Note: Descriptions are shown in the official language in which they were submitted.
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Case 22697
A method to reduce false positive results
Field of invention
The present invention provides a method and a compound for the decontamination
of liquids from certain molecules. In particular, the present invention is
directed to
a method and a compound that maintains reagents free from said molecules. More
particular, the present invention is directed to a method and a compound that
ensures a nucleic acid amplification reaction of a target molecule in a sample
avoiding false positive results.
Prior art background
Microbiological tests for diagnostics or research based on nucleic acid
analysis are
of still increasing importance. Since on the one hand, the nucleic acids are
often
present in very small concentrations and, on the other hand, they are often
found in
the presence of many other solid and dissolved substances e.g. after lysis of
cells,
they are difficult to isolate or to measure, in particular in biospecific
assays which
allow the detection of specific analytes. Therefore, in the majority of cases,
these
microbiological tests comprise at least one amplification step of the
characteristic
DNA molecules to be detected. A well-known assay which entails the selective
binding of two oligonucleotide primers is the polymerase chain reaction (PCR)
described in US 4,683,195. This method allows the selective amplification of a
specific nucleic acid region to detectable levels by a thermostable polymerase
in
the presence of deoxynucleotide triphosphates in several cycles. Other
possible
amplification reactions are the Ligase Chain Reaction (LCR, Wu, D.Y. and
Wallace, R.B., Genomics 4 (1989) 560-569 and Barany, Proc. Natl. Acad. Sci.
USA 88 (1991) 189-193); Polymerase Ligase Chain Reaction (Barany, PCR
Methods and Applic. 1 (1991) 5-16); Gap-LCR (PCT Patent Publication No. WO
90/01069); Repair Chain Reaction (European Patent Publication No. 439 182 A2),
3SR (Kwoh, D.Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177;
Guatelli, J.C. et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; PCT
Patent
Publication No. WO 92/0880A), and NASBA (U.S. Pat. No. 5,130,238). Further,
there are strand displacement amplification (SDA), transciption mediated
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amplification (TMA), and Q(3-amplification (for a review see e.g. Whelen, A.C.
and Persing,D.H., Annu. Rev. Microbiol. 50 (1996) 349-373; Abramson, R.D. and
Myers, T.W., Current Opinion in Biotechnology 4 (1993) 41-47).
Since these techniques result in a drastic amplification of nucleic acid
molecules,
even the slightest contamination of the sample with undesired nucleic acid
molecules from the reagents necessary for said amplification may result in a
huge
amount of false amplification products synonymous to e.g. a false positive
diagnosis. A prominent example of such a phenomenon may occur in a
microbiological test, where the existence of a bacterium in a sample should be
tested and some of the used reagents are contaminated with this bacterium or
with
nucleic acids derived from this bacterium as well. In this case, the target
nucleic
acid molecules from the sample under investigation are identical to the
undesired
molecules from the contaminated reagent.
Therefore, the requirements towards contamination-free environments of nucleic
acid amplification reactions are of utmost importance.
In the scientific literature and in patent applications several procedures for
the
decontamination of solutions from nucleic acids are described. These
procedures
include chemical treatments with e.g. Clorox (Prince, A.M. and Andrus, L.,
Biotechniques 12 (1992) 358-360), surfactants or oxidizing agents (WO
2002/060539). Other strategies utilize treatments with digestion enzymes (EP 0
585
660; Hilali, F. et al, Mol. Biotechnol. 7 (1997) 207-216) or UV illumination
(Fox,
J.C. et al, J. Virol. Meth. 33 (1991) 375-3823). Concerning the above
mentioned
strategies several review articles can be found in literature (Abravaya, K. et
al,
Nucleic Acid Ampl. Technol. Chapter 9 (1997) 125-133; Corless, C.E. et al, J.
Clin. Microbiol. 38 (2000) 1747-1752; Klaschik, S. et al, Mol. Biotechnol. 22
(2002) 231-242).
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An other approach, normally applied for the separation or isolation of e.g.
