U.S. patent application number 13/346449 was filed with the patent office on 2012-10-04 for marker solution to be applied by means of an inkjet printer.
This patent application is currently assigned to Secutech International Pte. Ltd.. Invention is credited to Andre Josten.
Application Number | 20120252136 13/346449 |
Document ID | / |
Family ID | 36581960 |
Filed Date | 2012-10-04 |
United States Patent
Application |
20120252136 |
Kind Code |
A1 |
Josten; Andre |
October 4, 2012 |
MARKER SOLUTION TO BE APPLIED BY MEANS OF AN INKJET PRINTER
Abstract
The invention relates to a marker solution which is to be
applied by means of an inkjet printer and contains (i) at least one
organic solvent that has a greater steam pressure than water at
20.degree. C. and a water content of less than 50 percent (v/v),
(ii) predefined first synthetically produced nucleic acids, and
(iii) a nucleic acid-complexing, organic auxiliary agent as
components.
Inventors: |
Josten; Andre; (Nurnberg,
DE) |
Assignee: |
Secutech International Pte.
Ltd.
Singapore
SG
|
Family ID: |
36581960 |
Appl. No.: |
13/346449 |
Filed: |
January 9, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11885243 |
Oct 6, 2008 |
8114207 |
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PCT/EP2006/001863 |
Mar 1, 2006 |
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13346449 |
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Current U.S.
Class: |
436/501 |
Current CPC
Class: |
C12Q 1/6816 20130101;
C12Q 1/6816 20130101; C09D 11/50 20130101; C09D 11/38 20130101;
C12Q 2563/185 20130101 |
Class at
Publication: |
436/501 |
International
Class: |
G01N 21/64 20060101
G01N021/64 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 4, 2005 |
DE |
10 2005 009 943.2 |
Mar 18, 2005 |
DE |
10 2005 012 567.0 |
Claims
1. A marker solution for application by means of an inkjet printer,
which contains as components (i) at least one organic solvent with
a higher vapor pressure than water at 20.degree. C. and a water
content of less than 50% (v/v), (ii) specified first synthetically
prepared nucleic acids and (iii) an organic auxiliary agent that
complexes the first nucleic acids.
2. The marker solution of claim 1, characterized in that the marker
solution has a higher vapor pressure than water at 20.degree.
C.
3. The marker solution of any of the preceding claims,
characterized in that the vapor pressure of the organic solvent or
of the marker solution at 20.degree. C. is higher than 0.025 bar,
in particular higher than 0.027 bar, and preferably higher than
0.03 bar.
4. The marker solution of any of the preceding claims,
characterized in that the organic solvent is methanol, ethanol,
propanol, isopropyl alcohol, butanone, acetone, ethyl ether,
benzene or a mixture of at least two of these solvents.
5. The marker solution of any of the preceding claims,
characterized in that the concentration of the organic solvent in
the marker solution is higher than 50% (v/v), preferably higher
than 80% (v/v), in particular higher than 90% (v/v).
6. The marker solution of any of the preceding claims,
characterized in that the concentration of water in the marker
solution is less than 20% (v/v), preferably less than 10% (v/v), in
particular less than 5% (v/v).
7. The marker solution of any of the preceding claims,
characterized in that the marker solution additionally contains a
dye, in particular a luminescent, especially preferably fluorescent
dye.
8. The marker solution of any of the preceding claims,
characterized in that the first nucleic acids are in
single-stranded form.
9. The marker solution of any of the preceding claims,
characterized in that it contains viscous polymers for adjusting
the viscosity of the marker solution, and/or charged molecules, in
particular tetrabutylammonium bromide, for adjusting the
conductivity of the marker solution.
10. The marker solution of any of the preceding claims,
characterized in that the first nucleic acids have a length of 5 to
300 nucleotides, in particular 10 to 100 nucleotides, preferably 15
to 25 nucleotides.
