U.S. patent application number 11/913094 was filed with the patent office on 2009-01-29 for decontamination solution and its use for denaturation, modification, degradation, solubilisation and removal of proteins, nucleic acid molecules and microorganisms.
This patent application is currently assigned to MULTIBIND BIOTEC GMBH. Invention is credited to Karlheinz Esser, Richard Lisowsky, Thomas Lisowsky.
Application Number | 20090028961 11/913094 |
Document ID | / |
Family ID | 36791064 |
Filed Date | 2009-01-29 |
United States Patent
Application |
20090028961 |
Kind Code |
A1 |
Lisowsky; Thomas ; et
al. |
January 29, 2009 |
Decontamination solution and its use for denaturation,
modification, degradation, solubilisation and removal of proteins,
nucleic acid molecules and microorganisms
Abstract
The invention concerns a three component system comprising
surface-active substances, vitamins and metal ions for efficient
destruction and removal of contaminating proteins, nucleic acids
and microorganisms from surfaces like for example laboratory
benches, floors, equipment and instruments. These non-corrosive and
non-toxic solutions for removal of proteins, nucleic acids and
microorganisms are applied by spraying, rubbing or immersion of
contaminated surfaces thereby destroying, solubilizing inactivating
and removing proteins and nucleic acids. In that way also
microorganisms are killed with high efficiency and at the same time
all genetic information is inactivated.
Inventors: |
Lisowsky; Thomas; (Monheim,
DE) ; Esser; Karlheinz; (Monchengladbach, DE)
; Lisowsky; Richard; (Kamen, DE) |
Correspondence
Address: |
JOYCE VON NATZMER;PEQUIGNOT + MYERS LLC
200 Madison Avenue, Suite 1901
New York
NY
10016
US
|
Assignee: |
MULTIBIND BIOTEC GMBH
Dortmund
DE
|
Family ID: |
36791064 |
Appl. No.: |
11/913094 |
Filed: |
May 2, 2006 |
PCT Filed: |
May 2, 2006 |
PCT NO: |
PCT/DE2006/000758 |
371 Date: |
June 30, 2008 |
Current U.S.
Class: |
424/617 ;
424/600; 424/722; 510/109 |
Current CPC
Class: |
A61P 31/02 20180101;
A01N 31/14 20130101; A01N 59/16 20130101; A61P 43/00 20180101; A01N
2300/00 20130101; A01N 2300/00 20130101; A01N 2300/00 20130101;
A01N 43/08 20130101; A01N 2300/00 20130101; A01N 43/08 20130101;
A01N 43/78 20130101; A01N 57/16 20130101; A01N 57/16 20130101; A01N
59/16 20130101; A01N 43/78 20130101 |
Class at
Publication: |
424/617 ;
424/600; 424/722; 510/109 |
International
Class: |
A01N 55/02 20060101
A01N055/02; C11D 3/02 20060101 C11D003/02; A01P 1/00 20060101
A01P001/00 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 30, 2005 |
DE |
10 2005 020 327.2 |
Claims
1. Decontamination solution comprising a synergistic mixture of a)
at least one vitamin in concentrations from 1 mM to 1000 mM, and b)
at least one metal ion in concentrations of 1 mM to 100 mM, and c)
at least on surface-active substance in concentrations of 0.1% to
10% (weight) in relation to the total solution.
2. Decontamination solution according to claim 1, wherein the
mixture has a pH value ranging between pH 2 and pH 8.5.
3. Decontamination solution according to claim 1, wherein the
mixture additionally comprises a buffer system including carbonates
and derivatives of succinic acid.
4. Decontamination solution according to claim 1, wherein said
vitamins, their related salts or acidic derivates are at least one
compound selected from the group of the water-soluble vitamins
having the characteristics of an antioxidant.
5. Decontamination solution according to claim 1, wherein the metal
ions are taken from metals from 4th group and sub-groups I, II, or
VIII of the periodic table of the elements.
6. Decontamination solution according to claim 5, wherein the
metals are provided as salts of their acids or bases.