DNA
from complex biological fluids, is the use of DNA binding materials. The most
prominent example of DNA binding material are glass surfaces due to their
ability
to reversibly bind DNA in the presence of chaotropic reagents (Vogelstein, B.,
and
Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-619). However, this
process does not distinguish between single- and double-stranded DNA. US
04/121336 descibes a method of binding nucleic acid to a multiplicity of solid
substrate binding units.
More general, cationic surfaces may be used to bind charged DNA molecules,
whereby e.g. EP 0 281390 describes a polycationic support for nucleic acid
isolation, WO 01/94573 charged membranes or WO 00/69872 a pH dependent ion
exchange matrix. WO 02/48164 discloses polymers with switchable charge on
solid supports for reversible binding of DNA. Similar to cationic surfaces,
polycationic entities have certain DNA-binding affinity, too. Stewart, et al.,
J.
Phys. Org. Chem. 5 ( 1992) 461-466 reports an increasing affinity of
polyamines in
solution for binding to DNA with increasing cationic charge. Dore et al JACS
126
(2004) 4240-4244) describes the selectivity of cationic compounds between
double-stranded and single-stranded nucleic acids.
Moreover, double-stranded (ds) DNA binding molecules are known in the art that
may distinguish between single- and double-stranded DNA. Here, one should
mention especially minor groove binders (MGBs), anti-ds DNA antibodies and
intercalators.
MGBs like the pyrrol amidine antibiotics Distamycin or Netropsin bind to the
minor groove by hydrogen bonding and do not interact by intercalation (Fish,
E.L.
et al, Biochemistry 27 (1988) 6026-6032). Boger, D.L. et al., J. Am. Chem.
Soc.
123 (2001) 5878-5891 studied the binding of Distamycin, Netropsin and 4',6-
diamidino-2-phenylindole (DAPI) to double-stranded DNA immobilized to
microtiter plates. US 2002/0095073 describes the use of MGBs as probes that
are
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immobilized on a solid support via amide or thiol bonds to bind target DNA in
order to determine a cause of one or more medical symptoms. Li, M. et al
Bioorg.
Med. Chem. Letters 12 (2003) 4351-4354 describes a DAPI derivative that may
bind by a covalent amide bond to amino groups of the surface.
Anti-ds DNA antibodies are known to have the ability to distinguish between
single- and double-standed DNA and therefore, they were used e.g. as a probe
for
DNA hybrids immobilized to a solid support, whereby the presence of the
antibody
was verified by colorimetric detection (Mantero, G. et al, Clin. Chem. 37
(1991)
422-429). WO 2002/074993 describes a method to immobilize an anti-DNA
antibody on gold covered glass slides using an activated thiol monolayer. The
patent EP 0 792 376 relates to the field of nucleic acid amplification
reactions and
discloses the immobilization of anti-hybrid antibodies to surfaces via a
biotin/streptavidin bond.
In the state of the art, immobilized antibodies are also used to reversibly
bind cells
or assemblies of cells, e.g. bacteria. For the separation of food pathogens
there are
already commercial products based on beads functionalized with antibodies
(DYNAL Biotech or MATRIX Microscience Ltd). The WO 91/19003 discloses a
process to detect contaminations using antibodies on surfaces. PROFOS GmbH
discloses the use of bacteriophage tail proteins coupled to a carrier to bind
bacteria
(WO 01/09370, WO 02/06117) or endotoxins (WO 04/01418). Koepsel and
Russell (Koepsel, R.R. and Russell, A.J., Biomacromolecules 4 (2003) 850-855)
disclose antibodies on bioplastic films to capture bacteria. For an unspecific
accumulation of bacteria, the WO 03/33698 describes a method with charged
polymers.