11. The marker solution of any of the preceding claims,
characterized in that it contains additional nucleic acids, in
particular having the same or almost the same length as the first
nucleic acids.
12. The marker solution of any of the preceding claims,
characterized in that in said solution, the quantitative ratio
between the first and the additional nucleic acids is at most 1:10,
preferably at most 1:20, in particular at most 1:100.
13. The marker solution of any of the preceding claims,
characterized in that the concentration of the first or of the
first and of the additional nucleic acids in the marker solution is
higher than 0.1% (w/v), in particular higher than 1% (w/v),
preferably higher than 2% (w/v).
14. The marker solution of any of the preceding claims,
characterized in that the auxiliary agent complexing the first
nucleic acids is a detergent with one or more positive charges or
partial charges.
15. The marker solution of claim 14, characterized in that the
concentration of the detergent in the marker solution is between
0.1% (w/v) and 10% (w/v), in particular between 0.5% (w/v) and 5%
(w/v), and preferably between 0.7% (w/v) and 3% (w/v).
16. The marker solution of any of the preceding claims,
characterized in that the auxiliary agent complexing the first
nucleic acids is a monomeric or polymeric amine such as spermdine
or polylysine.
17. The marker solution of any of the preceding claims,
characterized in that the auxiliary agent is a cationic
detergent.
18. The marker solution of any of the preceding claims,
characterized in that the cationic detergent is
hexadecyltrthylammonium bromide or dodecyltrimethylammonium
bromide.
19. The marker solution of any of the preceding claims,
characterized in that the first or the additional nucleic acids
form complexes with the auxiliary agent that are insoluble in
aqueous solution.
20. The marker solution of any of the preceding claims,
characterized in that the solubility of the first and of the
additional nucleic acids in the marker solution is higher by a
factor of 5, preferably by a factor of 10, especially preferably by
a factor of 100 than the solubility in an identical solution
without the detergent.
21. A method for the production of the marker solution of any of
claims 1 to 20, with the following steps: a) dissolving first
synthetically produced nucleic acids in an aqueous solvent or
providing first synthetically produced nucleic acids in an aqueous
solvent, b) bringing the first nucleic acids into contact with an
organic auxiliary agent that complexes the first nucleic acids,
wherein complexes of the first nucleic acids and the auxiliary
agent are formed, c) mixing the complexes with an organic solvent
that has a higher vapor pressure than water at 20.degree. C.
22. The method of claim 21, characterized in that complexes that
are precipitated in step b) are separated from the aqueous solvent
and are dissolved in the organic solvent in step c).
23. The method of claim 21 or 22, characterized in that the vapor
pressure of the organic solvent at 20.degree. C. is higher than
0.025 bar, in particular higher than 0.027 bar, preferably higher
than 0.03 bar.
24. The method of any of claims 21 to 23, characterized in that the
organic solvent is methanol, ethanol, propanol, isopropyl alcohol,
butanone, acetone, ethyl ether, benzene or a mixture of at least
two of these solvents.
25. The method of any of claims 21 to 24, characterized in that the
concentration of the organic solvent in the marker solution is
adjusted to a value higher than 50% (v/v), preferably higher than
80% (v/v), in particular higher than 90% (v/v).
26. The method of any of claims 21 to characterized in that the
concentration of water in the marker solution is adjusted to a
value less than 20% (v/v), preferably less than 10% (v/v), in
particular less than 5% (v/v).
27. The method of any of claims 21 to 26, characterized in that the
concentration of the auxiliary agent in the marker solution is
adjusted to a value between 0.1 (w/v) and 10% (w/v), in particular
between 0.5% (w/v) and 5% (w/v), preferably between 0.7% (w/v) and
3% (w/v).
28. The method of any of claims 21 to 27, characterized in that a
dye, in particular a fluorescent dye, is added to the marker
solution, to the aqueous solvent or to the organic solvent.
29. The method of any of claims 21 to 28, characterized in that,
the first nucleic acids are in single-stranded form.