7. Decontamination solution according to claim 1, wherein said
surface-active substance is at least one compound of the group of
the anionic, non-ionic, amphometeric or cationic tensides or
suitable mixtures thereof.
8.-10. (canceled)
11. Method for denaturation, modification, degradation,
solubilisation and removal of proteins, nucleic acid molecules and
microorganisms from surfaces comprising providing the
decontamination solution according to claim 1 and treating surfaces
for denaturation, modification, degradation, solubilisation and
removal of proteins, nucleic acid molecules and microorganisms from
said surfaces.
12. Buffer system for adjusting the pH value of a solution,
comprising carbonates and derivatives of succinic acid.
13. Buffer system according to claim 12, characterized in that said
carbonates and derivatives of succinic acid are each provided in
concentrations between 1 mM and 500 mM.
14. Buffer system according to claim 12 wherein said solution is
adjusted by the buffer system to a pH value ranging from pH 2 to
8.5.
15. Method for adjusting the pH value of a solution which comprises
at least a mixture of a) at least one vitamin, and b) at least one
metal ion, and c) at least one surface-active substance, wherein
said mixture is adjusted by the buffer system comprising carbonates
and derivatives of succinic acid to a pH value ranging from 2 to
8.5.
16. Decontamination solution according to claim 2, wherein the
mixture has a pH value ranging between pH 3 and 7.
17. Decontamination solution according to claim 2, wherein the
mixture has a pH value ranging between pH 4 and 6.
18. Decontamination solution according to claim 3, wherein said
carbonates and derivatives of succinic acid are each present in-a
concentration between 1 mM and 500 mM.
19. Buffer system according to claim 14, wherein said solution is
adjusted by the buffer system to a pH value ranging from pH 3 to
7
20. Buffer system according to claim 14, wherein said solution is
adjusted by the buffer system to a pH value ranging from pH 4 to
6
21. The method of claim 15, wherein said mixture is adjusted to a
pH value ranging from 3 to 7.
22. The method of claim 15, wherein said mixture is adjusted to a
pH value ranging from 4 to 6.
Description
BACKGROUND OF THE INVENTION
[0001] The invention concerns a decontamination solution for the
treatment of surfaces that are contaminated by unwanted proteins,
nucleic acid molecules or microorganisms. The invention further
concerns the use of said decontamination solution and a suitable
buffer system.
[0002] The dynamic developments in molecular biology stresses the
importance of new methods and techniques for detection and
amplification of DNA molecules or proteins. [Sambrook, J. et al.,
eds (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.]. Latest
examples concern medical diagnostics, forensic analysis and
biomedical research.
[0003] With the invention of the polymerase chain reaction (PCR) it
is even possible to detect single molecules. A new problem of the
extreme sensitivity of these new detection methods is the
contamination of surfaces in laboratories, on equipment or working
materials with unwanted nucleic acid molecules, proteins or
microorganisms.
[0004] In addition microbial contaminations cause severe problems
and commercial losses for example in food processing and
technology, in production facilities, in hospitals, Hygiene
institutes and also in the general household.
PRIOR ART
[0005] Therefore already for a longer time many different
decontamination solutions exist that use aggressive chemical agents
like for example formaldehyde, alcohols, phenols, sodium azide,
sodium hypochloride against microorganisms or strong oxidizing
agents like for example hypochloride, bleaching substances or
mineralic acids that denature proteins and modify nucleic acids
thereby rendering them inappropriate for amplification methods like
for example the polymerase chain reaction (PCR), nick translation
by the klenow polymerase, strand-displacement amplification, ligase
chain reaction, transcription-mediated amplification,
rolling-circle-amplification and many others more.
[0006] The aggressive chemicals and strongly oxidizing agents used
for decontamination cause permanent modifications and denaturations
of proteins and destabilize and modify the DNA double strand
thereby blocking amplification reactions. In general modifications
and oxidative damages are introduced at especially reactive
chemical groups of the target molecules.