The intercalating properties of some small organic molecules that bind between
stacked base pairs of double stranded DNA was intensively studied in the past
(Brana, M.F. et al, Curr. Pharm. Design 7 (2001) 1745-1780). WO 2002/82078
describes complexes out of intercalators and signaling molecules in order to
detect
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the presence of double-stranded DNA on a solid support. The use of
intercalators
attached to a support to separate single- and double-stranded DNA is described
in
US 2002/0006617. US 2001/0026921 discloses a nucleic acid hybridization assay
composition with intercalators bound to a surface via an amino functionality
and a
second intercalator bearing a fluorophore.
US 2003/0130499 discloses the use of e.g. Actinomycin D or ethidium bromide as
intercalators that may be incorporated into a solid phase in order to bind
nucleic
acids.
Brief descrintion of the invention:
Many alternatives for the binding of biological molecules, like e.g. nucleic
acids,
from a liquid solution to the surface of a solid support are known to someone
skilled in the art, but non of these alternatives are applicable as a
decontamination
method for special applications.
Therefore, the present invention is directed to a method and a compound for
the
detection of biological molecules in a sample that decontaminates reagents,
necessary for said detection, from and/or maintains said reagents free of
biological
molecules, whereby the decontamination process is permanently active in the
background of a certain experiment. More particular, the present invention is
directed to an improved method and a compound that ensures a nucleic acid
amplification performance without the amplification of non-target nucleic acid
molecules and/or target nucleic acid molecules potentially present in said
reagents
via a nucleic acid binding compound that is permanently present until the
actual
nucleic acid amplification.
One subject matter of the present invention is a method for the detection of
biological molecules in a sample that avoids the detection of biological
molecules
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potentially present in the reagents necessary to detect the biological
molecules in
the sample, comprising the steps of
a) providing a sample potentially comprising said biological molecules,
b) providing binding moieties coupled to the surface of a solid phase,
c) providing reagents necessary to detect the biological molecules,
d) adding said reagents to said sample,
e) detecting the biological molecules in said sample and
wherein in step c) or in step d) or in step c) and d) said reagents are in
physical
contact with said binding moieties under conditions, whereby said biological
molecules potentially present in said reagents bind to said binding moieties
coupled
to said surface of a solid phase.
The invention also concerns a method to amplify a target nucleic acid molecule
comprising the steps of
a) providing a sample potentially comprising target nucleic acid
molecules,
b) providing nucleic acid binding moieties coupled to the surface of a
solid phase,
c) providing reagents necessary to amplify said target nucleic acid
molecules,
d) adding said reagents to said sample
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e) amplifying said target nucleic acid molecules in said sample and
wherein in step c) or in step d) or in step c) and d) said reagents are in
physical
contact with said nucleic acid binding moieties under conditions, whereby said
nucleic acid molecules potentially present in said reagents bind to said
nucleic acid
binding moieties coupled to said surface of a solid phase.
In alternative embodiments of the invention, the steps a) to c) are performed
in any
other possible succession of these steps.
'Binding moieties' throughout this invention are molecular complexes that are
able
to reversibly bind biological molecules or biological compartments. If they
are able
to bind nucleic acids they are named nucleic acid binding moieties. If they
bind
double-stranded nucleic acids with a higher affinity than single-stranded
nucleic
acids they are named double-stranded nucleic acid binding moieties. The
phrases
double-stranded nucleic acid binding moiety or 'ds-NA binder' are used as
equivalents throughout this invention. In the context of this invention the
phrase
nucleic acid (NA) summarizes deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA) as well as nucleic acid analogues like peptide nucleic acids (PNA) or
locked
nucleic acids (LNA).
Biological compartments are cells or assemblies of cells. Since cells have
certain
proteins or polymers within their cell membrane, the binding moieties may have
an
affinity for these molecular structures in order to reversibly bind the cells
or
assemblies of cells. Additionally, the electrostatic interaction of the
negatively
charged cell membrane with cationic binding moieties can be utilized to
reversibly
bind biological compartments.
