
the Chen et al. or Ren et al. methods were combined in a final
volume of 20 .mu.l. PCR Primers for mouse ear punches & mouse
tails produced an 1181 bp amplicon from the Mouse Interleukin One
Beta (IL-1.beta.) gene (5'-TCTGGGGTTGATGTAGGA-3' [SEQ ID NO:1] and
5'-GGGCTGGAAAAATGGTC-3' [SEQ ID NO:2]). Reactions were assembled at
room temperature and placed in a GeneAmp PCR System 9700 (Perkin
Elmer/Applied Biosystems, Foster City, Calif.). PCR conditions for
mouse ear punch & mouse tail reactions were: 94.degree. C. for
3 min, then 35 cycles of 94.degree. C. for 45 sec, 58.degree. C.
for 60 sec, 72.degree. C. for 120 sec, then 72.degree. C. 7
min.
[0086] The completed reactions were analyzed by gel electrophoresis
(5 .mu.l/PCR reaction) on a 1.times.TBE 2% Seakem LE Plus
10.times.15 Latitude Precast agarose gel with 0.5 .mu.g/ml Ethidium
Bromide added from Biowhittaker Molecular Applications (Rockland,
Me.) (cat. No. 57266-lot No. LT0826). The marker used on the gel
was P9577 PCR Marker (Sigma-Aldrich, St. Louis, Mo.).
[0087] For quantitative PCR, which was done on an ABI PRISM.TM.
7700 Sequence Detection System (Perkin Elmer/Applied Biosystems,
Foster City, Calif.), a 96 well plate was prepared using 10 .mu.l
of REDExtract-N-AMP.TM. PCR Ready mix (R4775) for Extract-N-Amp
extracts or 12.5 .mu.l of JumpStart REDTaq ReadyMix PCR Reaction
Mix (P0982) for earlier methods, both forward and reverse primers
for IL-1.beta. at 0.4 .mu.M, SYBR.RTM.Green I dye (40 ppm
final)(Sigma-Aldrich), 0.6 .mu.l of Reference Dye for Quantitative
PCR 100X (Sigma-Aldrich), and aliquots of the extract, 4 .mu.l of
the extract using the present method or 1 .mu.l of the extract
using the earlier methods were combined in a final volume of 20
.mu.l. PCR Primers for mouse ear punches & mouse tails produced
an 1181 bp amplicon from the Mouse Interleukin One Beta (IL-1) gene
(5'-TCTGGGGTTGATGTAGGA-3' (SEQ ID NO: 1) and
5'-GGGCTGGAAAAATGGTC-3') (SEQ ID NO: 2). Reactions were assembled
at room temperature in a laminar flow hood. PCR conditions for
mouse ear punch & mouse tail reactions: 94.degree. C. for 3
min, then 40 cycles of 94.degree. C. for 30 sec, 58.degree. C. for
30 sec, 72.degree. C. for 90 sec.
[0088] Results are shown in FIG. 1. Duplicate samples were run side
by side. Row I shows the results of extraction at room temperature
for 10 minutes (lanes 1 and 2), 20 minutes (lanes 3 and 4) and for
30 minutes (lanes 5 and 6). Extraction at 55.degree. C. is shown
for 10 minutes (lanes 7 and 8), 20 minutes (lanes 9 and 10) and 30
minutes (lanes 11 and 12). As shown in the figure, a robust
extraction of DNA was achieved with the present method under all
conditions tested.
[0089] Row II illustrates the effect of the effect of not heating
to 95.degree. C. for 3 minutes by the lack of PCR amplification
product (lanes 1 and 2).
[0090] Row II lanes 3 and 4 show PCR amplification product in
absence of addition of the Neutralization Solution B.
[0091] Row II lanes 5-12 show PCR amplification product from fresh
mouse tails illustrating a robust DNA extraction for this tissue as
well.