[0007] Therefore currently such aggressive chemical solutions are
applied for washing and rinsing of equipment, instruments and
surfaces.
[0008] The major disadvantages of these solutions and methods are
the only selective actions against proteins, DNA or microorganisms
and the incomplete removal of all nucleic acids molecules, the
remaining modified molecules, the only partial degradation and the
corrosive effect of the applied chemicals against equipment,
instruments, surfaces and also against skin and mucous membranes of
the customer.
[0009] A limited improvement of the efficiency of these methods was
achieved by combining the agents in the solution with
surface-active chemicals like detergents. Still the problem of the
aggressive chemical substances and the incomplete destruction and
removal of nucleic acids, proteins and microorganisms remains.
[0010] The commercial interest in such solutions is stressed by the
already available and marketed products for decontamination
solutions against DNA and protein contaminations as well as
anti-microbial agents listed under a large number of different
trade marks.
[0011] The disadvantages of the currently known decontamination
solutions and methods are their only limited action against
different biological molecules like proteins or nucleic acids or
only anti-microbial actions and the highly corrosive and aggressive
chemical potentials in combination with harmful properties that
cause severe health problems. Especially for the decontamination of
microorganisms there are currently only solutions available that
are killing the microorganisms but that do not inactivate or
degrade the genetic information, extrachromosomal DNA or
proteins.
[0012] It is well known that physiological concentrations of
micromolar amounts of antioxidants in combination with divalent
metal ions result in partial damages and partial breakage of
strands in nucleic acids molecules (Podiatry S. J., Katz A., Wang
Y., Eck P., Kwon O., Lee J. H., Chen S., Corpse C, Data A, Data S K
and Levine M. (2003) Vitamin C as an antioxidant: evaluation of its
role in disease prevention. J. Am. Coll. Nut. 1, 18-35; Blocking
O., Virolainen E., Fagerstedt K. V. (2003) Antioxidants, Oxidative
Damage and Oxygen Depriviation Stress: a Review. Annals Botany
91:179-194; Veal J. M., Merchant K. & Rill R. L. (1991) The
influence of reducing agent and 1,10-phenanthroline concentration
on DNA cleavage by phenanthroline+copper. Nucl Acids Res Vol. 19,
No. 12, 3383-3388). These are only selected and isolated results
that can only be applied to specific single cases.
[0013] The latest findings in modern molecular biology and gene
technology demonstrate that already the genetic information alone
single genes or even fragments thereof as well as specific proteins
are sufficient to cause diseases or unwanted changes in the genetic
information.
[0014] Therefore in practice there is a need for new improved
protocols, agents, methods and solutions for efficient and at the
same time gentle complete decontamination of surfaces and equipment
from proteins, nucleic acids and microorganisms.
SUMMARY OF THE INVENTION
[0015] An object of the here described invention is therefore to
overcome the disadvantages of the prior art and to develop new
methods and solutions that do not use aggressive chemicals or
highly oxidizing agents and that in addition completely
decontaminate the treated substrates.
[0016] According to the invention, this object is achieved by a
decontamination solution comprising a synergistic mixture of
a) at least one vitamin and b) at least one metal ion and c) at
least one surface-active compound, wherein said mixture has a pH
value ranging from pH 2 to 8.5.
[0017] By applying natural anti-oxidants in combination with metal
ions and surface-active agents, surprisingly, it was found that
different vitamins in combination with metal ions and detergents
result in an extremely fast and massive strand breakages and
modifications in nucleic acid molecules and proteins. This
surprising effect leads to efficient killing of microorganisms by
inactivation and degradation of their genetic information and
proteins. Especially surprising and new is the finding that the
three component system of this invention shows inactivation and
degradation in the entire pH range of 2 to 8.5 with an essentially
comparable efficiency. Since one can work within a comparatively
mild range of pH, the solution according to the invention prevents
the surface to be treated from damage and is also skin-compatible
for the user. By spraying, rubbing or immersion in solutions of the
three components, proteins and nucleic acids are denatured,
solubilised, inactivated, degraded and removed and thereby also
microorganisms are killed with a high efficiency.