The binding moieties are coupled to the surface of a solid phase throughout
this
invention. The surface of this solid phase may be of arbitrary shape and
therefore,
includes planar surfaces of e.g. a cover slide, the curved surface of e.g. a
test tube, a
CA 02512599 2005-08-17
_g_
vessel or a pipette tip, the surface of small particles like e.g. magnetic
beads or the
surface of porous materials like e.g. glass fleeces. Throughout this invention
a
vessel is a single reaction vessel, like e.g. a Eppendorf cap or a
centrifugation tube.
As solid phase material all materials are possible within the scope of this
invention,
as far as the surface of this solid phase material comprises coupling groups
for said
binding moieties or as far as the surface of this solid phase material may be
functionalized with coupling groups for said binding moieties. In some cases
it is
necessary to provide said surface with a functional coating, before the
binding
moieties may be coupled. Such a functional coating is e.g. a polymer layer.
In the context of this invention the coupling of the binding moieties to the
surface
of the solid phase includes covalent bonds like e.g. silane coupling, amide
bonds or
epoxide coupling, coordinative bindings like e.g. between His-tags and
chelators,
bioaffine bindings like e.g. a biotin/streptavidin bond.
The 'biological molecules in the sample' are the target molecules potentially
present within said sample that should be detected and therefore, they are
also
named 'target molecules in the sample'. Additionally, certain non-target
molecules
may be present in said sample that may disturb the detection of the target
molecules. These non-target molecules initially present in the sample can not
be
avoided by the present invention. If the target molecules and the non-target
molecules are nucleic acid molecules, they are called target nucleic acid
molecules
and non-target nucleic acid molecules, respectively. A non-target nucleic acid
molecule is a molecule with a different sequence than the target nucleic acid
molecule.
Moreover, the reagents necessary to detect said target molecule are solutions
that
may contain non-target molecules or target molecules or both and both
molecules
may disturb the detection of the target molecules in the sample, e.g. within a
diagnostic test, if they are introduced in said sample as a contamination.
Within the
scope of this invention, non-target molecules potentially present in the
reagents
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also comprise cells containing target and/or non-target nucleic acid
molecules.
The non-target molecules and/or the target molecules potentially present in
the
reagents are summarized by the phrase 'biological molecules potentially
present in
the reagents' throughout this invention.
Regarding nucleic acids, the target molecules potentially present in the
reagents
comprise the nucleic acid molecule itself as well as corresponding amplicons
and
nucleic acids in cells. The phrase 'nucleic acid molecules potentially present
in the
reagents' is used throughout this invention to summarize all possible
combinations
of nucleic acid molecules potentially present in the reagents, namely just
target
nucleic acid molecules, just non-target nucleic acid molecules or both target
and
non-target nucleic acid molecules.
Reagents include all substances necessary to detect the target molecule in the
sample, namely e.g. buffers, enzymes, antibodies, primers, probes, labels,
nucleotides and/or oligonucleotides.
The contaminations of said reagents can either be present initially as e.g.
non-target
molecules and/or target molecules or they may be introduced during one or more
of
the steps of the detection procedures, e.g. by adding contaminated reagents or
simply by contact of the reagents with the surrounding atmosphere. The
contaminations of the reagents can introduce non-target molecules as well as
target
molecules to said sample leading to false positive results.
Within the present invention, possible contaminations of said reagent include
cells
or assemblies of cells as well and therefore, cells or assemblies of cells are
understood as non-target molecules within the scope of the present invention,
too.
As a prominent example the bacterial contamination of PCR reagents should be
mentioned here, since these bacterial contaminations of the reagents can add
double-stranded (ds) DNA to the solution as well. If the contaminating
bacterium
within the reagent is the same as the one that should be verified by detecting
a
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specific target molecule within the sample, this may produce false positive
results
in a diagnostic test based on a PCR amplification of the target nucleic acid.
In this
case, target nucleic acid molecules that do not originate from the sample
under
investigation will contribute to the amplification result of the PCR.
The decontamination of the reagents from non-target molecules and/or target
molecules occurs, when the binding moieties are in physical contact with the
reagents under certain conditions, whereby the non-target molecules and/or
target
molecules bind reversibly to the binding moieties.
