U.S. patent application number 10/909724 was filed with the patent office on 2005-02-10 for compositions and methods for using a solid support to purify dna.
Invention is credited to Bair, Robert Jackson, Benedict, Kristen Campbell, Kivens, Wendy J., Kwiatkowski, Robert W. JR., Paulsen, Kim, Strom, Daniel A., Wages, John M..
Application Number | 20050032105 10/909724 |
Document ID | / |
Family ID | 35640050 |
Filed Date | 2005-02-10 |
United States Patent
Application |
20050032105 |
Kind Code |
A1 |
Bair, Robert Jackson ; et
al. |
February 10, 2005 |
Compositions and methods for using a solid support to purify
DNA
Abstract
Reagents, methods and kits for the purification of DNA from
biological materials are provided.
Inventors: |
Bair, Robert Jackson;
(Plymouth, MN) ; Benedict, Kristen Campbell;
(Maple Grove, MN) ; Kivens, Wendy J.; (Edina,
MN) ; Kwiatkowski, Robert W. JR.; (Verona, WI)
; Paulsen, Kim; (Brooklyn Park, MN) ; Strom,
Daniel A.; (Minneapolis, MN) ; Wages, John M.;
(Tupelo, MS) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
3300 DAIN RAUSCHER PLAZA
60 SOUTH SIXTH STREET
MINNEAPOLIS
MN
55402
US
|
Family ID: |
35640050 |
Appl. No.: |
10/909724 |
Filed: |
August 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10909724 |
Aug 2, 2004 |
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10418194 |
Apr 16, 2003 |
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10418194 |
Apr 16, 2003 |
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09974798 |
Oct 12, 2001 |
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Current U.S.
Class: |
435/6.12 ;
435/270 |
Current CPC
Class: |
C12N 15/1003 20130101;
C12N 15/1006 20130101; C07H 21/04 20130101 |
Class at
Publication: |
435/006 ;
435/270 |
International
Class: |
C12Q 001/68; C12N
001/08 |
Goverment Interests
[0002] Work relating to this application was supported by National
Institutes of Health Grant 1 R43 CA106124-01. The government may
have certain rights in the invention.
Claims
We claim:
1. A formulation for isolating and purifying DNA comprising: a
lithium salt at a concentration of at least about 1 M, at least one
surfactant, and a buffer.
2. The formulation of claim 1, further comprising a chelating
agent.
3. The formulation of claim 1, wherein the chelating agent is EDTA
or citrate.
4. The formulation of claim 1, wherein the formulation lacks a
chaotrope and/or a strong chaotropic substance.
5. The formulation of claim 1, wherein the lithium salt is lithium
chloride.
6. The formulation of claim 1, wherein the lithium salts is at a
concentration of 2-10 M.
7. The formulation of claim 1, wherein the formulation has pH of
above about 7.
8. The formulation of claim 1, wherein the solution has a pH
between about 7 and about
9. The formulation of claim 1, wherein the surfactant is present at
a concentration of about 10-40% v/v.
10. The formulation of claim 1, wherein at least one surfactant is
a detergent.
11. The formulation of claim 10, wherein the detergent is an
anionic, cationic, zwitterionic or non-ionic detergent.
12. The formulation of claim 1, wherein the surfactant is diethyl
glycol monoethyl ether (DGME).
13. The formulation of claim 12, wherein the DGME is present at a
concentration of about 5% to about 35% v/v.
14. The formulation of claim 10, wherein the detergent is a mixture
of anionic and non-ionic detergents.
15. The formulation of claim 11, wherein the anionic detergent is
SDS.
16. The formulation of claim 14, wherein the SDS is present at a
concentration of between 0.05-0.2% v/v.
17. The formulation of claim 12, wherein the detergent is
Triton-X.
18. The formulation of claim 17, wherein the detergent is present
at a concentration of between 0.05-5.0% v/v.
19. A method for isolating substantially pure and undegraded DNA
from biological material, comprising the steps of: (a) contacting
the biological material with a DNA Lysing Solution to form a
mixture, wherein the DNA Lysing Solution is buffered at pH of
greater than 7, and wherein the DNA Lysing Solution comprises a
lithium salt and at least one surfactant; (b) contacting the
mixture with a DNA Spiking Solution; (c) contacting the mixture to
a solid support such that DNA present in the biological material
binds to the solid support; (d) washing the sold support with a
Wash Solution; and (e) eluting the DNA with a DNA Elution
Solution.
20. The method of claim 19, wherein the alcohol is isopropanol,
ethanol or methanol.
21. The method of claim 19, wherein the alcohol may be a mixture of
alcohols.
22. The method of claim 19, wherein the alcohol is 100%
isopropanol.
23. The method of claim 19, wherein the DNA Spiking Solution
contains an alkali-metal salt greater than 1M.
24. The method of claim 19, wherein the biological material is a
cervical cell sample.
25. The method of method of claim 19, wherein the biological
material is whole blood.
26. The method of claim 19, wherein the biological material is a
cultured cell, fixed cell, and/or tissue sample.
27. The method of claim 19, wherein the Wash Solution is buffered
at a pH of greater than about 7.
28. The method of claim 19, wherein the Lysing and/or Wash Solution
lack a chaotrope and/or a strong chaotropic substance.
29. The method of claim 19, wherein the DNA Lysing Solution further
comprises a chelating agent.
30. The method of claim 19, wherein the solid support comprises
components of silica, cellulose, cellulose acetate, nitrocellulose,
nylon, polyester, polyethersulfone, polyolefin, or polyvinylidene
fluoride, or combinations thereof.
31. The method of claim 19, wherein the solid support is
pre-treated with RNase solution prior to contacting the biological
material with the solid support.
32. The method of claim 19, wherein the lithium salt of the Lysing
Solution is lithium chloride or lithium bromide.
33. The method of claim 19, wherein the lithium salt of the Lysing
Solution is present at a concentration greater than about 1 M.
34. The method of claim 19, wherein the lithium salt of the Lysing
Solution is present at a concentration of between 2 M and 8 M.
35. The method of claim 19, wherein the Lysing Solution further
comprises a chelating agent.
36. The method of claim 35, wherein the chelating agent is EDTA or
citrate.
37. The method of claim 19, wherein the DNA Lysing Solution
comprises a surfactant at a concentration of between about 25-35%
v/v.
38. The method of claim 19, wherein the surfactant is a
detergent.
39. The method of claim 38, wherein the detergent is a non-ionic
detergent.
40. The method of claim 39, wherein the non-ionic detergent is a
Tween, Triton, Nonidet, Igepal or Tergitol.
41. The method of claim 38, wherein the detergent is an anionic
detergent.
42. The method of claim 41, wherein the anionic detergent is SDS
(sodium dodecyl sulfate) or N-lauroyl sarcosine.
43. The method of claim 19, wherein the surfactant is a mixture of
detergents, or a mixture of detergents with a solubilizing
surfactant.
44. The method of claim 43, wherein the solubilizing surfactant is
DGME.
45. A method for purifying substantially pure and undegraded DNA
from biological material, comprising the steps of: (a) contacting a
biological material containing DNA with a solid support pre-treated
with a DNA Lysing Solution, wherein the DNA Lysing Solution is
buffered at a pH of greater than about 7, and wherein the DNA
Lysing Solution comprises a surfactant and a lithium salt; (b)
adding a DNA Spiking Solution to the biological material; (c)
contacting the biological material to the solid support in order to
release nucleic acids comprising substantially undegraded DNA and
non-nucleic acid biological matter, wherein the nucleic acids
comprising substantially undegraded DNA bind to the solid support;
(d) washing the solid support with a Wash Solution to remove
biological materials other than bound nucleic acids comprising
undegraded DNA; and (e) eluting the bound undegraded DNA from the
solid support with a DNA Elution Solution.
46. A direct lysis method for purifying substantially pure and
undegraded DNA from biological material without using a red blood
cell lysis step, comprising: (a) contacting a biological material
containing DNA, with a first DNA Lysing Solution, wherein the first
DNA Lysing Solution is buffered at a pH of greater than about 7 and
comprises a surfactant at a concentration of between about 5-15%
v/v and a DNA-complexing salt at a concentration of greater than
1M, (b) contracting the biological material with a second DNA
Lysing Solution, wherein the second DNA Lysing Solution comprises a
surfactant and a DNA-complexing salt greater than 1M; (c)
contacting the mixture with a DNA Spiking Solution; (d) contacting
the mixture with a solid support, wherein nucleic acids comprising
substantially undegraded DNA from the biological material bind to
the solid support; (e) washing the solid support with a DNA Wash
Solution to remove biological materials other than bound nucleic
acids comprising substantially undegraded DNA, the DNA Wash
Solution comprising a DNA-complexing salt and an alcohol; and (f)
preferentially eluting the bound substantially undegraded DNA from
the solid support with a DNA Elution Solution.
47. The method of claim 46, wherein the second DNA Lysing Solution
further comprises a chelating agent.
48. A method for purifying substantially pure and undegraded DNA
from biological material comprising: (a) contacting a biological
material containing DNA with DNA Lysing Solution buffered at a pH
of greater than about 7, wherein the DNA Lysing Solution comprises
a surfactant and an DNA-complexing salt at a concentration greater
than 1 M; (b) contacting the biological material to a solid
support, wherein nucleic acids comprising substantially undegraded
DNA bind to the solid support; (c) contacting the solid support
with a DNA Spiking Solution; (d) washing the solid support with a
DNA Wash Solution to remove biological materials other than bound
nucleic acids comprising undegraded DNA, the DNA Wash Solution
comprising a DNA-complexing salt and an alcohol; and (e)
preferentially eluting bound substantially undegraded DNA from the
solid support with a DNA Elution Solution.