[0092] Row V shows the results using the method of Ren et al. and
Row VI shows the results using the method of Chen et al. As shown
in Row V, the method of Ren et al. produced very little PCR
amplification product following incubation at room temperature for
10 minutes (lanes 1 and 2) and 20 minutes (lanes 3 and 4)
suggesting that very little DNA was extracted from the sample. Some
possible extraction after 30 minutes incubation was suggested from
the corresponding amplification product (lanes 5 and 6) in at least
one of the duplicate runs. No more than slight improvement in the
extraction of DNA was suggested from the amplification product
produced following incubation at 55.degree. C. for 10 minutes
(lanes 7 and 8), 20 minutes (lanes 9 and 10) or 30 minutes (lanes
11 and 12).
[0093] The extraction method of Chen et al. at 55.degree. C.
produced a modest amount amplification product following incubation
for 30 minutes (lanes 3 and 4). Lanes 1 and 2 show the results of
extraction using a modification of the method of Ren et.al. in
which Neutralization Solution B is added to the preparation.
[0094] Thus, the present method showed substantially greater
extraction of DNA following incubation at room temperature for 10
minutes than was apparent with either the method of Chen et al. or
the method of Ren et al. even upon incubation at 55.degree. C. by
the earlier methods.
EXAMPLE 3
[0095] This example illustrates the effect of pH and KCl
concentration on extraction of DNA from tissue samples.
[0096] Frozen mouse tail cuttings were extracted and amplified as
described in Example 2. Results are shown in FIG. 1, Rows III and
IV.
[0097] PCR amplification following extraction at pH adjusted to pH
10.5 (lanes 1 and 2), adjusted to pH 9.5 (lanes 3 and 4), adjusted
to pH 8.5 (lanes 5 and 6) and adjusted to pH 7.5 (lanes 7 and 8)
with negative and positive controls in lanes 9 and 10. As can be
seen in the figure, little difference in the extraction efficiency
was seen from pH 8.5 to pH 10.5. At pH 7.5 one of the two
preparations did not show PCR amplification product which suggests
that extraction efficiency might begin to decrease as pH decreases
to 7.5 and below.
[0098] KCl concentration did not show any effect on extraction
efficiency at 200 mM KCl (lanes 1 and 2), at 100 mM KCl (lanes 5
and 6), at 50 mM KCl (lanes 9 and 10) or at 0 mM KCl (lanes 13 and
14). Lanes 3, 7, 11 and 15 and 4, 8, 12 and 16 are negative and
positive controls, respectively.
EXAMPLE 4
[0099] This example illustrates the extraction of seeds in absence
and presence of proteinase K.
[0100] The effect of the absence and presence of proteinase K on
extraction efficiency was tested using Soybean, Sorghum and
Arabidopsis seeds.
[0101] All materials were obtained from Sigma-Aldrich (St. Louis,
Mo.) unless otherwise noted. All PCR primers were obtained from
SigmaGenosys (The Woodlands, Tex.). The primers used were as
follows: TABLE-US-00001 Soybean 0513 bp: Forward- 5'-TTG GGA ACC
TCT ACA TTC-3' (SEQ ID NO: 3) Reverse- 5'-CGG GCA GAA ACT AAA TC-3'
(SEQ ID NO: 4) Sorghum 0503 bp: Forward- 5'-ATT GGC GAT GGC AAG
T-3' (SEQ ID NO: 5) Reverse- 5'-GAA TCT GCT GGG GTT CAG-3' (SEQ ID
NO: 6) Arabidopsis 2110 bp: Forward- 5'-ACT CTA CCT CGC CAC CAT-3'
(SEQ ID NO: 7) Reverse- 5'-GCC CAC TCT GCT TCA AAC-3' (SEQ ID NO:
8)
[0102] Seeds were placed into wells of a 96 square well block. 1
seed was placed into each of two separate wells for Soybean and
Sorghum. Around 50 Arabidopsis seeds were placed into each of two
separate wells. A single Stainless Steel grinding ball was placed
into each seed containing well. 800 .mu.l of water was pipetted
into the wells containing soybean seeds. 200 .mu.l of water was
pipetted into the wells containing sorghum seeds. 100 .mu.l of
water was pipetted into the wells containing arabidopsis seeds. The
plate was sealed using a rubber sealing mat. The seeds were ground
using a 2000 Geno/Grinder (Spex Certiprep, Inc (Metuchen, N.J.) for
10 minutes at 1500 strokes/minute.