Throughout this invention, the phrase physical contact shall be understood as
any
contact between said binding moieties and said reagents under certain
conditions of
buffer, pressure, temperature, irradiation or mechanical stress. As known to
someone skilled in the art, the binding between organic molecules is in
general
reversible and depending on the conditions of the surrounding buffer solution.
Therefore, one has to provide certain conditions in order to guarantee the
binding
of the non-target molecules and/or target molecules to the binding moieties.
Throughout this invention, the physical contact includes immersion of slides
or
particles in the reagents, filling of e.g. tubes or the flow through of e.g.
pipette tips
by the reagents.
In the scope of this invention all detection formats are possible for the
detection of
target molecules as far as they are capable of detecting organic molecules.
In case of DNA, suitable detection methods are known to the expert in the
field and
are described in standard textbooks as of Sambrook et al.: Molecular Cloning,
A Laboratory Manual (2nd Addition, Cold Spring Harbour Laboratory Press, Cold
Spring Harbour, NY) or of Ausubel et al.: Current Protocols in Molecular
Biology
(1987) J. Wiley and Sons, NY).
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In case of proteins, suitable assays are described in the text books of
Tijssen:
Practice and theory of enzyme immunoassays (1985, Elsevier, Amsterdam,
Netherlands) and Aslam & Dent: Bioconjugation (2000, Macmillian Reference,
London, GB).
For the amplification of target nucleic acid molecules all amplification
techniques
known to someone skilled in the art are possible within the scope of the
present
invention, like e.g. PCR, LCR or the replicase amplification.
Another aspect of the invention concerns a solid phase material, whereas
double-
stranded nucleic acid binding moieties are coupled to the surface of said
solid
phase material. The complex of the solid phase material and a certain number
of
binding moieties that are coupled to its surface can be any combination of a
solid
phase material and binding moieties according to the invention.
Another subject matter of the present invention is the use of said solid phase
material for a target nucleic acid amplification in a sample avoiding the
amplification of nucleic acid molecules potentially present in the reagents
necessary to amplify said target nucleic acid molecules in said sample. The
amplification of a target nucleic acid molecule includes any possibility to
enlarge
the number of a nucleic acid molecule within a sample. Examples are the PCR or
LCR amplification providing an exponential growth of the nucleic acid copy
number against the cycle number or the replicase amplification providing a
linear
growth of the nucleic acid copy number against the reaction time.
The invention also concerns a kit comprising the reagents necessary to perform
a
target nucleic acid amplification and said solid phase material according to
the
invention.
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Description of the Figures:
Figure 1: Biotinylated ds-NA binder (MGB)
Figure 2: Characterization of ds-NA binder (MGBs, peptides &
intercalator)
Figure 3: Decontamination efficiency from bacterial DNA using ds-NA
binders coupled to Dynabeads~ determined by PCR
amplification.
Figure 4: Decontamination efficiency from hg DNA using ds-NA binders
coupled to Dynabeads~ determined by PCR amplification.
Figure 5: Decontamination efficiency from plasmid DNA using three ds-
NA binders coupled to Dynabeads~ determined by PCR
amplification.
Figure 6: Selectivity evaluation of the Bi-PEG-Distamycin/SA-magnetic
bead complex with respect to double-stranded and single-
stranded oligonucleotides in a time dependent manner using UV-
absorbance.
Figure 7: Selectivity evaluation of three ds-NA binders with respect to
double-stranded and single-stranded oligonucleotides in a time
dependent manner using PCR amplification.
Figure 8: Decontamination of solutions from bacterial DNA using the Bi-
PEG-Distamycin/SA-magnetic bead complex and quantification
by PCR amplification.
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Figure 9: Decontamination of solutions from bacterial DNA using the Bi-
PEG-Distamycin/SA-coated tubes and quantification by PCR
amplification.
Figure 10: Decontamination efficiency for two bacteria concentrations using
several bacteria binder/SA-magnetic bead complexes determined
by PCR amplification.