[0018] In an advantageous embodiment of the invention, it is
provided that the mixture has a pH value ranging between pH 3 and
7, preferably between pH 4 and 6. In such pH ranges the solution
according to the invention is stable over a long period of time and
allows for very efficient degradation of nucleic acids.
Additionally, the skin-compatibility of the solution according to
the invention is optimal in the range between pH 4 and 6.
[0019] Especially preferred is an advantageous embodiment in which
the mixture additionally comprises a buffer system including
carbonates and derivatives of succinic acid, each preferably in a
concentration between 1 mM and 500 mM. Using this buffer system
according to the invention with the decontamination solution
according to the invention, the pH value of the solution, which is
in an acidic range due to the dissolved components, in particular
the acidic vitamins, can be increased up to, for example, a neutral
or weak basic range without precipitation of the dissolved metal
ions.
[0020] In a preferred embodiment of the decontamination solution
according to the invention, the vitamins or their respective salts
or acidic derivates contained in the solution according to the
invention are one or several compounds and/or their related salts
selected from the group of the water-soluble vitamins with the
properties of antioxidants, like preferably vitamin C, riboflavine
and niacin. Preferably, they are used in concentrations of 1 mM to
1000 mM in relation to the total volume of the solution, in
particular in a concentration of 10 mM to 100 mM.
[0021] In a further preferred embodiment of the decontamination
solution according to the invention, the metal ions contained
according to the invention are di- and/or tri-valent ions of metals
found in the 4th group and/or sub-group I, II and VIII of the
periodic table of the elements. They are used as salts in
combination with their organic and/or inorganic acids and bases.
According to the invention, one or several compounds selected from
sub-group VIII, especially iron, cobalt, nickel, copper or zinc,
are preferred. They are preferably used in concentrations of 1 mM
to 100 mM, in relation to the total volume of the solution, in
particular in concentrations of 5 mM to 10 mM.
[0022] The surface-active substances contained in the solution
according to the invention may be, for example, anionic, non-ionic,
amphoteric or cationic inert tensides or suitable mixtures thereof
or thereunder. Especially, alkylethersulfate, alkyl- and/or
arylsulfonate, alkylsulfate, amphotensides, betaines,
alkylamidoalkylamines, alkyl substituted amino acids, alkyl
substituted imino acids, acylated amino acids, and amphotenside
combinations can be used. In principle all inert tensides are
suitable. Inert means, that they do not influence the synergistic
solution and the experimental outcome. According to the invention,
anionic and non-ionic tensides are preferred. They are preferably
used in concentrations of 0.1% to 10% (weight), in relation to the
total volume of the solution, in particular in concentrations of
0.2% to 0.5% (weight).
[0023] The decontamination solutions according to the invention may
comprise additional common inert adjuvants and additives like for
example suitable buffer substances for adjusting a specific pH
value, like, for example, Tris (Tris(hydroxymethyl)-aminomethan),
MES (2(Morpholino)ethansulfonic acid), HEPES
(2-[4-(2-Hydroxyethyl)-1-piperazinyl]-ethansulfonic acid, and/or
MOPS (3-(N-Morpholino)propansulfonic acid). The buffer systems are
used in concentrations of 1 mM to 500 mM in relation to the total
volume of the solution.
[0024] The efficient action of the new three component system is
even more surprising as it is proven that the different isolated
substances alone do not exhibit a special degradation effect and
also the mixtures of the components outside the range of the
invention are not effective or do only show an unsatisfactory
effect. Only the combination of vitamins with metal ions and
detergents, preferably in an appropriate mixture, results in a
synergistic effect and in a rapid and massive degradation of the
biomolecules. In particular, by keeping the correct preferred
concentrations, an efficient activity of the decontamination
solution according to the invention is ensured.
[0025] In principle thereby all kind of surfaces can be treated in
a very gentle way for removal of proteins, nucleic acids
contaminations and microorganisms.