[0103] Two sets of tubes were prepared for the extractions. The
first set was labeled for the sample type and that the extraction
contained proteinase K. The second set of tubes was labeled with
the sample type and that no proteinase K was used in the
extraction. In the first set of tubes 45 .mu.l of Extraction
Solution and 5 .mu.l of Sample Preparation Solution, which contains
Proteinase K was pipetted and mixed together. In the second set of
tubes 50 .mu.l of Extraction Solution was pipetted in. To each
extraction tube 5 .mu.l of the appropriate ground seed material was
pipetted into the extraction mixture, and pipetted up and down to
mix. Each mixture was incubated at 55.degree. C. for 10 minutes to
extract the DNA. Each extract was then incubated at 95.degree. C.
for 3 minutes to stop the extraction. To each stopped extraction 50
.mu.l of Neutralization Solution B was pipetted in. The extractions
were then vortexed to mix.
[0104] For standard PCR amplification, 10 .mu.l of REDExtract-N-amp
PCR.TM. mix (Sigma-Aldrich), both forward and reverse primers at
0.4 .mu.M., and 4 .mu.l of extract in a final volume of 20 .mu.l.
PCR Primers for Soybean were 513 bp, Sorghum, 503 bp, and
Arabidopsis 2110 bp. Reactions were assembled at room temperature.
PCR conditions for Soybean and Sorghum PCRs: 94.degree. C. for 3
min, then 35 cycles of 94.degree. C. for 30 sec, 44.degree. C. for
30 sec, 68.degree. C. for 45 sec, and 68.degree. C. 7 min. For
Arabidopsis PCR: 94.degree. C. for 3 min, then 35 cycles of
94.degree. C. for 30 sec, 49.degree. C. for 30 sec, 68.degree. C.
for 2 min 15 sec, and 68.degree. C. 7 min. 5 .mu.l of PCR products
were analyzed by gel electrophoresis on a 1% agarose gel in
TBE.
[0105] As is seen in FIG. 2, proteinase K improves extraction of
DNA from the seeds. The intensity of the bands from the soybean
preps are brighter for the samples that included proteinase K in
the extraction. This can be seen in both the PCR's on the neat
extract and the PCR's on the 1:10 dilutions of the neat extract.
There does not seem to be any effect on the sorghum preps whether
or not proteinase K is included. A very large difference is seen in
the PCR's of the Arabidopsis preps (FIG. 3). It is very apparent
from FIG. 3 that the samples that did not have proteinase K in the
extraction did not perform as well as those that had proteinase K
in the extraction.
[0106] Thus, proteinase K improved extraction of DNA from soybean
and Arabidopsis seeds, but it did not appear to improve extraction
from the sorghum seeds tested.
EXAMPLE 5
[0107] This example compares the extraction efficiency of the
method here and that reported earlier (Guidet, Nucleic Acids Res.
21:4153-4154, 1994).
[0108] Extractions were performed using seeds from soybean,
sorghum, canola, wheat, and Arabidopsis. All materials were
obtained from Sigma-Aldrich (St. Louis, Mo.) unless otherwise
noted. All PCR primers were obtained from SigmaGenosys (The
Woodlands, Tex.). Primers used for Soybean, Sorghum and Arabidopsis
were as described in Example 4. Primers used for Canola and Wheat
were as follows: TABLE-US-00002 Canola 0762 bp: Forward- 5'-CTT TTC
CTC CCG CAC CTT-3' (SEQ ID NO: 9) Reverse- 5'-GCC GCC GAC TTG ATT
TC-3' SEQ ID NO; 10) Wheat 0500 bp: Forward- 5'-AGT GGC GAG AGG AGG
TTC-3' (SEQ ID NO: 11) Reverse- 5'-TGG TTG GCG ATT GTG C-3' (SEQ ID
NO: 12)
[0109] Seeds were placed into wells of a 96 square well block. 1
seed was placed into 2 separate wells for Soybean, Sorghum, Canola,
and Wheat. Around 50 Arabidopsis seeds were placed into 2 separate
wells. A single Stainless Steel grinding ball was placed into each
seed containing well. 800 .mu.l of water is pipetted into the wells
containing soybean seeds. 200 .mu.l of water is pipetted into the
wells containing sorghum, canola, and wheat seeds. 100 .mu.l of
water is pipetted into the wells containing arabidopsis seeds. The
plate is sealed using a rubber sealing mat. The seeds are ground
using a 2000 Geno/Grinder (Spex Certiprep, Inc (Metuchen, N.J.) for
10 minutes at 1500 strokes/minute.