30. The method of any of claims 21 to 29, characterized in that
viscous polymers for adjusting the viscosity of the marker solution
and/or charged molecules, in particular tetrabutylammonium bromide,
for adjusting the conductivity of the marker solution, are added to
the marker solution, to the aqueous solvent or to the organic
solvent.
31. The method of any of claims 21 to 30, characterized in that the
first nucleic acids have a length of 5 to 300 nucleotides, in
particular 10 to 100 nucleotides, preferably 1.5 to 25
nucleotides.
32. The method of any of claims 21 to 31, characterized in that
additional nucleic acids, in particular having the same or almost
the same length as the first nucleic acids, are dissolved in the
aqueous solvent in addition to the first nucleic acids, or
additional nucleic acids are provided in the aqueous solvent in
addition to the first nucleic acids.
33. The method of any of claims 21 to 32, characterized in that the
quantitative ratio between the first and the additional nucleic
acids is at most 1:10, preferably at most 1:20, in particular at
most 1:100.
34. The method of any of claims 21 to 33, characterized in that the
concentration of the first or of the first and of the second
nucleic acids in the marker solution is adjusted to a value higher
than 0.1% (w/v), in particular higher than 1% (w/v) , preferably
higher than 2% (w/v).
35. The method of any of claims 21 to 34, characterized in that he
auxiliary agent is a detergent.
36. The method of any of claims 21 to 35, characterized in that the
detergent is a cationic detergent.
37. The method of any of claims 21 to 36, characterized in that the
cationic detergent is hexadecyltrimethylammonium bromide or
dodecyltrimethylammonium bromide.
38. The method of any of claims 21 to 37, characterized in that the
auxiliary agent is a monomeric or polymeric amine, preferably
spermidine or polylysine.
39. The method of any of claims 21 to 38, characterized in that the
first and additional nucleic acids are dissolved in the organic
solvent at a concentration at which they would not be completely
soluble in the organic solvent without the detergent.
40. Use of a marker solution, which contains as components (i) at
least one organic solvent with higher vapor pressure than water at
20.degree. C. and a water content of less than 50% (v/v), (ii)
specified first nucleic acids and (iii) an organic auxiliary agent
that complexes the first nucleic acids, for the marking of a
surface of an object by means of a printer.
41. The use of claim 40, characterized in that the printer is an
inkjet printer.
42. The use of claim 40 or 41, characterized in that the surface is
smooth.
43. The use of any of claims 40 to 42, characterized in that the
surface consists of hydrophobic plastic.
44. The use of claim 43, characterized in that the plastic is
polyethylene or polypropylene.
45. The use of any of claims 40 to 44, characterized in that a
marking is formed by applying the marker solution to defined areas
and another solution to other areas, the other solution, having the
same composition as the marker solution, except that it does not
contain any first nucleic acids.
46. The use of claim 45, characterized in that the other solution
contains second nucleic acids.
Description
[0001] The invention relates to a marker solution that is suitable
for the production of forgery-proof markings and can be applied
with ordinary printers conventionally used in commerce and
industry, e.g. inkjet printers. In particular the invention relates
to a marker solution that contains at least one organic solvent
with a vapor pressure higher than water at 20.degree. C. Such
marker solutions can be used in the form of inks, in particular in
inkjet printers that are used industrially. They have the advantage
that they dry quickly and also permit precisely defined marking on
hydrophobic surfaces. The invention further relates to a method for
the production of the marker solution and the use of the marker
solution for the marking of objects.
[0002] A method of extracting nucleic acid from biological material
is known from US 2000229222 A1. In that method the biological
material is vigorously shaken with small particles and with a
two-phase mixture of an aqueous solution containing complexing
agents and quaternary ammonium salts and an organic solvent. After
extraction, the nucleic acid is obtained from the aqueous phase by
ethanol precipitation. The organic solvent permits efficient
purification, because contaminants contained in the biological
material are easily taken up in the organic solvent.