[0026] In general decontamination is achieved by spraying or
rubbing the inventive solutions onto contaminated surfaces or by
immersion. A residence time of 0.5 to 2 minutes at room temperature
or slightly higher temperatures is normally sufficient for complete
denaturation, modification, degradation, solubilisation and removal
of proteins, nucleic acids and microorganisms from surfaces. The
applied methods are however variable and can be adjusted to the
different tasks.
[0027] Another purpose of this invention is the use of the
inventive decontamination solutions for denaturation, modification,
degradation, solubilisation and removal of proteins, nucleic acid
molecules and microorganisms from surfaces.
[0028] The new and advantageous buffer system with carbonate and
derivatives of succinic acid is especially suitable for the
decontamination solution of the present invention. The different
mixtures with pH values between pH 2 and 8.5 each provide clear
solutions of a light yellow to light brown color that are stable
over longer periods of time and that also show in particular in the
pH range of pH 4.5 to pH 6 a very efficient degradation of DNA
molecules as demonstrated in comparison to the strong mineralic 0.5
M phosphoric acid of pH 1.5, as shown in FIG. 5.
[0029] In an advantageous embodiment of the buffer system according
to the invention, it is provided that the carbonates and
derivatives of succinic acid are each provided in concentrations
between 1 mM and 500 mM.
[0030] The solution comprising the buffer system may be
advantageously adjusted to a pH value ranging from pH 2 to 8.5, in
particular from 3 to 7, preferably from 4 to 6. Thereby it is
ensured that the further components dissolved in the solution are
not impaired and additionally a skin-compatible solution can be
prepared.
[0031] Therefore, the invention also concerns a method for
adjusting the pH value of a solution which comprises a mixture
of
a) at least one vitamin, and b) at least one metal ion, and c) at
least one surface-active substance, wherein the mixture can be
adjusted by the buffer system according to the invention to a pH
value ranging from 2 to 8.5, in particular from 3 to 7, preferably
from 4 to 6.
BRIEF DESCRIPTION OF THE FIGURES
[0032] The invention is illustrated by nonrestrictive figures,
examples and tables shown in the following part:
[0033] In that case FIG. 1 to 5 show the efficient degradation of
DNA molecules by the three component system in comparison with
known other solutions of prior art.
[0034] FIG. 6 demonstrates the blockage of PCR amplification of DNA
molecules after the treatment with the new three component
system.
[0035] FIG. 7 shows the standard test with RNaseA for enzyme
inactivation with the new three component system.
[0036] FIG. 8 shows the efficient degradation of genomic DNA and
extra chromosomal genetic material inside microorganisms by the new
three component system.
[0037] Table 1 shows a test for the anti-microbial action of the
new three component system.
[0038] Table 2 shows the preferred basic composition and the
preferred mixtures for the three component system containing
detergents, vitamins and metal ions.
DESCRIPTION OF ADVANTAGEOUS AND PREFERRED EMBODIMENTS OF THE
INVENTION
[0039] FIGS. 1 to 5 show the efficient degradation of DNA molecules
by the new three component system in comparison with known other
solutions. Identical aliquots of DNA plasmids (YEp351) were treated
for 2 minutes with the listed solutions. Afterwards the DNA samples
were denatured and the single-stranded DNA molecules were separated
by gel electrophoresis on an agarose gel (1%). After staining with
ethidium bromide the listed pictures were produced. The control
shows intact plasmid DNA after treatment with sterile water.
Introduction of nicks into the DNA strand results in a reduction of
the size and molecular weight of the respective DNA molecules. This
effect can be identified in the gel by comparison with the control
and the molecular weight marker. In each sample 5 .mu.g DNA were
present in 5 .mu.l sterile Tris buffer (1 mM; pH 8.0) and were
treated for 2 minutes at room temperature with 5 .mu.l of the
listed solutions. Subsequently the samples were mixed with 5 .mu.l
100 mM Tris (pH 12) and bromphenol blue marker and were denatured
for 5 minutes at 95.degree. C. The denatured samples were
immediately cooled to 4.degree. C. and identical aliquots of 1
.mu.g DNA were loaded per gel lane. DNA molecules were stained with
ethidium bromide after gel electrophoresis in a 1% agarose gel and
photographed.