[0110] For DNA extractions using the methods herein, two sets of
tubes were prepared. The first set was labeled for the sample type
and with method and the second set of tubes was labeled with the
sample type, method and dilution for the 1:10 dilutions of the
first set. In the first set of tubes 45 .mu.l of Extraction
Solution and 5 .mu.l Sample Preparation Solution containing
proteinase K were pipetted and mixed together. Into the second set
of tubes 18 .mu.l of a 50:50 mixture of Extraction Solution and
Neutralization Solution B was pipetted. To each extraction tube 5
.mu.l of the appropriate ground seed material was pipetted into the
extraction mixture, and pipetted up and down to mix. Each mixture
was incubated at 55.degree. C. for 10 minutes to extract the DNA.
Each extract was then incubated at 95.degree. C. for 3 minutes to
stop the extraction and denature the proteinase K. 50 .mu.l of
Neutralization Solution B was pipetted into each of the extraction
mixtures. The extractions were then vortexed to mix. 2 .mu.l of the
neutralized extract was pipetted into the second set of tubes and
vortexed to mix.
[0111] For DNA extractions using the method of Guidet (1994, supra)
a solution was prepared containing 10 mM Tris-HCl, ph 8.0, 0.45 M
EDTA, 1% lauryl sarkosyl, and 1 mg/ml Proteinase K as described in
the paper. 100 .mu.l of the Extraction Buffer were pipetted into a
set of tubes labeled with the sample type and with a G to signify
that they are the preparations of the method reported earlier. 5
.mu.l of the ground seed material was pipetted into the correct
tubes. The entire set of samples was then incubated at 50.degree.
C. for 1 hour. After the incubation the extracts were diluted with
150 .mu.l of milliQ H.sub.2O. 20 .mu.l of the diluted extract was
pipetted into tubes containing 80 .mu.l of milliQ H.sub.2O with 10
.mu.g of RNase A. Samples were then incubated at 100.degree. C. for
5 minutes. After the samples were allowed to cool to room
temperature, 10 .mu.l were pipetted into a new tube containing 240
.mu.l of milliQ H.sub.2O. The samples were vortexed to mix
thoroughly.
[0112] PCR amplifications of extractions obtained using the methods
herein, were performed using 10 .mu.l of REDExtract-N-Amp PCR.TM.
mix (Sigma-Aldrich, St. Louis, Mo.), both forward and reverse
primers at 0.4 .mu.M., and 4 .mu.l of extract in a final volume of
20 .mu.l. For the samples extracted using the method reported
earlier by Guidet, the PCR was performed using 10 .mu.l of
REDExtract-N-Amp PCR.TM. mix (Sigma-Aldrich, St. Louis, Mo.), both
forward and reverse primers at 0.4 .mu.M, 4 .mu.l of a 50:50
Extraction Solution and Neutralization Solution B mixture, and 5
.mu.l of extract obtained using the method of Guidet, in a final
volume of 20 PI. For the samples set up according to the method of
Guidet, 1 unit of Jumpstart taq (Sigma-Aldrich, St. Louis, Mo.),
0.1 mM of each dNTP (Sigma-Aldrich, St. Louis, Mo.),1X of Taq
polymerase buffer (Sigma-Aldrich, St. Louis, Mo.), 0.4 .mu.M of
each primer and 5 .mu.l of extract obtained using the method of
Guidet in a final volume of 20 .mu.l. PCR Primers for Soybean were
513 bp, sorghum, 503 bp, canola 762 bp, wheat 500 bp and
Arabidopsis 2110 bp. Reactions were assembled at room temperature.