[0003] A marker solution for the forgery-proof marking of a
valuable item is known from WO 03/038000 A1. The marker solution
comprises an aqueous solution with single stranded nucleic acids
and glycerol and polyethylene glycol. The glycerol acts as a
hygroscopic substance and permanently keeps the marking moist. This
leads to a good ability of hybridization with a complementary
nucleic acid that is to be used for detecting the marking. The
marker solution can for example be applied by stamping. It is not
suitable for use in an inkjet printer on account of the high
viscosity caused by the glycerol an polyethylene glycol, as well as
the resultant slow and incomplete drying.
[0004] A medium or an ink solution that contains nucleic acid is
known from US 2004/0054160 A1. The medium contains between 30 and
80 vol. % of an organic solution comprising dimethylsulfoxide,
ethylene glycol, formamide or a combination thereof. The medium is
also suitable for stamping. Because of a reduced water content, the
medium evaporates more slowly than traditional ink. Thus, the
change in the composition of the ink caused by evaporation takes
place more slowly, so that more constant printing results are
achieved. A quick-drying medium is not disclosed by US 2004/0054160
A1.
[0005] A "bubble-jet" method of applying samples on solid supports
is known from EP 0 895 082 B1. The fluid applied can contain a
nucleic acid and 5 to 10 wt. % urea, 5 to 10 wt. % glycerol, 5 to
10 wt. % thiodiglycol and 1% of an acetylenic alcohol. This fluid
is not quick-drying either. The glycerol causes an applied sample
to remain permanently moist.
[0006] In industrially used inkjet printers, use of a quick-drying
ink having an organic solvent with a higher vapor pressure than
water at 20.degree. C. is known. Owing to the slow drying,
water-based inks without organic solvents are only suitable for
application on absorbent materials or for use in marking processes
in which a sufficiently long drying time is possible. For fast
marking processes or for application on hydrophobic and/or
non-absorbent surfaces, for example high-gloss surfaces or, in
particular, smooth surfaces of plastics, inks based on organic
solvents are required. Conventional nucleic acids can only be
dissolved in such inks at low concentration, if at all. The amount
of nucleic acid that can be applied with such an ink is too small
for it to be detected without an amplification reaction.
[0007] The aim of the present invention is to provide a
quick-drying marker solution containing nucleic acid that is
suitable for application by means of a printer. In particular, with
the marker solution, it should be possible to apply nucleic acid,
e.g., by means of an inkjet printer, in an amount such that direct
detection is possible without carrying out an amplification
reaction on the marked surface. A further aim of the invention is
to provide a nucleic acid-containing marking fluid whose nucleic
acids, in the printed state, are directly accessible for
hybridization with complementary nucleic acids.
[0008] This aim is achieved by the features of claims 1, 21 and 40.
Advantageous embodiments can be seen from the features of claims 2
to 20, 22 to 39 and 41 to 46.
[0009] According to the invention, a marker solution is provided
for application by means of an inkjet printer, which contains as
components (i) at least one organic solvent with a higher vapor
pressure than water at 20.degree. C. and a water content of less
than 50% (v/v), (ii) specified first synthetically prepared nucleic
acids and (iii) an organic auxiliary agent that complexes the first
nucleic acids.
[0010] The present inventors recognized that by providing the
auxiliary agent that complexes the first nucleic acids, it is also
possible to dissolve first nucleic acids, in particular DNA, at
relatively high concentration in a marker solution based on an
organic solvent. It is then possible, by means of the marker
solution and, e.g., an inkjet printer, to apply DNA on a surface in
an amount such that it can be detected directly, i.e., without
prior amplification, using an in-situ test. It was also
surprisingly found that, after drying on the surface, the nucleic
acids complexed with the auxiliary agent, despite the drying and
despite the presence of the auxiliary agent, are in a form such
that they are directly accessible for hybridization, with a
complementary nucleic acid present in aqueous solution. Marking
produced using the marker solution according to the invention can
thus be detected quickly and simply, directly on the marked
surface. For detection, it is not necessary remove the nucleic
acids from the surface. Detection of the first nucleic acids thus
present can for example be carried out using a method that is known
from WO 01/51652 A2. The first nucleic acid preferably is a
synthetically prepared nucleic acid. Precisely defined coding can
be provided by this means.