[0040] FIG. 1 shows the comparison of nucleic acid degradation by
vitamins and metal ions alone and by the three component system
containing vitamins, metal ions and detergents. (M: Marker DNA 1 kb
ladder; C: Control: DNA+5 .mu.l sterile H.sub.2O; 1: 5 mM
FeCl.sub.3; 2: 1 mM FAD; 3: 1 mM FAD+1 mM FeCl.sub.3; 4: 100 mM
NAD; 5: 100 mM NAD+5 mM FeCl.sub.3; 6: 100 mM thiamin; 7: 100 mM
thiamin+5 mM FeCl.sub.3; 8: 100 mM vitamin C; 9: 100 mM vitamin C+5
mM FeCl.sub.3; 10: 100 mM Na-ascorbate; 11: 100 mM Na-ascorbate+5
mM FeCl.sub.3; 12: 100 mM ascorbic acid+5 mM FeCl.sub.3). All
samples contained 0.2% Triton X-100 and 0.2% Tween 20.
[0041] FIG. 2 shows the test for nucleic acid degradation by
mixtures containing vitamin C, metal ions and detergents. L: 1 Kb
Ladder; M: Marker DNA Lambda EcoRI/HindIII; 1: 10 mM vitamin C; 2:
100 mM vitamin C; 3: 10 mM FeCl.sub.3; 4: 100 mM vitamin C+10 mM
FeCl.sub.3; 5: 10 mM ZnCl.sub.2; 6: 100 mM vitamin C+10 mM
ZnCl.sub.2; 7: DNA-OFF.TM.; C: Control: 5 .mu.l sterile H.sub.2O).
All samples contained 0.2% Triton X-100 and 0.2% Tween 20.
[0042] FIG. 3 shows the comparison of nucleic acid degradation by
mixtures containing ascorbic acid or Na-ascorbate. (M: Marker DNA
Lambda EcoRI/HindIII; C: Control: 5 .mu.l sterile H.sub.2O; 1:
DNA-OFF.TM.; 2: 100 mM HAc+10 mM FeCl.sub.330 0.2% Triton X-100; 3:
100 mM ascorbic acid+0.2% TritonX-100; 4: 100 mM ascorbic acid+10
mM FeSO.sub.4+0.2% TritonX-100; 5: 100 mM ascorbic
acid+ZnCl.sub.2+0.2% TritonX-100; 6: 100 mM Na-ascorbate+0.2%
TritonX-100; 7: 100 mM Na-ascorbate+10 mM FeCl.sub.3+0.2% Triton
X-100; 8: 100 mM Na-ascorbate+ZnCl.sub.2+0.2% Triton X-100).
[0043] FIG. 4 shows the comparison of nucleic acid degradation by
mixtures containing mineralic acids, ascorbic acid or Na-ascorbate
(pH 6 to 8.5). (M: Marker DNA Lambda EcoRI/HindIII C: Control:
DNA+5 .mu.l sterile H.sub.2O; 1: RNase-OFF.TM. (sample 1); 2:
RNase-OFF.TM. (sample 2); 3: DNA-OFF.TM. (sample 1); 4: DNA-OFF.TM.
(sample 2); 5: 0.5 M H.sub.3PO.sub.4; 6: 0.5 M HNO.sub.3; 7: 100 mM
ascorbic acid+10 mM FeCl.sub.3; 8: 10 mM ascorbic acid+10 mM
FeCl.sub.3; 9: 100 mM Na-ascorbate+10 mM FeCl.sub.3; 10: mixture
like in 9 only with pH 6; 11: mixture like in 9 only with pH 7.1;
12: mixture like in 9 only with pH 8; 13: mixture like in 9 only
with pH 8.5). The samples number 5 to 13 contained 0.2% Triton
X-100 and 0.2% Tween 20.