49. A method for purifying substantially pure and undegraded DNA
fixed cervical cell samples, comprising the steps of (a) contacting
a biological material containing DNA with DNA Lysing Solution
buffered at a pH of greater than about 7, wherein the DNA Lysing
Solution containing a surfactant at a concentration of between
about 25-35% V/v, a chelating agent, and a DNA-complexing salt of
greater than 1 M; (b) adding to the mixture, an optional DNA
Spiking Solution containing alcohol; (c) contacting the biological
material to the solid support in order to release nucleic acids
comprising substantially undegraded DNA and non-nucleic acid
biological matter causing nucleic acids comprising substantially
undegraded DNA to bind to the solid support; (d) washing the solid
support with a DNA Wash Solution to remove biological materials
other than bound nucleic acids comprising undegraded DNA; and (e)
preferentially eluting the bound undegraded DNA from the solid
support with a DNA Elution Solution in order to obtain
substantially pure and undegraded DNA.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 10/418,194, filed Apr. 16, 2003, which is a
continuation of U.S. application Ser. No. 09/974,798, filed Oct.
12, 2001, which is incorporated by reference herein.
BACKGROUND OF THE INVENTION
[0003] Nucleic acids such as deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) are used extensively in the field of
molecular biology for research and clinical analyses. There are
numerous nucleic acid purification methods that fall into two
general categories, liquid phase and solid phase purification. In
liquid phase purification, the DNA remains in the liquid phase
while impurities are removed by precipitation and/or
centrifugation. In solid phase purification, the DNA is bound to a
solid support while impurities are selectively eluted. Conventional
liquid phase and solid phase purification strategies require many
steps and hazardous reagents.
SUMMARY OF THE INVENTION
[0004] The present invention provides a formulation for isolating
and/or purifying nucleic acids. The formulation contains a lithium
salt at a concentration of at least about 1 M, at least one
surfactant, and a buffer. The formulation may further contain a
chelating agent, such as EDTA or citrate. The formulation of the
present invention may lack a chaotrope and/or a strong chaotropic
substance. Examples of strong chaotropic substances are guanidinium
salts, urea, ammonium, cesium, rubidium, potassium, or iodide salt.
The lithium salt present in the formulation may be lithium
chloride. The lithium salt may be present at a concentration of 2 M
to 10 M, or at a concentration of 2 M to 6 M. In certain
embodiments, the concentration of the lithium salt may be at 2 M, 3
M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M or 10 M, or any range between (such
as 5.5 M). The formulation may have a pH of above about 7, such as
between about 7 and about 9 (e.g., a pH of about 8, about 8.5, or
about 9). In certain embodiments, the nucleic acid that is isolated
and/or purified is DNA.
[0005] The surfactant of the present formulation may be present at
a concentration of about 10% to about 40% (or any percentage in
between) of the total volume of the formulation. The surfactant or
surfactants in the formulation of the present invention may be a
detergent. The detergent may be an anionic, cationic, zwitterionic
or non-ionic detergent. In one embodiment, the surfactant is a
non-ionic detergent. Examples of appropriate non-ionic detergents
include a Tween, Triton, Nonidet, Igepal or Tergitol. For instance
the detergent may be Triton-X.
[0006] Further, the surfactant may be diethyl glycol monoethyl
ether (DGME). The DGME may be present at a concentration of about
5% to about 35% (or any percentage in between) of the total volume
of the formulation. In general, as the concentration of other
components of the formulation increase, the amount of DGME is
increased so as to increase the solubility of the other components.
In certain embodiments, the surfactant is a mixture of Triton-X and
DGME. In one embodiment the surfactant is a mixture of 5% v/v
Triton-X and 5% v/v DGME. In one embodiment, the surfactant is
present at a concentration of about 10% v/v of the final volume of
the solution.
[0007] In one embodiment, the surfactant is an anionic detergent.
Examples of appropriate detergents include sodium dodecyl sulfate
(SDS) or N-lauroyl sarcosine. In one embodiment, SDS is the anionic
detergent. In one embodiment, the detergent is be present at a
concentration of between about 0.05-0.2% (or any percentage in
between). In one embodiment the concentration is about 0.1%.
[0008] In certain embodiments, the formulation contains a mixture
of more than one surfactant at a concentration of 0.1% SDS and 30%
DGME.
[0009] The buffer of the formulation of the present invention may
have a pKa of at least about 8. The chelating agent may be EDTA or
citrate.
[0010] In one embodiment of the present invention the formulation
for isolating and purifying nucleic acids contains a lithium salt
at a concentration of at least about 1 M, a surfactant, a buffer,
and an optional chelating agent, wherein the solution has pH of
above about 7.
[0011] In another embodiment of the present invention, the
formulation for isolating and purifying nucleic acids consists
essentially of a lithium salt at a concentration of at least about
1 M, at least one surfactant, a buffer, and an optional chelating
agent.
[0012] In yet another embodiment, the formulation of the present
invention consists essentially of a lithium salt, at least one
surfactant, a buffer, and an optional chelating agent, wherein the
solution has pH of above about 7.
[0013] A further embodiment of the present invention consists
essentially of a lithium salt at a concentration of at least about
1 M, a surfactant, a buffer, and an optional chelating agent,
wherein the solution has pH of above about 7.
[0014] The present invention also provides methods for purifying
substantially pure and undegraded DNA from biological material. The
biological material used in the method of the present invention may
be a crude sample or a partially purified mixture of nucleic acids.
Examples of biological materials include a sample of eukaryotic
cells, prokaryotic cells, microbial cells, bacterial cells, plant
cells, mycoplasma, protozoa, bacteria, fungi, virus, yeast, or
rickettsia, or homogenates thereof. Additional examples of
biological materials include whole blood, bone marrow, cervical
swabs, blood spot, blood serum, blood plasma, buffy coat
preparation, saliva, cerebrospinal fluid, or solid animal tissue.
Further examples of biological materials include feces, urine,
tears, or sweat. The biological material may also be an
environmental sample taken from air, water, sediment or soil. The
biological material may be of a variety of sample types including,
for example, cultured cells, fixed cells, and/or tissues. In one
embodiment, the biological sample is a cervical cell sample. In
another embodiment the biological sample is whole blood.
[0015] The solid support used in the methods of the present
invention include components of silica, cellulose, cellulose
acetate, nitrocellulose, nylon, polyester, polyethersulfone,
polyolefin, or polyvinylidene fluoride, or combinations thereof.
The solid support may be contained in a vessel, wherein the vessel
is a centrifuge tube, spin tube, syringes, cartridge, chamber,
multiple-well plate, or test tube, or combinations thereof. The
solid support may be pre-treated with RNase solution prior to
contacting the biological material with the solid support.
[0016] The lithium salt of the DNA Lysing Solution used in the
method of the present invention is a DNA-complexing salt. Examples
of lithium salts that may be used include lithium chloride or
lithium bromide. The DNA-complexing salt of the DNA Lysing Solution
may be present at a concentration greater than about 1 M. In one
embodiment, the DNA-complexing salt may be present at a
concentration of between 2 M and 8 M. In certain embodiments, the
concentration of the DNA-complexing salt is at a concentration of
about 2 M, 3 M, 4M, 5 M, 6 M, 7 M, or 8 M, or any concentration in
between (such as at about 5.5 M).
[0017] The Lysing Solution may optionally contain a chelating
agent, such as EDTA or citrate.
[0018] The present invention provides a method involving the steps
of contacting a biological material with a DNA Lysing Solution to
form a mixture, wherein the DNA Lysing Solution is buffered at pH
of greater than 7, and wherein the DNA Lysing Solution contains a
lithium salt and at least one surfactant; contacting the mixture
with a DNA Spiking Solution; contacting the mixture to a solid
support such that DNA present in the biological material binds to
the solid support; washing the solid support with a wash solution
to remove biological materials other than bound substantially
undegraded DNA; and eluting the DNA with a DNA Elution Solution in
order to obtain substantially pure and undegraded DNA.
"Substantially undegraded DNA binds to the solid support" means
that the substantially undegraded DNA binds to the solid support
while other cellular or non-cellular components found in the
biological sample (such as cellular membranes or proteins) largely
do not bind to the solid support.
[0019] The present invention provides a method for purifying
substantially pure and undegraded DNA from biological material,
involving the steps of contacting a biological material containing
DNA with a solid support pre-treated with an DNA Lysing Solution,
wherein the DNA Lysing Solution is buffered at a pH of greater than
about 7, and wherein the DNA Lysing Solution contains a surfactant
and a lithium salt; adding a DNA Spiking Solution to the biological
material; contacting the biological material to the solid support
in order to release nucleic acids comprising substantially
undegraded DNA and non-nucleic acid biological matter, wherein the
nucleic acids comprising substantially undegraded DNA bind to the
solid support; washing the solid support with a Wash Solution to
remove biological materials other than bound nucleic acids
comprising undegraded DNA; and eluting the bound undegraded DNA
from the solid support with an DNA Elution Solution. The Wash
Solution may be buffered at a pH of greater than about 7. In
certain embodiments the Wash Solution may be buffered between about
7 and about 9 (e.g., a pH of about 8, about 8.5, or about 9). The
Lysing and/or Wash Solution used in the methods of the present
invention formulation of the present invention may lack a chaotrope
and/or a strong chaotropic substance.
[0020] The DNA Spiking Solution used in the methods of the present
invention may be an alcohol. The alcohol may be isopropanol,
ethanol, methanol or the like. The alcohol may be a mixture of
alcohols. In one embodiment, the DNA Spiking Solution is 100%
isopropanol.
[0021] The surfactant used in the methods of the present invention
may be a detergent. The detergent may be a non-ionic detergent,
such as a Tween, Triton, Nonidet, Igepal or Tergitol. The detergent
may be an anionic detergent, such as SDS (sodium dodecyl sulfate)
or N-lauroyl sarcosine. The surfactant may be a mixture of
detergents, or a mixture of detergents with a solubilizing
surfactant such as DGME.