[0011] It is possible to detect the marking directly on the surface
by contact with dissolved complementary detection nucleic acids at
the site or marking. Hybridization can be detected, e.g., based on
observation of altered optical properties of the detection nucleic
acids. The detection nucleic acids used can be, e.g., molecular
beacons, which are applied to the marking using a pencil or a
dosing device. The fluorescence of the molecular beacons, altered
by hybridization, can be detected using a hand scanner. Owing to
these properties, identification of the marking can be carried out
easily, even, by untrained personnel, in situ, i.e., without
laboratory equipment.
[0012] The marker solution according to the invention has the
additional advantage that it does not contain any particles that
could clog a nozzle of a printhead of an inkjet printer. Another
advantage of the marker solution according to the invention is that
such large amount of nucleic acid can be dissolved in the marker
solution, that in addition to the specific, first nucleic acid that
serves for marking, a large amount of nonspecific nucleic acid can
be dissolved. Accordingly, in a manner of speaking, the specific
nucleic acid can be concealed in the nonspecific nucleic acid. It
is then very difficult to analyze and copy the specific nucleic
acid, in order to apply a forged marking. This makes the marker
solution very forgery-proof. It was found that 3 wt. % nucleic acid
can be dissolved in the marker solution according to the invention.
Another advantage of the marker solution according to the invention
is that in the dried state it is almost invisible. As a result, the
place where the marking has been applied to the marked object is
not immediately recognizable. The marking can only be found with
great difficulty, so that it can be analyzed and possibly copied.
Accordingly, very forgery-proof markings can be produced with the
marker solution according to the invention.
[0013] Furthermore, the marking applied with the marker solution
according to the invention adheres well especially to smooth and
even hydrophobic surfaces, without the latter having to undergo a
corresponding pretreatment. In addition, the organic solvent
ensures good wettability of hydrophobic surfaces.
[0014] Since organic solvents like isopropanol or ethanol are
usually employed in molecular biology for the precipitation of
nucleic acid, it is very surprising that a comparatively large
amount of DNA can be dissolved in these solvents with the aid of an
auxiliary agent that complexes the first nucleic acids, for example
a cationic detergent.
[0015] In order to increase the solubility in organic solvents, the
first nucleic acids could also be modified. For example, methyl or
cholesteryl groups could be provided on the first nucleic acids
during nucleic acid synthesis or by subsequent modification.
However, chemical modification of the first nucleic acids has the
disadvantage of increased costs and the unwanted possibility of
isolating the first nucleic acids on the basis of the modification.
The method disclosed here offers the possibility of dissolving
first nucleic acids at high concentrations in organic solvents,
without having to rely on modification of the first nucleic
acids.
[0016] It is especially advantageous if the marker solution as a
whole has a higher vapor pressure than water at 20.degree. C. This
ensures even faster drying of the applied marker solution. The
vapor pressure of the organic solvent, or of the marker solution at
20.degree. C. is preferably above 0.025 bar, in particular above
0.027 bar, and more preferably above 0.03 bar. The organic solvent
can be methanol, ethanol, propanol, isopropyl alcohol, butanone,
acetone, ethyl ether, benzene or a mixture of at least two of these
solvents.
[0017] Preferably the concentration of the organic solvent in the
marker solution is greater than 50% (v/v), preferably greater than
80% (v/v), in particular greater than 90% (v/v). (v/v) means
(volume/volume). The higher the proportion of organic solvent in
the marker solution, the quicker the marker solution dries. The
concentration of water in the marker solution preferably is less
than 20% (v/v), especially preferably less than 10% (v/v), and in
particular less than 5% (v/v).