[0044] FIG. 5 shows an example for the new buffer system containing
Na-carbonate and malic (hydroxy-succinic) acid in comparison with
mineralic acid. The basic mixture contains 50 mM ascorbic acid, 5
mM FeCl.sub.3 and 0.2% Triton X-100 and 0.2% Tween 20. (M: Marker
DNA 1 kb Ladder), C: Control: DNA+5 .mu.l sterile H.sub.2O; 1:
mixture with pH 10; 2: mixture with pH 9; 3: mixture with pH 7; 4:
mixture with pH 6; 5: mixture with pH 4; 6: 0.5 M H.sub.3PO.sub.4
(pH 1.5) with 0.2% Triton X-100 and 0.2% Tween 20.
[0045] FIG. 6 shows the blockage of PCR amplification for DNA
molecules after treatment with the new three component system
(mixture A: 100 mM vitamin C+10 mM FeCl.sub.3+0.2% Triton X-100 and
0.2% Tween 20). Different amounts (0.1 to 1 ng) of a DNA sample
were dried in PCR tubes. PCR tubes containing the dried DNA were
treated for 20 seconds with the listed solutions. Subsequently the
tubes were washed two-times with 100 .mu.l of sterile, distilled
water. Finally the tubes were filled with a 50 .mu.l PCR reaction
mixture and the PCR reaction was performed. The PCR reaction
mixture contained pair of primers for the amplification of the
control DNA (scIMP2 gene of yeast) and the test DNA (scPCP1 gene of
yeast). The control DNA (1 ng) indicates a successful PCR reaction.
A band of the test DNA demonstrates that intact DNA molecules for
this gene are still present. In case of complete removal or
blockage of the test DNA there shall not be any amplified DNA band
in the gel.
[0046] DNA was stained with ethidium bromide after gel
electrophoresis in a 1% agarose gel and the gel was photographed.
As a positive control the conventional DNA-OFF.TM. was used.
Sterile water (H.sub.2O) served as a negative control.
[0047] FIG. 7 shows the test of prior art for RNaseA enzyme
inactivation in comparison with the new three component system.
Identical aliquots of 10 .mu.g RNaseA were dried in Eppendorf
tubes. Afterwards each tube was treated with 1 ml of the listed
solutions, vortexed for 20 seconds and finally incubated for 5
minutes at room temperature. Subsequently each tube was washed
two-times with 1 ml of sterile water. Afterwards 5 .mu.g of total
RNA from E. coli were added into the tubes and incubated for 30
minutes at 37.degree. C. Subsequently total RNA samples were mixed
with formamide/bromphenol blue buffer and denatured at 95.degree.
C. for 5 minutes. Afterwards the complete 5 .mu.g total RNA sample
was loaded onto an agarose gel (1.2%) and separated by gel
electrophoresis. After staining with ethidium bromide the
documented picture was taken. The untreated controls represent
intact total RNA. In the presence of active RNaseA these RNA
molecules are degraded. In case of successful, complete
inactivation of RNaseA the total RNA molecules will also remain
intact.
[0048] (C: Control of total RNA; 1: RNase-OFF (A); 2: RNase-OFF
(B); 3: ascorbic acid mixture with 100 mM vitamin C+10 mM
FeCl.sub.3+0.5% SDS; 4: empty lane; 5: H.sub.2O; 6: 10 .mu.g RNase
A; 7: empty lane; C: Control of total RNA)
[0049] FIG. 8 shows the efficient degradation of genomic DNA and
extra-chromosomal genetic material inside microorganisms by the new
three component system. A recombinant Escherichia coli strain with
an extra-chromosomal plasmid (YEp351) was grown over night in LB,
amp medium. Aliquots of 5 .mu.l from the E. coli suspension were
treated with 5 .mu.l lysozyme solution (1 mg/ml) for 5 minutes and
subsequently incubated with 5 .mu.l of the listed solutions (1-5)
for additional 5 minutes. After addition of bromphenol blue the
samples were loaded into the gel slots and separated by
electrophoresis of the DNA molecules. Only for sample number 5 with
the three component system a massive degradation of the DNA
molecules is identifiable. For the control with sterile water (C)
and for sample 3 and 4 lysis of the cells is observed and therefore
the extra-chromosomal plasmid DNA is released and can migrate into
the gel. For samples 1 and 2 one observes a precipitation of the
lysate and the DNA and therefore consequently all DNA molecules
remain at the bottom of the gel slot.