[0022] The DNA Spiking Solution may contain an alkali-metal salt,
such as lithium salt. The DNA Spiking Solution may be buffered at a
pH greater than 7. In certain embodiments the DNA Spiking Solution
may be buffered at a value between about 7 and about 9 (e.g., a pH
of about 8, about 8.5, or about 9). The DNA Spiking Solution may
contain a surfactant. In one embodiment, the surfactant is
DGME.
[0023] The present invention further provides a method for
purifying substantially pure and undegraded DNA from biological
material, involving the steps of (a) contacting a biological
material containing DNA with a solid support pre-treated with a DNA
Lysing Solution buffered at a pH of greater than about 7 such that
the DNA Lysing Solution is bound to the solid support, wherein the
DNA Binding Solution contains a DNA-complexing salt; (b) adding an
optional DNA Spiking Solution to the mixture; (c) contacting the
biological material to the solid support such that nucleic acids
comprising substantially undegraded DNA bind to the solid support;
(d) washing the solid support with a DNA wash solution to remove
biological materials other than bound nucleic acids comprising
substantially undegraded DNA; and (e) preferentially eluting the
bound substantially undegraded DNA from the solid support with an
DNA Elution Solution in order to obtain substantially pure and
undegraded DNA.
[0024] The present invention further provides a method for
purifying substantially pure and undegraded DNA from biological
material, involving the steps of (a) contacting a biological
material containing DNA with a solid support pre-treated with a DNA
Lysing Solution buffered at a pH of greater than about 7 such that
the DNA Lysing Solution is bound to the solid support, the DNA
Lysing Solution containing a surfactant and an DNA-complexing salt;
(b) adding to the biological sample an optional DNA Spiking
Solution; (c) contacting the biological material to the solid
support in order to release nucleic acids comprising substantially
undegraded DNA and non-nucleic acid biological matter, causing
nucleic acids comprising substantially undegraded DNA to bind to
the solid support; (d) washing the solid support with to remove
biological materials other than bound nucleic acids comprising
undegraded DNA; and (e) preferentially eluting the bound undegraded
DNA from the solid support with an DNA Elution Solution in order to
obtain substantially pure and undegraded DNA.
[0025] The present invention further provides a direct lysis method
for purifying substantially pure and undegraded DNA from biological
material, for instance whole blood, without using a red blood cell
(RBC) lysis step, involving the steps of contacting a biological
material containing DNA with a first DNA Lysing Solution, wherein
the first DNA Lysing Solution is buffered at a pH of greater than
about 7 and contains a surfactant at a concentration of between
about 5-15% v/v and a DNA-complexing salt at a concentration of
greater than 1 M, contacting the biological material with a second
DNA Lysing Solution, wherein the second DNA Lysing Solution
comprises a surfactant and a DNA-complexing salt greater than 1M;
contacting the mixture with a DNA Spiking Solution; contacting the
mixture with a solid support, wherein nucleic acids comprising
substantially undegraded DNA from the biological material bind to
the solid support; washing the solid support with a DNA Wash
Solution to remove biological materials other than bound nucleic
acids comprising substantially undegraded DNA, the DNA Wash
Solution containing a DNA-complexing salt and a surfactant; and
preferentially eluting the bound substantially undegraded DNA from
the solid support with an DNA Elution Solution. In certain
embodiments, the second DNA Lysing Solution is buffered at a pH of
greater than about 7. In certain embodiments, the concentration of
the surfactant in the second DNA Lysing Solution is between about
25-35% v/v. In certain embodiments, the second DNA Lysing Solution
also contains a chelating agent.
[0026] The present invention further provides a method for
purifying substantially pure and undegraded DNA from biological
material, for instance fixed cervical cell samples, involving the
steps of contacting a biological material containing DNA with DNA
Lysing Solution buffered at a pH of greater than about 7, the DNA
Lysing Solution containing a surfactant and a DNA-complexing salt
of greater than 1 M; adding to the mixture, an optional DNA Spiking
Solution containing alcohol; contacting the biological material to
the solid support in order to release nucleic acids comprising
substantially undegraded DNA and non-nucleic acid biological
matter, causing nucleic acids comprising substantially undegraded
DNA to bind to the solid support; washing the solid support with a
DNA Wash Solution to remove biological materials other than bound
nucleic acids comprising undegraded DNA; and preferentially eluting
the bound undegraded DNA from the solid support with a DNA Elution
Solution in order to obtain substantially pure and undegraded DNA.
In certain embodiments, the concentration of the surfactant in the
DNA Lysing Solution is between about 25-35% v/v. In certain
embodiments, the DNA Lysing Solution also contains a chelating
agent.
[0027] As used herein, "substantially pure" means substantially
free of RNA, carbohydrate, protein, lipid impurities, such that the
DNA can be used in subsequent analyses known to those with skill in
the art such as nucleic acid quantification, restriction enzyme
digestion, DNA sequencing, hybridization technologies, such as
Southern Blotting, etc., and amplification methods such as
Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR),
Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained
Sequence Replication (SSR or 3SR), Strand Displacement
Amplification (SDA), and Transcription Mediated Amplification
(TMA), Quantitative PCR (qPCR), or other DNA analyses.
[0028] As used herein, "substantially undegraded" DNA means
nondigested or intact DNA, which can be readily determined by one
of skill in the art using standard techniques. "Substantially
undegraded" DNA is not damaged by enzymatic, physical or chemical
means during the purification methods of the present invention.
[0029] The reagents, methods and kits of the present invention may
be used to isolate substantially pure and undegraded DNA over a
wide range of biological sources, and life forms, all of which can
be recovered over a wide molecular weight range. The substantially
pure and undegraded DNA obtained from practicing the invention can
be evaluated for purity, yield, size, reverse transcriptase or
other hybridization processes, amplification, hybridization
ability, etc. The biological samples include, for example, cell or
viral suspensions and pellets thereof, body fluids, cervical cell
swabs and tissue homogenates, etc. If the biological sample
consists of cells or viruses, the cells or viruses may be
enumerated. The enumeration may be conducted using standard cell
counting methods such as an electronic cell counter (e.g., CBC5
Coulter Counter, Coulter Corp., Hialeah, Fla.) or a visual counting
chamber (e.g., a hemacytometer, Bright Line, American Optical,
Buffalo, N.Y.).
[0030] It should be noted that the indefinite articles "a" and "an"
and the definite article "the" are used in the present application,
as is common in patent applications, to mean one or more unless the
context clearly dictates otherwise. Further, the term "or" is used
in the present application, as is common in patent applications, to
mean the disjunctive "or" or the conjunctive "and."
BRIEF DESCRIPTION OF THE FIGURES
[0031] FIG. 1 is a graph depicting the percentage of theoretical
DNA yield comparing QIAamp Blood DNA Midi Kit (Qiagen, Inc.
Germany) and Versagene Blood DNA Kit (Gentra Systems, Inc.,
Minnesota).
[0032] FIG. 2 is a graph depicting the percentage of theoretical
DNA yield comparing lithium salts and pH of the lysis solution.
DETAILED DESCRIPTION
[0033] The use of chaotropic salts for the binding and purification
of nucleic acids is well known in the art. For example, Kuroita et
al. (U.S. Pat. No. 5,990,302) disclose that biological material may
be lysed in an acidic solution containing a lithium salt and a
chaotropic agent such as guanidinium isothiocyanate (GITC), after
which the RNA is brought into contact with a nucleic acid-binding
carrier such as silica. The RNA is subsequently purified by eluting
from the silica in a low ionic-strength buffer. This method is
disadvantageous in its use of hazardous substances such as the
chaotropic salt, guanidine isothiocyanate.
[0034] Combinations of chaotropic substances such as guanidine
isothiocyanate, guanidine hydrochloride, sodium iodide, and urea
mixtures at ionic strengths greater than 4 M in conjunction with
silica-based carriers have been taught in the art for RNA
purification. For example, Hillebrand et al. (WO 95/34569)
describes a one-step method involving a slurry of silica beads to
which chaotropic substances are added in order to cause RNA to
bind.
[0035] The apparent opposite approach to the use of chaotropes is
the use of antichaotropes (also known as "kosmotropes" in the art)
to isolate RNA. Hillebrand et al., (US 2001/0041332) describes the
use of "antichaotropes," such as ammonium chloride (also cesium,
sodium and/or potassium salts are mentioned), in combination with
PVP (polyvinyl pyrrolidone) to lyse the starting sample and bind to
the solid support with a detergent/alcohol mixture. Besides the
fact that it is generally known that cesium and potassium are
clearly considered to be chaotropes, due to their low charge
density and weak hydration characteristics, while ammonium is
considered to be a marginal chaotrope (Collins, K. Sticky Ions in
Biological Systems, Proc. Natl. Acad. Sci. USA, 92 (1995),
5553-5557; Wiggins, P. M. High and Low Density Intracellular Water,
Cellular and Molecular Biology 47 (5), 735-744), several
disadvantages to the methods of Hillebrand exist. First, the
methods use PVP, which has been investigated as a tumorigen.
Secondly, heating steps of 65-70.degree. C. are required for lysis
and elution. Such heating may cause damage to the nucleic acids by
nonspecific degradation or digestion resulting in limited
downstream applications, such as incompatibility with restriction
digests or blot analysis.
[0036] It should be noted that the method of selectively
precipitating nucleic acids from a solution containing nucleic
acids and other biological materials is physically different than
that of using a solid phase to selectively bind the DNA or RNA
molecules in a solution. A "precipitation" event is the reverse of
a "solution" event. Solution involves the dissolving of a solute,
such as DNA, by separation of that solute into molecules that are
surrounded by solvent. Precipitation involves the removal of
solvent and coalescence of individual DNA molecules into a solid
that separates from the solvent. These Precipitation and Solution
events occur within a solution environment and do not depend upon a
separate and distinct solid phase on which DNA purification and
separation occurs.