[0018] In one embodiment of the invention the marker solution
additionally contains a dye, in particular a fluorescent dye. In
this way the marking can be located more easily for
identification.
[0019] It is especially favorable if the first nucleic acids in the
marker solution are in single-stranded form. Then the direct
detection of the first nucleic acids does not require a denaturing
step. It was found, surprisingly, that the first nucleic acids are
directly accessible for hybridization in the dried, printed
marking. This is all the more surprising because detection takes
place by hybridization in the presence of the complexing auxiliary
agent, e.g. a cationic detergent, that is present in the marking
and complexes the first nucleic acids. Detection of a marking
produced by means of the marker solution can thus be carried out
quickly and simply on the marked surface.
[0020] The first nucleic acids contained in the marking can be
detected by adding a small amount of, e.g., 1 to 10 .mu.l of an
identification solution, said identification solution containing
detection nucleic acids, which are complementary to the first
nucleic acids. Addition can be performed, e.g., using a pen, which
is brought into contact with the marking and, as a result of
capillary forces, releases a small amount of the identification
solution into the marking. Of course, the identification solution
can also be applied to the marking by means of a pipetting device.
Hybridization is preferably detected by means of complementary
molecular beacons, based on the fluorescence altered by
hybridization. The altered fluorescence can be detected, e.g., by
means of a hand-held fluorescence scanner. In this embodiment,
in-situ detection of the marking is possible without laboratory
equipment and without personnel with scientific training.
[0021] To adjust the viscosity of the marker solution it can
contain viscous polymers. Adjustment of viscosity may be necessary
in order to adapt the marker solution to the requirements of
different printheads and their nozzles. To adjust the conductivity
of the marker solution it can contain charged molecules, in
particular tetrabutylammonium bromide. Adjustment of conductivity
may be necessary for inkjet printers in which the point of ink
application is determined by deflection of an ink jet in an
electric field. A particular conductivity of the marker solution
must be provided to obtain the desired deflection of the ink jet at
a given field strength.
[0022] The first nucleic acids can have a length of 5 to 300
nucleotides, in particular 10 to 100 nucleotides, preferably 15 to
25 nucleotides. The greater the length of the nucleic acids, the
larger the quantity of codings that can be provided. At the same
time, however, with increasing length, it becomes more laborious to
provide error-free defined nucleic acids. The stated lengths have
proved to be favorable.
[0023] The security of a marking provided by means of the marker
solution can be further increased if it contains additional nucleic
acids, especially if they have a similar length to the first
nucleic acids. With the additional nucleic acids it is possible to
conceal the first nucleic acids within a quantity of additional
nucleic acids. Consequently, it is not possible to determine the
sequence of the first nucleic acid and thus fake the marker
solution and/or the marking applied with it.
[0024] The ratio of the amounts of the first and the additional
nucleic acids is preferably at most 1:10, especially preferably at
most 1:20, in particular at most 1:100. The smaller this ratio, the
more forgery-proof is the marking applied using the marker
solution, because it becomes more and more difficult to isolate and
identify the first nucleic acids among all of the first and
additional nucleic acids that are present.
[0025] The concentration of the first or of the first and of the
additional nucleic acids in the marker solution is preferably above
0.1% (w/v), in particular above 1% (w/v), more preferably above 2%
(w/v). A high concentration. of the fir it nucleic acids in the
marker solution facilitates amplification-free detection of the
first nucleic acid in the printed marking.
[0026] In the context of the present invention, the auxiliary agent
that complexes the first nucleic acids is an organic compound that
binds to the first nucleic acids, and carries at least one positive
charge or positive partial charge and has organic residues. The
auxiliary agent can also be a polymer, carrying more than one
positive charge per molecule. Typical representatives of the
auxiliary agents are cationic detergents, for example quaternary
ammonium compounds with various alkyl residues. The concentration
of the detergent in the marker solution can be between 0.1% (w/v)
and 10% (w/v), in particular between. 0.5% (w/v) and 5% (w/v),
preferably between 0.7% (w/v) and 3% (w/v). (w/v) signifies
(weight/volume). Organic, amphiphilic compounds, such as organic
amines, can also be used as auxiliary agents. Preferably, auxiliary
agents such as spermidine, spermine or palyiysme, which form
insoluble complexes with nucleic acids in aqueous solutions, can be
used.