[0050] (M: Marker 1 Kb Ladder; C: Control with H.sub.2O; 1: 70%
Ethanol; 2: 0.5% Bacillozid.TM.; 3: 0.5% SDS; 4: 0.5% Na-Azide+0.5%
SDS; 5: 100 mM vitamin C+10 mM FeCl.sub.3+0.5% SDS; C: Control: 5
.mu.l sterile H.sub.2O)
[0051] Table 1 shows the test for antimicrobial action of the new
three component system.
[0052] Freshly grown cultures of the listed microorganisms were
adjusted to a cell number of 10 in a 50 .mu.l volume and mixed in a
ratio of 1:1 with 50 .mu.l of water, 70% ethanol or the three
component system (100 mM ascorbic acid, 10 mM FeCl.sub.3 and 0.5%
SDS). After an incubation time of 2 minutes the 100 .mu.l samples
containing the microorganisms were plated onto the respective
growth media. After an incubation period of 1-3 days at 28.degree.
C. (Saccharomyces cerevisiae and Candida parapsilosis) or
37.degree. C. (Escherichia coli and Bacillus subtilis) the number
of grown colonies was determined. In test samples with sterile
water all microorganisms survived. Test samples with 70% ethanol or
the three component system did not show any living cell colony,
indicating that under these conditions all microorganisms were
killed.
[0053] Table 2 summarizes the preferred composition of the solution
with the three component system comprising surface-active
substances, vitamins and metal ions for removal of DNA molecules
from surfaces and equipments.
LIST WITH EXPLANATION OF THE ABBREVIATIONS IN THE FIGURES
[0054] amp: ampicillin [0055] Bacillozid.TM.: commercial
anti-bacterial solution [0056] DNase-OFF.TM.: commercial solution
for inactivation of DNA [0057] EtBr: ethidium bromide [0058] FAD:
Flavine adenine dinucleotide [0059] C: Control [0060] M: Molecular
weight marker [0061] PCR: Polymerase Chain Reaction [0062]
RNase-OFF.TM.: commercial solution for inactivation of RNases
[0063] RNaseA: Ribonuclease A (from bovine pancreas) [0064] RT:
Room Temperature [0065] sc: Saccharomyces cerevisiae [0066] scIMP2:
Saccharomyces cerevisiae gene for Inner Membrane Protease 2 [0067]
scPCP1: Saccharomyces cerevisiae gene for Processing of Cytochrome
c Peroxidase [0068] SDS: Sodiumdodecylsulfate [0069] TX:
TritonX-100 (non-ionic detergent) [0070] YEp351: Yeast Episomal
plasmid
TABLE-US-00001 [0070] TABLE 1 Test for the anti-microbial action of
the new three component system. 70% three component H.sub.2O
ethanol system microorganisms Escherichia 10.sup.6 0 0 coli
Bacillus 10.sup.6 0 0 subtilis Saccharomyces 10.sup.6 0 0
cerevisiae Candida 10.sup.6 0 0 parapsilosis
TABLE-US-00002 TABLE 2 Preferred composition of solutions
containing the three component system comprising surface-active
substances, vitamins and metal ions for the removal of DNA
molecules from surfaces and equipment. composition of the solutions
pH range: 2.0 to 8.5 vitamins: 1 mM to 100 mM metal ions: 1 mM to
50 mM detergents: 0.1% to 5%
* * * * *