[0037] To advance the field of DNA sample preparation there is a
need for solid phase DNA purification strategies. There is also a
need for reagents and methods that are adaptable to solid phase
purification strategies and are not only simple and rapid but also
general in scope to maximize adaptability for automation. There is
a need for reagents that are of generally low concentration, stable
at room temperature (i.e., 20-25.degree. C.), less hazardous (i.e.,
less corrosive, flammable or toxic), nonparticulate to eliminate
the need for mixing, and protective of DNA quality. There is also a
need for methods with few steps that can be performed using a
variety of biological starting materials, whether hydrated or
dried, especially as applied to routine testing as found in
clinical laboratories. The reagents must not inhibit subsequent DNA
analysis procedures by interfering with the buffering capacity of
PCR buffers, or cause degradation of polymerase, primers or
oligonucleotides used in DNA amplification. There is also a need
for methods with few steps that can be performed using a variety of
biological starting materials, whether hydrated or dried,
especially as applied to routine testing as found in clinical
laboratories. The reagents and methods used in the solid phase
purification strategy must also not interfere with standard
experimental and/or diagnostic methods of nucleic acid
manipulation.
[0038] Additionally, isolating and purifying nucleic acids has
become more challenging with the discovery of more challenging
sample types. For example, isolating and purifying DNA from
cervical cells for use in molecular-based tests for HPV (human
papilloma virus) have been rapidly adopted in clinical laboratories
as the role of HPV infection in cervical cancer has become evident.
Molecular diagnostic labs now perform over 6 million HPV diagnostic
tests per year. However, preparation of DNA from cervical samples
for molecular testing has not kept pace with the growing demand. In
standard practice, the exfoliated cervical epithelial cells are
harvested in a liquid media containing preservative, such as
SurePath.TM. Preservative Fluid (TriPath Imaging, Burlington, N.C.)
or the ThinPrep.RTM. Pap Test.TM. (Cytyc, Boxborough, Mass.), and
the samples are often heavily contaminated with a variety of
cellular and non-cellular components making isolation and
purification a challenge. These contaminants can include mucus,
white blood cells, red blood cells, and proteins. The current
manual DNA purification methods have several significant
disadvantages that impede further adoption of molecular-based HPV
diagnostics in the clinical laboratory. There is now an urgent need
for more efficient and effective methods for the purification of
DNA from samples containing exfoliated cervical cells.
[0039] The present invention provides reagents, methods, and kits
that incorporate a solid support for isolating substantially pure
and undegraded DNA from fresh, frozen, and dried biological
samples. The purified DNA is suitable for use in widely used
analytical and diagnostic methods such nucleic acid quantification,
restriction enzyme digestion, DNA sequencing, hybridization
technologies, such as Southern Blotting, etc., and amplification
methods such as Polymerase Chain Reaction (PCR), Ligase Chain
Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA),
Self-sustained Sequence Replication (SSR or 3SR), Strand
Displacement Amplification (SDA), and Transcription Mediated
Amplification (TMA), Quantitative PCR (qPCR), or other DNA
analyses.
[0040] Biological Materials and/or Samples
[0041] The present invention provides reagents, methods and kits
for purifying DNA from biological samples. Such biological samples
include biological material, typically in an aqueous mixture or
dried, that contains DNA, including complex biological mixtures of
prokaryotic or eukaryotic cells. Typically, the biological material
also contains DNA, carbohydrates, proteins, and lipids. Biological
materials include, but are not restricted to the following: body
fluids such as whole blood, bone marrow, blood spots, blood serum,
blood plasma, buffy coat preparations, saliva and cerebrospinal
fluid, buccal swabs, cultured cells, fixed cells, cervical cell
swabs, cell suspensions of bacteria or tissue homogenates, solid
animal tissues such as heart, liver and brain, body waste products,
such as feces and urine, environmental samples taken from air,
water, sediment or soil, plant tissues, yeasts, bacteria, viruses,
mycoplasmas, fungi, protozoa, rickettsia, and other small microbial
cells. Lysates, homogenates, or partially purified samples of these
biological materials may also be used. In one embodiment, the
biological material is crude or partially purified mixtures of
nucleic acids.
[0042] Reagents of the Present Invention
[0043] The present invention discloses four categories of reagents.
These are DNA Lysing Solutions, DNA Spiking Solutions, DNA Wash
Solution, and DNA Elution Solution. These reagents, used in
conjunction with an appropriate solid support, are used to generate
undegraded DNA, which is substantially pure and contaminant-free.
The reagents that may be used to purify DNA from a variety of
biological materials without the use of hazardous substances such
as phenol, and chloroform, or hazardous chaotropic substances such
as guanidinium salts, urea, etc.
[0044] (1) DNA Lysing Solutions: A DNA Lysing Solution enables
efficient lysis of the biological sample to release the nucleic
acids, and effectively inhibits DNase activity. A DNA Lysing
Solution of the present invention has the following components: a
lithium salt; a buffer; a surfactant, such as a detergent or
detergent/surfactant mixture; and optionally a chelating reagent. A
DNA Lysing Solution of the present invention is unique in that it
requires no added strong chaotropic substances such as guanidinium
salts, urea, etc. Guanidinium salts and urea are strong chaotropic
salts that disrupt the structure of water and thus tend to decrease
the strength of hydrophobic interactions resulting in a drastic
effect on other solute molecules. For example, urea, when dissolved
in water, disrupts the secondary, tertiary, and quaternary
structures of proteins, and subsequently causes dissociation of
proteins from DNA. Guanidinium salts and urea dissolve in water
through endothermic reactions. Both guanidinium salts and urea are
considered to be strongly chaotropic salts as defined by the
Hofineister series, a widely used system that ranks cations and
anions according to relative chaotropic strength (F. Hofineister,
On the understanding of the effects of salts, Arch. Exp. Pathol.
Pharmakol. (Leipzig) 24 (1888) 247-260).
[0045] Unlike strong chaotropic salts, the reaction of lithium
salts (such as lithium chloride and lithium bromide) in water is an
exothermic reaction and is indicative of the tremendous ion-dipole
interaction exhibited by the strong kosmotropic lithium ion and the
resulting large solubility. Differences such as these are
indicative of the differences between the strong chaotropic
substances, such as guanidinium salts, and the alkali-metal salts,
especially lithium chloride, of the present invention.
[0046] The present invention involves the use of lithium salts,
including for example, lithium chloride and lithium bromide. The
lithium ion is considered very kosmotropic, due to its high surface
charge density and strong hydration characteristics. The lithium
ion is unique in that it has a small radius, in comparison to that
of sodium, potassium, rubidium and cesium ions also found in the
alkali metal group. This causes its surface charge density to be
larger than the other ions in this group. The larger surface charge
density is responsible for the tremendous interaction of the
lithium ion with water molecules. This causes water molecules to
organize around the ion and to maintain this structured effect even
past the first hydration shell.
[0047] Suitable DNA-complexing salts for the present invention
include those that contain the alkali metal ions such as lithium,
sodium, potassium, rubidium and cesium, since all of these cations
complex specifically to the phosphate groups of the DNA molecules.
This complexation and subsequent neutralization of the DNA molecule
cause the DNA molecules to become less stable in the aqueous
environment and promotes binding to the solid phase. One embodiment
of the present invention is to use a lithium salt. Lithium salts
used to practice the present invention include, but are not limited
to, lithium chloride and lithium bromide. Lithium fluoride and
lithium iodide are less desirable alkali salts because their cost
is about five times the cost of the lithium chloride and bromide
salts. In addition, lithium ion is the only clearly kosmotropic ion
in the aforementioned list. The sodium ion is a borderline
kosmotrope, while potassium, rubidium and cesium ions are
chaotropic ions (Collins, K. Sticky Ions in Biological Systems,
Proc. Natl. Acad. Sci. USA, 92 (1995), 5553-5557). Cesium chloride
costs about five times more than the other alkali metal chloride
salts (Table 1) and has more limited solubility behavior than the
lithium chloride and bromide salts. In addition, sodium, potassium
and ammonium chloride salts have much more limited solubility
behavior as compared to the lithium chloride and bromide salts, as
exhibited by the large exothermic heats of solution exhibited by
lithium salts in water (CRC Handbook of Chemistry and Physics, 62nd
edition, CRC Press, Boca Raton, Fla.).
[0048] Binding of DNA to a solid support is enhanced by high
concentrations of alkali metal salts in the Lysing and/or Spiking
Solutions. The alkali-metal salt may be at a concentration of
between 2-10 M. In Example 2, it is observed that in the Lysing
Solution, ammonium and potassium chloride salts have maximum
solubility of 3 M and <3 M respectively, while cesium and sodium
chloride salts readily dissolve to 4 M. As can be seen in Table 2,
these salt values approximately match those expected in aqueous
solution, except for that of ammonium chloride (CRC Handbook of
Chemistry and Physics, 62nd edition, CRC Press, Boca Raton,
Fla.).
[0049] Alkaline earth metal salts containing the kosmotropic
magnesium and calcium ions, although also having properties of
forming salt complexes with DNA, however they are not soluble at
the high concentrations needed to bind DNA to the solid support. In
addition, for example, the alkaline earth metal beryllium is about
20 times more expensive than for the alkaline metal salts lithium
chloride or lithium bromide, and therefore is not as practical for
use in the present invention.
[0050] The DNA Lysing Solution of the present invention contains a
lithium salt so that DNA binds to a distinct solid phase through an
adsorption mechanism. The use of lithium salt to cause adsorption
of DNA to a solid phase differs from the use of lithium for the
precipitation of DNA. In the adsorption process, the solvent
molecules are separated from the DNA molecules. The interaction
between the DNA and the solid phase is energetically more favorable
than that of DNA molecular interactions, so that adsorption to the
solid phase occurs instead of precipitation. An example of an
appropriate solid phase is borosilicate.