[0027] In a preferred embodiment of the marker solution according
to the invention, the cationic detergent hexadecyltrmethylammonium
bromide or dodecyltrimethylammonium bromide is used as auxiliary
agent. Preferably the first and the additional nucleic acids are
dissolved in the marker solution at a concentration at which they
would not be completely soluble in the otherwise unaltered marker
solution without the auxiliary agent. The concentration of the
nucleic acids in the marker solution is preferably higher by
greater than a factor of 10, preferably a factor of 100 above the
concentration at which they would be soluble in the otherwise
unaltered marker solution without the auxiliary agent. The higher
the concentration of the first and additional nucleic acids in the
marker solution, the greater is the quantity of these nucleic acids
that can be applied per area unit by means of an inkjet printer.
The greater the quantity applied per area unit, the easier it is to
detect these nucleic acids, and the more secure the marking can be
made, because a quantity of first nucleic acids that is detectable
offhand can be concealed in a large quantity of additional nucleic
acids.
[0028] The marker solution according to the invention can be
contained in a printhead of an inkjet printer, in a reservoir or in
a channel.
[0029] The invention further relates to a method for the production
of the marker solution according to the invention with the
following steps:
[0030] a) dissolving first synthetically produced nucleic acids in
an aqueous solvent or provision of first synthetically produced
nucleic acids in an aqueous solvent,
[0031] b) bringing the first nucleic acids into contact with an
organic auxiliary agent that complexes the first nucleic acids,
whereby complexes of the first nucleic acids and the auxiliary
agent are formed,
[0032] c) mixing the complexes with an organic solvent that has a
higher vapor pressure than water at 20.degree. C.
[0033] Preferably the contacting of the first nucleic acids with
the auxiliary agent in step b) takes place by first providing a
solution of the auxiliary agent, to which an aqueous solution
containing the first, nucleic acids is added. In this way it is
possible to ensure that the auxiliary agent is present in excess
during contact with the first nucleic acids, and nucleic acid
complexes saturated with the auxiliary agent can form.
[0034] Mixing of the complexes with the organic solvent can also
carried out by mixing aqueous solvents containing the first nucleic
acids and the auxiliary agent with the organic solvent.
[0035] Preferably the auxiliary agent and the aqueous solvent are
selected so that in step "b" the complexes from first nucleic acids
and auxiliary agent are precipitated. The precipitated complexes
can be separated from the aqueous solvent and dissolved in the
organic solvent in step "c". The precipitated complexes can be
separated for example by centrifugation or by filtration. By
precipitating and separating the complexes, concentration of the
nucleic acids can be achieved.
[0036] Furthermore, the invention relates to a use of a marker
solution, which contains as components (i) at least one organic
solvent with a higher vapor pressure than water at 20.degree. C.
and a water content of less than 50% (v/v), (ii) specified first,
in particular synthetically prepared, nucleic acids and (iii) an
organic auxiliary agent that complexes the first nucleic acids, for
the marking of a surface of an object by means of a printer, in
particular an inkjet printer.
[0037] Preferably the surface is smooth and/or consists of plastic,
in particular a hydrophobic plastic. The plastic can be
polyethylene or polypropylene. To increase the level of security
against forgery, a marking can be formed by applying the marker
solution to defined areas and another solution to other areas. The
other solution has the same composition as the marker solution,
except that it does not contain first nucleic acids. As a result it
is difficult for a forcer to ascertain which of the areas contain
the first nucleic acids. Moreover, the additional area can serve as
a reference area during detection of the marking.