PCR conditions for soybean and sorghum PCRs: 94.degree. C. for 3
min, then 35 cycles of 94.degree. C. for 30 sec, 44.degree. C. for
30 sec, 68.degree. C. for 45 sec, and 68.degree. C. 7 min. For
canola and wheat PCRs: 94.degree. C. for 3 min, then 35 cycles of
94.degree. C. for 30 sec, 55.degree. C. for 30 sec, 68.degree. C.
for 1 min, and 68.degree. C. 7 min. For Arabidopsis PCR: 94.degree.
C. for 3 min, then 35 cycles of 94.degree. C. for 30 sec,
49.degree. C. for 30 sec, 68.degree. C. for 2 min 15 sec, and
68.degree. C. 7 min. 5 .mu.l of all red PCR product and 6 .mu.l of
the clear product, with 4 .mu.l of loading buffer (G2526) added to
the entire PCR volume, were analyzed by gel electrophoresis on a 1%
agarose gel in TBE. The gel was run at 125 V for 1 hour.
[0113] As seen in FIG. 4, the extraction method herein produced
substantially more PCR amplification product from all the seeds
tested than was the method reported by Guidet. Furthermore, the
decreased time of 10 minutes required for the method herein was a
substantial shortening of the one hour extraction time required by
the earlier method of Guidet.
EXAMPLE 6
[0114] This example illustrates the effects of varying the
different components of the Extraction Solution and Sample
Preparation Solution on effectiveness of extraction.
[0115] Frozen mouse tails were used to test the individual
components of the extraction composition, which was made up of the
Extraction Solution and Sample Preparation Solution. A factorial
experimental design evaluated all possible combinations of the
components the extraction composition. Table 1 below shows the
combinations tested. TABLE-US-00003 TABLE 1 Test No. KCl EDTA
Tris-HCl Pro K 1 0 0 0 0 2 0 10 mM 0 0 3 0 0 100 mM 0 4 0 10 mM 100
mM 0 5 0 0 0 4 ug/ml 6 0 10 mM 0 4 ug/ml 7 0 0 100 mM 4 ug/ml 8 0
10 mm 100 mM 4 ug/ml 9 250 mM 0 0 0 10 250 mM 10 mM 0 0 11 250 mM 0
100 mM 0 12 250 mM 10 mM 100 mM 0 13 250 mM 0 0 4 ug/ml 14 250 mM
10 mM 0 4 ug/ml 15 250 mM 0 100 mM 4 ug/ml 16 250 mM 10 mM 100 mM 4
ug/ml
[0116] All materials were obtained from Sigma-Aldrich (St. Louis,
Mo.) unless otherwise noted. All PCR primers were obtained from
SigmaGenosys (The Woodlands, Tex.). All reagents were obtained from
Sigma-Aldrich (St. Louis, Mo.).
[0117] DNA extractions were performed as follows. Mouse tail snips
(0.5 cm tail tip) were collected earlier and kept frozen at
-20.degree. C. for up to several months before use. Each extraction
was setup by placing 1 mouse tail into a 1.5 ml microcentrifuge
tube. The method used was as described in Example 1.
[0118] One tube was prepared for the extraction of each condition.
In each tube 100 .mu.l of each extraction mixture and 25 .mu.l
Sample Preparation Solution or water was pipetted and mixed
together in each tube, which already contained a mouse tail. Each
mixture was incubated at room temperature, i.e. approximately
22.degree. C. for 10 minutes to extract the DNA. Each extraction
was then incubated for 3 minutes at 95.degree. C. To each
extraction mixture, 100 .mu.l of Neutralization Solution B was
added. The extractions were then vortexed to ensure thorough mixing
of reagents. The extracts were then ready for PCR
amplification.