[0051] The DNA Lysing Solution of the present invention achieves
binding of DNA, as compared to other materials in the biological
material, to a solid support by the presence of the DNA-complexing
lithium salt, such as lithium chloride or lithium bromide, in a
buffer, and a surfactant, without the use of hazardous chaotropic
substances such as guanidinium salts, urea, etc. The lithium ion
binds to the charged phosphate backbone of nucleic acids such as
DNA, causing the DNA to be less soluble at high lithium ion
concentrations (Kazakov S. A., Nucleic Acid Binding and Catalysis
by Metal Ions, in Bioorganic Chemistry: Nucleic Acids, Ed. Hecht,
S. M., Oxford University Press, NY & Oxford, 1996). Thus, the
DNA-complexing salt confers unique binding properties to nucleic
acids, such as DNA, so that the nucleic acids can bind to the solid
support over other contaminants such as proteins, phospholipids,
etc.
[0052] The second component of the DNA Lysing Solution is a buffer
that maintains the pH of the solution. The present invention also
teaches the use of a unique neutral to high pH DNA Lysing Solution
for maximum DNA yield from various sample types. For example, the
DNA Lysing Solution may be buffered to maintain the pH at least
about 7, at least about 8, at least about 8.5, or even at least
about 9. The buffer may have a pKa of at least about 8, and may be
used at a concentration of 10-100 mM. An example of an appropriate
buffer is tris(hydroxymethyl)aminomethane (Tris). Optionally, a
base may be used to adjust the pH of the DNA Lysing Solution. The
base may be one that can raise the pH of the solutions to no less
than 7. The base may be an alkali-metal hydroxide. Such
alkali-metal hydroxides include sodium hydroxide, potassium
hydroxide, and lithium hydroxide.
[0053] The DNA Lysing Solution additionally comprises one or more
surfactants. A surfactant comprises a molecule that reduces the
surface tension of a liquid and by reducing attractions between
molecules of similar polarity and structure to allow for
solubilization between molecules of differing polarity and
structure. In one embodiment, the surfactant is a detergent, or a
detergent/surfactant mixture, that aids in lysing the biological
material. The detergent is present in order to solubilize membrane
components, such as lipids and proteins, in order to facilitate the
lysis of cell membranes and the homogenization process. The
surfactant is present in order to assist in the solubility of the
solution as well as help increase shelf-life of the solution.
Anionic, cationic, nonionic and zwitterionic detergents may all be
used. In certain instances, DNA isolation is optimally achieved
through the use of a non-ionic detergent, while in other instances,
DNA isolation is optimally achieved through use of an anionic
detergent. Although any nonionic or anionic detergents may be used,
examples of non-ionic detergents are those from the Tween class
(Tween-20, Tween-40, Tween-60, Tween-80, etc.), the Triton class
(X-100, X-114, XL-80N, etc), Tergitols (XD, TMN-6, etc.) and
Nonidets or Igepal (NP-40, etc.), and examples of anionic
detergents are SDS (sodium dodecyl sulfate) or N-lauroyl sarcosine.
The nonionic detergent may be used at a concentration of 5-15%,
such as at about 10%. The anionic detergent may be used at a
concentration of 0.05-0.2%. In another embodiment, a combination of
detergents and surfactants may be used. In one embodiment the
surfactant is DGME (diethyl glycol monoethyl ether). In one
embodiment, a combination of the detergent Triton-X and surfactant
DGME is used. The combination may be at a concentration of 5-15%,
such as at about 10%. In one example, the combination is 5%
Triton-X and 5% DGME. In yet another embodiment, the combination
may be at a concentration of 25-35%, such as about 30%. In one
example, the combination is 0.1% SDS and 30% DGME.
[0054] The combination of the lithium salt and a detergent or
surfactant in a neutral to high pH buffer, also serve to denature
enzymes such as DNases, which are generally associated with
biological material. Optionally, the DNA Lysing Solution may also
contain a chelating agent to complex extraneous metal ions. The
chelating agent may be present at a concentration of 1-100 mM, or
at a concentration of 1-10 mM. Examples of chelating agents are
EDTA or citrate. The DNA Lysing Solution of the present invention
possesses significant advantages over other described reagents. The
unique combination of the DNA-complexing lithium salt and detergent
in a neutral- to high-pH buffer inactivates enzymes harmful to DNA
(such as DNases) without the use of such reagents as phenol,
chloroform, and guanidinium salts, while allowing for complete
lysing of biological material and facilitation of the binding
process when used in combination with the DNA Spiking
Solutions.
[0055] (2) DNA Spiking Solutions: The present invention also
teaches of the use of DNA Spiking Solutions that can be used to
dehydrate the DNA molecules such as to cause quantitative binding
of DNA to the solid phase. The DNA Spiking Solution can be an
alcohol. Examples of alcohols are isopropanol, ethanol, or
methanol. In one embodiment, the DNA Spiking Solution is 100%
alcohol, such as 100% isopropanol. The DNA Spiking Solution can
alternatively comprise an alkali-metal salt. The alkali-metal salt
DNA Spiking Solution may be buffered or not, such as at pH greater
than 7. The alkali-metal salt DNA Spiking Solution may additionally
contain optional detergent or surfactant. In one embodiment, the
surfactant is DGME.
[0056] The DNA Spiking Solution dehydrates the DNA molecules such
as to cause quantitative binding of DNA to the solid phase. It has
been observed that LiCl will precipitate DNA out of solution at
extremely high concentrations (13 molal), where other salts are not
sufficiently soluble (Emanuel, C. F. Some Physical Properties of
Deoxyribonucleic Acids Dissolved in a High-Salt Medium: Salt
Hyperchromicity, Biochim. Biophys. Acta, 42, 91-98 (1960)). Higher
concentrations of salt or alcohol result in higher yields of DNA
bound to glass fiber. At increasingly higher concentrations of
LiCl, water molecules are pulled away from nucleic acid hydration
shells, to instead bind preferentially to Li.sup.+ in solution. One
study indicates that at high enough concentrations of LiCl,
eventually no water molecules are left bound to the DNA structure
(Chattoraj, D. K. & Birdi, K. S. Adsorption of Water Vapor by
Biopolymers, in Adsorption and the Gibbs Surface Excess, Plenum
Press, NY & London, 1984). The dehydration effect and
subsequent neutralization of the surface causes the DNA molecules
to be forced out of the highly ordered water solution, due to their
increased hydrophobicity, and to bind to the solid phase. DNA
adsorption to the silica solid phase is also partly driven
energetically by the increase in entropy that occurs when water
molecules are released from both the DNA molecules and the silica
solid phase surface during the dehydration process (Melzak, K. A.
et al. Driving Forces for DNA Adsorption to Silica in Perchlorate
Solutions, J. Colloid Interface Science, 181, 635-644 (1996)). The
use of a DNA Spiking Solution consisting of either a high salt
concentration or high alcohol composition will more completely
dehydrate or salt out the DNA molecules from the solution causing
adsorption to the solid phase.
[0057] In one embodiment, the DNA Spiking Solution contains a high
concentration of an alkali metal salt, such as a lithium salt. In
one embodiment, the alkali metal salt is at a concentration of
10-15M. The DNA Spiking Solution may be buffered to a pH of at
least 7. The DNA Spiking Solution may contain a surfactant to
assist in the solubility of the salt and buffer. The surfactant may
be DGME.
[0058] (3) DNA Wash Solution: The present invention also teaches a
DNA Wash Solution to wash the solid support to which nucleic acids
are bound so as to rid it of non-nucleic acid contaminants or
impurities such as proteins and phospholipids, while allowing the
nucleic acids to remain bound to the solid support. The Wash
Solution contains an alcohol, and a buffer, salt or chelator
(EDTA). The buffer composition may be Tris HCl, such as at pH 6-8.
The buffer concentration may be at 50-150 mM (e.g., at 100 mM). The
alcohol may be ethanol. The alcohol concentration may be at
25-100%. The EDTA concentration may be at 1-20 mM (e.g., at 5-10
mM).
[0059] (4) DNA Elution Solution: DNA bound to the solid support may
be eluted using a DNA Elution Solution. The simplicity of the
reagents used in lysing the biological material and binding of the
DNA to the solid support, and in washing the solid support taught
by the present invention lends itself to a simple DNA Elution
Solution. Other DNA Elution Solutions known to those skilled in the
art may also be used. For example, a DNA Elution Solution that may
be used is Versagene.TM. DNA Elution Solution (Gentra Systems,
Inc., Minneapolis, Minn.). Alternatively, Tris-EDTA (TE) may be
used.
[0060] Solid Supports
[0061] A variety of solid supports may be used in the present
invention. Suitable solid supports include silica-based supports
such as glass fiber, or other materials such as cellulose,
cellulose acetate, nitrocellulose, nylon, polyester,
polyethersulfone, polyolefin, polyvinylidene fluoride, and
combinations thereof. The solid support may be encased or
immobilized in a vessel to enable plug-flow or continuous-flow DNA
isolation methods. Alternatively, the material of the solid support
may be packed so as to create a free-standing solid support such as
a membrane, disk, or cylinder that may be immobilized or encased in
a suitable vessel, such as a tube or plate. In one embodiment, the
solid support may be fibrous or particulate to allow optimal
contact with the biological material. The size of the solid support
suitable for use with the reagents of this invention may vary
according to the volume of biological material. For example, glass
fiber membranes may be cut to different sizes, in order to allow
for the binding, purification and elution of different quantities
of DNA.