[0038] Security can be further enhanced if the other solution
contains second nucleic acids, because a forger must then
additionally ascertain whether the first or the second nucleic
acids provide the specific coding.
[0039] For identification of a marking applied by means of the
marker solution on a surface of an object, a specific hybridization
of the first nucleic acids can be carried out with complementary,
i.e. specific, third nucleic acids. The third nucleic acids can be
designed so that properties of the third nucleic acid are altered
by the hybridization. These altered properties can give rise to a
signal that can be detected. For this purpose, the third nucleic
acids can for example be molecular beacons. The detectable signal
can for example be a color change or a change in fluorescence. The
sequences and the sequence lengths of the regions of the first
nucleic acids and of the third nucleic acids complementary to each
other are preferably selected so that a specific hybridization is
possible at room temperature. This facilitates specific detection
of the marking. It is especially preferred if detection of the
marking can take place in a single-step reaction, i.e. without a
washing step. Such a method of detection is for example known from
WO 01/51652 A2. When the marking is formed by applying the marker
solution to defined areas and the other solution to other areas,
during identification of the marking it is advantageous if the
additional areas are also brought into contact with the third
nucleic acids, in order to see that no specific hybridization takes
place there. If, however, a specific hybridization does take place
there, this indicates a forged marking. The security of the method
against forgery can be further increased with such a procedure.
[0040] The invention is explained in more detail below with an
example of application.
[0041] 1) Preparation of the Marker Solution
[0042] First a solution is prepared from 27.5 g DNA comprising 2.5
g of first nucleic acid (Sequence 1=5'-tg gagggatgat actttgcgct
tgg-3') and 25 g of ultrasonically sheared herring sperm DNA
(Sigma, Ordering No. D3159) in 500 ml water. Stirring vigorously,
this solution is added to 1500 ml of a solution containing 100
mmol/l of dodecyltrimethylammonium bromide (Sigma, Ordering No.
519151) as auxiliary agent in water. A precipitate consisting of
DNA-dodecyltrtimethylammonium bromide complexes forms. The
precipitate is separated by filtration and, after drying at room
temperature, it is taken up in 1 l ethanol. The resultant
DNA-containing ethanolic solution is clear and can be used as a
marker solution itself or it can be used for the preparation of
other marker solutions.
[0043] 2) Production of the Marking by Inkjet Printing
[0044] 800 ml of the marker solution are transferred to a
container, which is connected by a pipe to the printhead of a
Metronic inkjet printing device (Veitshochheim, Germany). By means
of the inkjet printing device, circular markings with a diameter of
approx. 2 mm are applied to a plastic film that is transported past
the printhead. At a distance of approx. 5 mm from the first
marking, control markings, which are produced with marker solution
without first nucleic acids, are applied to the film.
[0045] 3) Detection of the Marking
[0046] For identification of the marking, two weeks after printing
the marking, detection solution is applied to the marking and to
the control marking by means of a pen. For this, the pen is brought
into contact with the marking for approx. 2 seconds. The detection
solution consists of an aqueous solution containing the molecular
beacon (Sequence 2=5'-6FAM-ccaagcgcaa agtatcatcc ctccaggctt
gg-Dabcyl-3'), which is partially complementary to the first
nucleic acids in the marking. Immediately after contact of the
marking and the control marking with the pen, the fluorescence of
the marking and of the control marking is determined with a
hand-held fluorescence scanner from the company identif GmbH
(Erlangen, Germany). The marking is identified on the basis of the
increased fluorescence of the marking relative to the control
marking.
Sequence CWU 1
1
2125DNAArtificial sequenceA synthetic oligonucleotide 1tggagggatg
atactttgcg cttgg 25232DNAArtificial sequenceA synthetic 5'-6FAM-
and 3'dt(C2-DABCYL)-labeled oligonucleotide 2ccaagcgcaa agtatcatcc
ctccaggctt gg 32
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