[0119] For standard PCR in 8 tube strips, 10 .mu.l of
REDExtract-N-amp PCR.TM. Ready mix (Sigma-Aldrich, St. Louis, Mo.),
both forward and reverse primers at 0.4 .mu.M., and 4 .mu.l of
extract, in a final volume of 20 .mu.l. Two master mixes were
prepared because half of the extracts did not contain KCl, which is
essential for the proper performance of the kit PCR mixes, so extra
KCl was added to one mix and not the other. PCR Primers for mouse
tails produced an 1181 bp amplicon from the Mouse Interleukin One
Beta (IL-1.beta.) gene. The primers were as follows: forward
primer: 5'-TCTGGGGTTGATGTAGGA-3' (SEQ ID NO:13) and reverse primer:
5'-GGGCTGGAAAAATGGTC-3'). (SEQ ID NO:14) Reactions were assembled
at room temperature and placed in a GeneAmp PCR System 9700 (Perkin
Elmer/Applied Biosystems, Foster City, Calif.). PCR conditions for
mouse tail reactions: 94.degree. C. for 3 min, then 35 cycles of
94.degree. C. for 45 sec, 58.degree. C. for 60 sec, 72.degree. C.
for 120 sec, then 72.degree. C. 7 min. The completed reactions were
analyzed by gel electrophoresis (5 .mu.l/PCR reaction) on a
1.times.TBE 2% Seakem LE Plus 10.times.15 Latitude Precast agarose
gel with 0.5 .mu.g/ml Ethidium Bromide added from BioWhittaker
Molecular Applications (Rockland, Me.) (Cat. No. 57231-lot No.
LT0902). The gel was run in 0.5.times.TBE running buffer, which is
a dilution of Tris-Borate-EDTA Buffer 5.times. Concentrate (T6400).
Using a submarine gel electrophoresis rig (E1138) run at 125 volts
for 45 minutes. The marker used on the gel was P9577 PCR Marker
(bands of 2000, 1500, 1000, 750, 500, 300, 150 & 50 bp).
[0120] DNA positive controls were prepared from 1.2 cm of mouse
tail with GeneElute.TM. Mammalian Genomic DNA Kit (Sigma-Aldrich,
St. Louis, Mo.). The purified DNA was diluted to 57.5 ng/.mu.l, and
2-fold serial dilutions were prepared from the 57.7 ng/.mu.l
dilution until a dilution .about.12.5 ng/.mu.l was reached, then 1
.mu.l is used in the positive control for the PCR mixes. Negative
controls consist of 4 .mu.l of a 50/50 mixture of Extraction
Solution and Neutralization Solution B in the PCR reaction.
[0121] As seen in FIG. 5, the presence of proteinase K and the
Tris-HCl buffer improve extraction efficiency. Extraction Solution,
E7526 was as identified in Example 1.
[0122] The study was repeated to confirm the results using
duplicate samples. Table 2 below shows all combinations tested in
the follow-up study. TABLE-US-00004 TABLE 2 Test No. KCl EDTA
Tris-HCl Pro K 1 0 0 100 mM 4 ug/ml 2 0 10 mm 100 mM 4 ug/ml 3 250
mM 0 100 mM 4 ug/ml 4 250 mM 10 mM 100 mM 4 ug/ml
[0123] Materials and methods were as described above.
[0124] As seen in FIG. 6, the E7526 Extraction Solution as
identified in Example 1, worked well if not better than the other 3
combinations of components in the factorial study. The Tris-HCl
component and proteinase K component were present in all samples
and all showed excellent extraction. Taken together with the first
factorial study, the buffering component, Tris-HCl and proteinase K
are believed to be the most important factors. The proteinase K
works well when a constant pH is maintained.
[0125] The duplicate samples in the second part of the study help
show consistency of the results for each set of conditions. The
E7526 Extraction Solution with kinase K was slightly brighter than
the other combination (FIG. 6). The KCl seems to make little
difference in the extractions, although its presence is needed for
the subsequent PCR mix.
[0126] As various changes could be made in the above methods and
compositions without departing from the scope of the invention, it
is intended that all matter contained in the above description be
interpreted as illustrative and not in a limiting sense.
[0127] All references cited in this specification are hereby
incorporated by reference. The discussion of the references herein
is intended merely to summarize the assertions made by their
authors and no admission is made that any reference constitutes
prior art relevant to patentability. Applicant reserves the right
to challenge the accuracy and pertinency of the cited
references.
* * * * *