[0062] In one embodiment, the solid support may be a material that
permits the binding of nucleic acids to the solid support instead
of other biological contaminants in the presence of the
aforementioned DNA Lysing Solution described above. Such a solid
support may be a silica-based or borosilicate glass fiber material.
Glass fiber materials provide a better yield because of the
specific binding properties to the electropositive silicon and
boron atoms, and because of hydrogen bonding properties of the
silicate surface. Because of the specificity of silica for nucleic
acids, more DNA is bound relative to other contaminants and the
eluted product is made more substantially pure.
[0063] The shape of the solid support suitable for use with the
reagents of this invention may be, for example, a sheet, a precut
disk, cylinder, single fiber, or a solid support composed of
particulates. The material of the solid support may be packed so as
to create a free-standing solid support such as a membrane, disk,
or cylinder that may be immobilized or encased in a suitable
vessel. If necessary, the solid support is contained in an
appropriate vessel, e.g., a paper form (such as a Guthrie card), a
microcentrifuge tube, a spin tube, a 96-well plate, a chamber, or a
cartridge. If the solid support comprises fibers, it may be encased
in a suitable vessel so as to pack the fibers appropriately, allow
for optimal nucleic acid binding, and the washing away of
contaminants such as protein, phospholipids, etc.
[0064] The solid support may be pre-treated with RNase solution in
order to degrade RNA present in the biological sample.
Additionally, using the pre-treated columns eliminates the need for
a separate RNase digestion step, as is typically required in
conventional methods.
[0065] Optionally, purification may be improved by the use of
RNase-treated columns (Gentra Systems, Inc.). The RNase-treated
columns degrade RNA present in the biological sample. Additionally,
using the pre-treated columns eliminates the need for a separate
RNase digestion step, as is typically required in conventional
methods. In another embodiment of the invention, the DNA Lysing
Solution may be added directly to the material (e.g., fibers, etc.)
used in making the solid support and may be allowed to dry before
it is made into the final user-ready form (e.g., paper, swab, disk,
plug, column, etc.). The use of RNase-treated columns (Gentra
Systems, Inc.) reduces the number of steps in the purification
process as well as time to process DNA samples.
[0066] In order that the invention may be better understood,
specific embodiments for vessels that contain the solid support
will now be described in more detail. In one embodiment of this
invention, the vessel is a cartridge equipped with one or more
inlet ports or pierceable septa at the top. The inlet ports are
attached to vessels upstream containing the sample or reagents
through a connector, such as a female Luer-Lock. One inlet, the
sample port, is used for the application of the biological sample
to the solid support. An optional feature on the sample port is a
self-sealing mechanism that seals the sample port after sample has
been transferred through it. The second inlet serves as a reagent
port. An optional feature on both inlet ports is a protective
breakaway seal. Furthermore, the inlet ports, breakaway seals and
diffuser may be housed in an optional screw-cap. The versatility
and effectiveness of the DNA Lysing Solution lends itself to two
viable alternative methods for DNA isolation. In the first method,
the biological material is contacted with the DNA Lysing Solution
before it is contacted with the solid support. In one embodiment,
when the biological material comprises cellular or viral material,
the DNA Lysing Solution is used to lyse the cells and release the
nucleic acids, including DNA. In the second method, the DNA Lysing
Solution is added directly to the solid support and allowed to bind
to the solid support, thereby eliminating a step, and further
simplifying the method. In this latter method, the DNA Lysing
Solution is directly applied to the solid support and then dried on
the solid support before contacting the biological material with
the treated solid support.
[0067] At the bottom of the solid support is an optional diffuser
with a pore size suitable for the dispersion and passage of
cellular debris, proteins and lipid molecules. The diffusers allow
for a uniform traversal of biological material across the cross
section of the cartridge, and prevent unequal buildup of biological
material anywhere above or below the solid support. The outlet of
the cartridge comes equipped with a protective cap that fits neatly
over the tapered barrel. The purified DNA is collected in a
collection tube that consists of a conical tube with a snap cap for
easy and contamination-free storage. The entire vessel can be
scaled in size depending on the size of the samples to be processed
and the yields needed for subsequent analysis.
[0068] In another embodiment of this invention, the vessel is a
spin tube designed to hold an insert into which the solid support
is packed. The solid support may be silica-based, cellulose,
cellulose acetate, nitrocellulose, nylon, polyester,
polyethersulfone, polyolefin, polyvinylidene fluoride, and
combinations thereof. In one embodiment, the support is a
silica-based borosilicate glass fiber membrane. The insert has a
flanged top to hold it in the spin tube and a perforated bottom to
allow fluids to pass through while supporting the solid support. A
cap tethered to the spin tube may be used to cover the insert.
Solutions, for instance, DNA Lysing Solution containing non-nucleic
acid contaminants, DNA Wash Solutions, or DNA Elution Solution
containing DNA, pass through the perforated bottom and are
collected at the bottom of the spin tube by centrifugal forces that
draw out the solutions.
[0069] In yet another embodiment, the vessel may be multiple well
plates, for example, 6, 12, 24, 48, 96, or 384 well plates where a
solid support is packed into each well. The bottom of each well has
an exit port through which solutions containing contaminants or
purified DNA can pass.
[0070] The unique combination of the solid support of choice with
the unique reagents--DNA Lysing Solutions, DNA Spiking Solutions,
DNA Wash Solution, and DNA Elution Solution--results in the
isolation of substantially pure, undegraded DNA. The properties of
the DNA Lysing and Spiking Solutions as described above permit
superior lysing and binding of the nucleic acids to the solid
support, while the DNA Elution Solution and optional RNase-treated
column permits the preferential elution of the DNA from the solid
support.
[0071] Kits
[0072] The present invention also provides kits for purifying DNA
that contain instruction means for preparing substantially pure and
undegraded DNA from a biological sample and one or all of the
following: DNA Lysing Solution, either as a separate solution or
pretreated onto a solid support, a solid support either untreated
or treated with a DNA Lysing Solution, a DNA Spiking Solution, a
DNA Wash Solution, a DNA Elution Solution or any combination
thereof. In addition, the kit can include auxiliary components such
as a proteinase K solution, a vessel to contain the solid support,
vessels to contain substantially pure and undegraded DNA, and
combinations thereof. Substantially pure, undegraded DNA is DNA
that is suitable for use in subsequent analyses known to those with
skill in the art, for example, nucleic acid quantification,
restriction enzyme digestion, DNA sequencing, hybridization
technologies, such as Southern Blotting, etc., and amplification
methods such as Polymerase Chain Reaction (PCR), Ligase Chain
Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA),
Self-sustained Sequence Replication (SSR or 3SR), Strand
Displacement Amplification (SDA), and Transcription Mediated
Amplification (TMA), Quantitative PCR (qPCR), or other DNA
analyses.
[0073] The reagents, methods and kits of the present invention
provide substantially pure and undegraded DNA with relatively
little contaminating RNA or other impurities such that the DNA may
be used in downstream processes such as nucleic acid
quantification, restriction enzyme digestion, DNA sequencing,
hybridization technologies, such as Southern Blotting, etc., and
amplification methods such as Polymerase Chain Reaction (PCR),
Ligase Chain Reaction (LCR), Nucleic Acid Sequence Based
Amplification (NASBA), Self-sustained Sequence Replication (SSR or
3SR), Strand Displacement Amplification (SDA), and Transcription
Mediated Amplification (TMA), Quantitative PCR (qPCR), or other DNA
analyses.
[0074] Methods
[0075] The present invention also provides methods for purifying
DNA from biological material. The reagents and solid supports
taught in the invention lend themselves to alternate isolation
methods.
[0076] In one embodiment of a method of the present invention, the
biological material is contacted with the DNA Lysing Solution
before it is contacted with the solid support. The DNA Lysing
Solution is used to lyse the biological material and release the
DNA before adding it to the solid support. Additionally, the DNA
Lysing Solution prevents the deleterious effects of harmful enzymes
such as DNAses. The DNA Lysing Solution may be successfully used to
lyse cultured cells or white blood cells in pellets, or to lyse
cells adhering to or collected in culture plates, such as standard
96-well plates. If the biological material is composed of tissue
chunks or small particles, the DNA Lysing Solution may be
effectively used to grind such tissue chunks into a slurry because
of its effective lysing capabilities. The DNA Lysing Solution
volume may be scaled up or down depending on the cell numbers or
tissue size. Once the biological material is lysed, a DNA Spiking
Solution is added to the lysate and then added to the solid
support.
[0077] In another embodiment, the DNA Lysing Solution may be added
directly to the solid support, thereby eliminating a step, and
further simplifying the method. In this latter method, the DNA
Lysing Solution may be applied to the solid support and then dried
on the solid support before contacting the biological material with
the treated solid support.
[0078] RNase may be added either directly to the solid support to
pre-treat the column or added to the Lysing Solution to degrade RNA
present in the biological sample. Using the pre-treated columns
and/or RNase added to the Lysing Solution, eliminates the need for
a separate lysis and/or RNase digestion steps, as is typically
required in conventional methods.
[0079] When the biological materials comprise cellular or viral
materials, direct contact with the DNA Lysing Solution, or contact
with the solid support pre-treated with the DNA Lysing Solution
and/or an RNase solution, causes the cell and nuclear membranes, or
viral coats, to solubilize and/or rupture, thereby releasing the
nucleic acids as well as other contaminating substances such as
proteins, phospholipids, etc.
[0080] In a third embodiment, the reagents of the present invention
could be used in a direct-lysis method, which may be useful for
example, with whole blood. This method eliminates the need to
perform a red blood cell lysis step common in most other whole
blood purification methods. In this method, a first DNA Lysing
Solution is added to the biological material. The first DNA Lysing
Solution comprising an alkali metal salt and a non-ionic detergent.
A second DNA Lysing Solution comprising an alkali metal salt and an
anionic detergent is added to the biological material. The use of
two lysis solutions is helpful in order to successfully lyse and
solubilize all blood cells during the direct lysis of large volumes
of blood.
[0081] In a fourth embodiment of the method of the present
invention, DNA can be effectively purified from, for example, fixed
cells or cervical swab media or fixed cervical cells. The Lysing
Solution comprising an anionic detergent is simply added to the
biological material, pipetted up and down to lyse the cells and
denature proteins. Following the lysis step, using proteinase K
solution may be essential for certain types of sample, for example,
cervical swab media. For these samples, proteinase K is added and
the sample mixed by vortex. Samples may be incubated for 2-3 hours
at 65.degree. C.
[0082] After, the lysis steps of any of the above methods, a DNA
Spiking Solution may be used to dehydrate the DNA molecules such as
to cause quantitative binding of DNA to the solid phase. Next, the
biological material is optionally removed by suitable means such as
centrifugation, pipetting, pressure, vacuum, or by the combined use
of these means with a DNA Wash Solution such that the nucleic acids
are left bound to the solid support. The remainder of the
non-nucleic acid biological material that includes proteins,
phospholipids, etc., may be removed first by centrifugation. By
doing this, the unbound contaminants in the lysate are separated
from the solid support.
[0083] Subsequently, the bound DNA may be eluted using an adequate
amount of a DNA Elution Solution known to those skilled in the art.
The solid support may then be centrifuged, or subjected to pressure
or vacuum, to release the DNA from the solid support and can then
be collected in a suitable vessel.
[0084] As another aspect of this invention, a kit is provided that
includes specific protocols, which in combination with the reagents
and optionally the solid supports described herein, may be used for
purifying DNA from biological materials according to the methods of
the invention.
[0085] This invention will be further described by reference to the
following detailed examples. These examples are offered to further
illustrate the various specific and illustrative embodiments and
techniques. It should be understood, however, that many variations
and modifications may be made while remaining within the scope of
the present invention.
[0086] All of the raw materials mentioned below are readily
available from commercial sources such as Sigma Chemical Company,
St. Louis, Mo. All percentages are in volume per volume, based on
the total volume of the reagent, unless specified otherwise.
EXAMPLES
Example 1
Cost Analysis
[0087] In order to produce the best quality solid phase DNA
purification product, the product must function exceptionally well
in several respects. The solid phase DNA purification product must
effectively isolate a pure DNA sample from a variety of sample
types and result in the highest possible yields of DNA. It must be
user friendly, meaning the steps must not be too onerous, and the
components must not be toxic and can be disposed of easily.
Further, the product must be economical for the user. Therefore,
finding cost effective components for the solutions was essential.
Table 1 shows the cost for each of the salts evaluated herein.
1TABLE 1 Cost Salt Amount (grams) Cost ($) BeCl.sub.2 25 600.00
CaCl.sub.2 500 105.00 CsCl 500 340.00 KCl 500 30.00 LiBr 500 65.00
LiCl 500 60.00 LiF 50 400.00 LiI 250 330.00 MgCl.sub.2 500 50.00
NaCl 500 24.00 NH.sub.4Cl 500 22.00
[0088] Although the lithium salts work well for the methods of the
present invention, the lithium salts LiF and LiI are expensive, and
additionally, LiF is quite hazardous. LiCl and LiBr both work well
with the methods of the present invention and cost about the same
at $60-65 per 500 grams. KCl, NaCl, and NH.sub.4Cl are all
economical, but do not result in the DNA yields desired.
Example 2
Solubility and Heat of Solution Data for Chloride Salts and
Compared to LiCl and LiBr Salts for Solid Phase DNA Purification
Procedure
[0089] The solubility and performance of several chloride salts was
examined and compared to two lithium salts, lithium chloride and
lithium bromide. The DNA Lysing Solution was prepared using other
chloride salts, in order to examine maximum solubility obtainable
in both the buffer and detergent based DNA Lysing Solution. Table 2
shows the approximate maximum solubility as measured in this work
and compares it to tabulated solubility data extrapolated to
20.degree. C., as well as tabulated heat of solution data as
obtained from the Handbook of Chemistry and Physics (62nd edition,
CRC Press, Boca Raton, Fla.).
[0090] The solubility of most of the chloride salts studied in the
Lysing Solution, were comparable to the lithium chloride and
bromide salts at a concentration of 4 M, except for those of
potassium chloride and ammonium chloride. The expected solubility
of potassium chloride was low, when compared to the solubility of
the other salts, at only approximately 3 M in aqueous solution. The
other salts were expected to be more soluble than 4 M in aqueous
solution, however it was observed that the ammonium chloride salt
is only soluble to approximately 3 M, when compared to the expected
solubility of about 7 M. This could be due to the presence of
surfactant, for example Triton X-100 (5%) and DGME (5%) in
solution, since the combination for these components in solution
was 10%. Except for the salts, the remainder of the DNA Lysing
Solution was kept constant within all solutions and contained, 5%
Triton X-100, 5% DGME (diethylene glycol monoethyl ether), 10 mM
EDTA, 10 mM TCEP, 1% sodium tungstate, in 100 mM TRIZMA at pH
8.8.
[0091] The large exothermic heats of solution for lithium chloride
and lithium bromide in water provided substantiation as to the
large solubilities demonstrated by lithium salts in general. The
other chloride salts in this example exhibited endothermic heats of
solution, indicating that solvation of these salts was not as
favorable in aqueous solution as for the lithium salts.
2TABLE 2 Heat of Measured Reported Aqueous Solubility Reported
Solubility Solution in Lysing Solubility in (kcal/ Type of Salt
Solution in Water* Alcohol(s) mole) Heat LiCl at least 18M soluble
in -8.85 exothermic 4M alcohol LiBr at least 19M soluble in -11.67
exothermic 4M alcohol & ethanol CsCl at least 11M very +4.25
endothermic 4M soluble in alcohol NH4Cl 3M 7M insoluble +3.53
endothermic ethanol NaCl at least 6M slightly sol +0.93 endothermic
4M in alcohol KCl less than 3M slightly sol +4.12 endothermic 3M in
alcohol, soluble in ethanol *Note: Data extrapolated to 20.degree.
C. from tabulated data in Handbook of Chemistry and Physics.
Example 3
Evaluation of Detergents for DNA Purification
[0092] Much time and effort was spent screening detergents that
would adequately lyse cells and also stay soluble in the DNA Lysing
Solution of the present invention. The Tween, Triton, Tergitol,
Nonidet and Igepal family of detergents were examined along with a
number of surfactant compounds.
[0093] It was discovered that many of these detergent formulations,
when mixed with the other ingredients of the DNA Lysing Solution,
saponified or degraded over time and precipitated out of solution.
For example, this occurred with 10% Tween-20 (polyoxylene sorbitan
monolaurate). Similar performance was found using an equal mixture
of Triton X-100 (t-octylphenoxy polyethoxyethanol) and Tween-20,
though this mixture was stable for longer periods of time. This
mixture, however, eventually saponified and separated. DGME
(diethylene glycol monoethyl ether) was chosen for testing as a
surfactant to help solubilize the salt and detergent combinations.
A mixture of Triton-X at 5% and DGME at 5% was found to work
particularly well to maintain solubility of the first DNA Lysing
Solution.
[0094] DNA extraction from cervical cells proved to be particularly
challenging, as Proteinase K digestion is required for robust
collection of DNA from this sample type. The presence of sodium
dodecyl sulfate (SDS) worked best in the Lysing Solution in order
to obtain the highest Proteinase K activity. The optimal
concentration was determined by systemically testing a variety of
DNA Lysing Solution with Proteinase K treatment. Higher
concentrations of SDS (greater than 0.1% SDS final concentration)
precipitate out of the Lysing Solution at temperatures above or
below room temperature, causing the Proteinase K activity to be
lower than required. Lower concentrations of SDS (less than 0.1%
final concentration) disallow full Proteinase K activity, which is
helpful for complete lysis of fixed cell preparations like cervical
cells. DGME was also included in this second DNA Lysing Solution to
allow for the SDS and LiCl salt to remain soluble through the
lysing, spiking and binding processes.
[0095] The major technical hurdle for the development of the direct
lysis process for whole blood was the lysis efficiency of
detergents in whole blood. The initial testing focused on the use
of the non-ionic detergent Triton X-100 for cell lysing. The use of
this detergent resulted in low DNA recovery in the direct lysis
method, probably due to the interference of red blood cells and red
blood cell lysate being present in the solution. Triton X-100 is
not a protein denaturant and due to the high levels of cellular
contamination it was decided to try a detergent that was a proven
protein denaturant. To increase the lysis efficiency, the anionic
detergent sodium dodecyl sulfate (SDS) was tested in place of the
non-ionic detergent. This increased DNA recovery but resulted in
clogging of the purification membrane. Previous work with cell
pellets had shown that a combination of both detergents resulted in
increased DNA recovery. The use of both detergents in the direct
lysis method resulted in an increase in DNA recovery and the
elimination of membrane clogging. Testing the order of detergent
addition (anionic/non-ionic vs. non-ionic/anionic) showed that for
optimal DNA recovery, the non-ionic detergent had to be added
before the anionic detergent.
Example 4
Isolation of DNA from Blood by Direct Lysis Method
[0096] An equal volume of the first DNA Lysing Solution (6 M LiCl,
5% Triton X-100, 5% DGME (diethylene glycol monoethyl ether), 10 mM
EDTA, in 100 mM Tris at pH 8.8) was added to the whole blood
samples. The resulting solution was pipetted up and down five
times, pulse vortexed five times at high speed, pipetted up and
down 20 times, and then pulse vortexed again five times, in order
to achieve complete homogenization. After homogenization, two blood
volumes of the second DNA Lysing Solution (25 mM Citrate, 2 M LiCl,
0.1% SDS, 30% DGME, in 25 mM Tris at pH 9.1) were added to the
samples. The samples were mixed following the same process as
described for the First DNA Lysing Solution. After homogenization
and lysis with the two lysis solutions, four blood volumes of DNA
Spiking Solution (10 M LiCl, 10% DGME, 100 mM Tris at pH 7.9) were
added to each sample. The resulting solution was pipetted up and
down five times to mix.
[0097] After homogenization, 600 .mu.L of each homogenized lysate
was pipetted onto each purification column. The purification column
contained a borosilicate glass fiber membrane (Whatman D glass
fiber membrane) within a basket and placed inside a vacuum elution
station. The lysates were pulled through the membrane using vacuum
filtration. The remaining lysates were added, 600 .mu.L at a time,
to the column and removed using vacuum filtration.
[0098] After vacuum filtration of each lysate and subsequent
binding of DNA to the borosilicate membrane surface, 400 .mu.L of
DNA Wash Solution (5 mM EDTA, 70% ethanol, in 100 mM Tris HCl at pH
7.6) was added to the column material and removed with vacuum for
30 seconds. The DNA Wash Solution addition and vacuum filtration
steps were repeated once into the same elution tube.
[0099] To elute the DNA from the solid support, the basket
containing the membrane was transferred to a microfuge tube and 50
.mu.L of DNA Elution Solution (1 mM EDTA in 10 mM Tris at pH 7.5)
solution was added to the column material, incubated for five
minutes and spun at maximum speed for one minute. The addition of
DNA Elution Solution, the five minute incubation and centrifugation
steps were repeated once. Purifications from five blood volumes
were performed (300 mL, 500 mL, 1 mL, 1.5 mL and 3 mL), and were
electrophoresed and compared to 200 ng of Lambda Hind III ladder.
The results indicated that intact, high molecular weight DNA could
be purified from whole blood using the direct lysis method from a
large range of whole blood input volumes.
Example 5
Isolation of DNA from Blood Using RBC Lysis Method
[0100] One embodiment of the invention used for the isolation of
DNA from a whole blood sample was to first lyse the red blood cells
in the blood sample, followed by pelleting of the white blood cells
from the resulting red blood cell lysate using centrifugation.
After the red blood cell lysate supernatant was poured off and the
white blood cell pellet was washed and repelleted, 200 .mu.L of DNA
Lysing Solution (6 M LiCl, 5% Triton X-100, 5% DGME (diethylene
glycol monoethyl ether), 10 mM EDTA in 100 mM Tris at pH 8.8) was
added to the pellet, to lyse the white blood cells by vortexing and
pipetting in the DNA Lysing Solution to thoroughly homogenize the
sample. After homogenization, 400 .mu.L of DNA Spiking Solution (10
M LiCl, 10% DGME, 100 Tris at pH 7.9) was added to the white blood
cell lysate and the mixture was added to the purification column
containing a borosilicate glass fiber membrane (Whatman D glass
fiber membrane) within a basket and placed inside a 2 mL microfuge
tube. The microfuge tube was then spun at 7000.times.g for 1
minute. After lysate centrifugation, 400 .mu.L of DNA Wash Solution
(5 mM EDTA, 70% ethanol, in 100 mM Tris HCl at pH 7.6) was added to
the column material and spun at maximum speed in a microcentrifuge
for one minute. The DNA Wash Solution addition and centrifugation
steps were repeated once. To elute the DNA from the solid support,
the basket containing the membrane was transferred to a new
microfuge tube and 50 .mu.L of DNA Elution Solution (1 mM EDTA in
10 mM Tris at pH 7.5) was added to the column and spun at maximum
speed in a microcentrifuge for one minute. The DNA Elution Solution
addition and centrifugation steps were repeated once into the same
elution tube.
[0101] In a direct comparison with the QIAamp Blood DNA Midi Kit
(Qiagen Inc., Germany) the Versagene Blood DNA mean yield is
approximately double the QIAamp mean yield value for 2 mL blood
samples as shown in FIG. 1. The percent of theoretical yield
calculation is based on the assumption that six pg of genomic DNA
are present per white blood cell. Yields as high as 70-80% of
expected theoretical yields were obtained in some cases using the
RBC Lysis Method as shown in FIG. 2 using two mL blood samples,
where DNA Lysing Solution formulations were varied for pH and LiCl
concentration.
Example 6
Isolation of DNA from Cervical Cells
[0102] Since it is typically difficult to extract nucleic acid from
cervical cell swab media or fixed cervical cells, it can be used
effectively to illustrate the efficiency of the compositions and
methods of the invention. The skilled artisan will understand,
however, that the disclosed compositions and methods may also be
effectively employed using a broad range of biological samples and
that the invention is not to be limited to use with any sample
type.
[0103] Cervical cell samples were collected in ThinPrep.RTM. Pap
TeSt.TM. (Cytyc, Boxborough, Mass. and spun down 2,200.times.g for
5 minutes and then resuspended in PBS to wash cells. The
resuspended cells were placed into microfuge tubes and spun down at
14,000.times.g for 15 seconds. The PBS supernatant was removed from
the cervical cell pellet. Each tube was vigorously vortexed to
resuspend the cells; this greatly facilitated cell lysis. 200 .mu.L
of Lysing Solution (25 mM Citrate, 2 M LiCl, 0.1% SDS, 30% DGME, in
25 mM Tris at pH 9.1) was added to the sample to the resuspended
cells and pipetted up and down to lyse the cells and denature
proteins. 1.0 .mu.L of Proteinase K Solution (20 mg/ml) was added
to the cell lysate and mixed by brief vortex; then incubated the
samples at 65.degree. C. for 2-3 hours. 400 .mu.L of 100%
Isopropanol DNA Spiking Solution was added to the sample. The
samples were pulse vortexed for 30 seconds and then allowed to sit
for two minutes prior to loading on to columns. The sample was
loaded on to a pre-treated RNase A treated purification column
(Gentra Systems, Inc.). The sample was centrifuged at 7,000.times.g
for one minute. The basket containing the purification column was
transferred to a new clear tube. 200 .mu.L of DNA Wash Solution (5
mM EDTA, 70% ethanol, in 100 mM Tris HCl at pH 7.6) was added to
each purification column. The purification columns were then
centrifuged at 7,000.times.g for one minute. An additional 200
.mu.L of DNA Wash Solution was added to each purification column.
The purification columns were then centrifuged at 7,000.times.g for
two minutes. The basket containing the purification column was
carefully transferred to a new clear tube. 50 .mu.L of DNA Elution
Solution (1 mM EDTA in 10 mM Tris at pH 7.5) was added to each
purification column and allowed to incubate at room temperature for
five minutes. The purification columns were then centrifuged at
14,000.times.g for one minute.
[0104] In a direct comparison with the Puregene Liquid Chemistry
(Gentra Systems, Inc.), the Versagene Solid Phase DNA Chemistry
(Gentra Systems, Inc.) mean yield is similar regardless of starting
cervical sample type, as shown in the Tables below.
3TABLE 3 % Yield of Pooled Puregene Versagene Versagene SurePath
260/280 260/280 Compared Pellets Puregene Ratio Versagene Ratio to
Puregene Apr. 30, 2004 16.88 1.78 4.94 1.82 29% May 3, 2004 14.78
1.82 2.48 1.74 17% May 6, 2004 23.40 1.85 3.45 1.82 15% May 20,
2004 10.63 1.81 4.93 1.79 46% May 24, 2004 22.72 1.82 3.17 1.70 14%
May 27, 2004 13.44 1.81 1.53 1.62 11% Jun. 2, 2004 31.62 1.79 6.23
1.79 20% Jun. 4, 2004 15.31 1.83 8.72 1.78 57% Jun. 8, 2004 39.98
1.82 21.37 1.82 53% Jun. 9, 2004 23.10 1.83 7.58 1.82 33% Jun. 15,
2004 41.20 1.81 6.68 1.76 16% Jun. 16, 2004 18.30 1.82 4.66 1.77
25% Jun. 17, 2004 14.74 1.79 2.89 1.73 20% Jun. 18, 2004 12.05 1.79
2.22 1.39 18% Jun. 29, 2004 18.30 1.85 4.33 1.76 24% Jun. 30, 2004
8.88 1.77 3.10 1.62 35% Jul. 2, 2004 34.67 1.81 7.86 1.77 23% Jul.
8, 2004 7.03 1.82 2.66 1.85 38% Average 20.39 1.81 5.49 1.74 27%
StdDev 10.30 0.02 4.49 0.11 NA
[0105]
4TABLE 4 % Yield of Pooled Puregene Versagene Versagene ThinPrep
260/280 260/280 Compared Liquid Puregene Ratio Versagene Ratio to
Puregene Jun. 18, 2004 7.71 1.80 8.30 1.73 108% Jul. 13, 2004 6.45
1.82 5.64 1.77 87% Average 7.08 1.81 6.97 1.75 98% StdDev 0.89 0.01
1.88 0.03 NA
[0106]
5TABLE 5 % Yield of Pooled Puregene Versagene Versagene SurePath
260/280 260/280 Compared Liquid Puregene Ratio Versagene Ratio to
Puregene Jun. 18, 2004 5.75 1.60 4.07 1.47 71% Jun. 21, 2004 6.00
1.82 4.72 1.81 79% Average 5.88 1.71 4.40 1.64 75% StdDev 0.18 0.16
0.46 0.24 NA NA = Not applicable
[0107] All publications, patents and patent applications are
incorporated herein by reference. While in the foregoing
specification this invention has been described in relation to
certain preferred embodiments thereof, and many details have been
set forth for purposes of illustration, it will be apparent to
those skilled in the art that the invention is susceptible to
additional embodiments and that certain of the details described
herein may be varied considerably without departing from the basic
principles of the invention.
* * * * *