Detailed descriution of the invention:
One subject matter of the present invention is a method for the detection of
biological molecules in a sample that avoids the detection of biological
molecules
potentially present in the reagents necessary to detect the biological
molecules in
the sample, comprising the steps of
a) providing a sample potentially comprising said biological molecules,
b) providing binding moieties coupled to the surface of a solid phase,
c) providing reagents necessary to detect the biological molecules,
d) adding said reagents to said sample,
e) detecting the biological molecules in said sample and
wherein in step c) or in step d) or in step c) and d) said reagents are in
physical
contact with said binding moieties under conditions, whereby said biological
molecules potentially present in said reagents bind to said binding moieties
coupled
to said surface of a solid phase.
Another subject matter of the present invention is a method for the detection
of
biological molecules in a sample that avoids the detection of biological
molecules
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potentially present in the reagents necessary to detect the biological
molecules in
the sample, comprising the steps of
a) providing binding moieties coupled to the surface of a solid phase,
b) providing reagents necessary to detect the biological molecules,
c) adding said reagents to the sample potentially comprising said target
molecule,
d) detecting the biological molecules in said sample and
wherein in step b) or in step c) or in step b) and c) said reagents are in
physical
contact with said binding moieties under conditions, whereby said biological
molecules potentially present in said reagents bind to said binding moieties
coupled
to said surface of a solid phase.
Reagents throughout the present invention are all reagents necessary to detect
said
target molecule and comprise substances like e.g. buffer solutions, enzymes,
antibodies, primers, probes, labels, nucleotides and/or oligonucleotides.
Since these
reagents are added to the sample, it is important to assure that the used
reagents are
free of contaminations with non-target molecules as well as target molecules
in
order to avoid false positive results detecting the target molecule within the
sample.
The detection methods mentioned above for detecting a target molecule in a
sample
that avoids the detection of biological molecules potentially present in said
reagents, reduce the risk of false positive results due to contaminations.
The biological molecules potentially present in said reagents are selected
from the
group consisting of target molecules, non-target molecules and target and non-
target molecules together. The binding of non-target molecules and/or target
molecules to said binding moieties is preferably reversible and therefore, the
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binding occurs under certain conditions and can be released by changing said
conditions. It is preferred that the binding and the release of non-target
molecules
and/or target molecules can be switched by altering the conditions in terms of
buffer concentration, temperature or mechanical stress. Most preferably, the
binding of non-target molecules and/or target molecules occurs at room
temperature at moderate salt concentrations.
The conditions for binding and the kind of interaction between organic
molecules
are known to people skilled in the art. In case of proteins, interactions are
described
in the text books of Tijssen: Practice and theory of enzyme immunoassays
(1985,
Elsevier, Amsterdam, Netherlands) and Aslam & Dent: Bioconjugation (2000,
Macmillian Reference, London, GB). The interaction of DNA with other organic
molecules is described in standard textbooks as of Sambrook et al.: Molecular
Cloning, A Laboratory Manual (2nd Addition, Cold Spring Harbour Laboratory
Press, Cold Spring Harbour, NY) or of Ausubel et al.: Current Protocols in
Molecular Biology (1987, J. Wiley and Sons, NY).
Binding of ds DNA to ds-NA binders like minor groove binders or intercalators
can
be performed in salt containing solutions like sodium chloride, potassium
chloride,
magnesium chloride or Tris buffer, whereas the binding affinity is higher at
lower
salt concentrations, preferably a salt concentration of 10 - 100 mM is used.
Moreover, the binding step is preferably performed at room temperature at non-
denaturing pH, preferably at pH 6 - 8 (Zimmer, G. et al., Prog. Biophys.
Molec.
Biol. 47 (1986) 37-112; Pauluhn, J. et al., Ber. Bunsenges. Phys. Chem. 82
(1978)
1265-1278).
For the binding of ds DNA to ds-DNA binding antibodies TBS buffer can be used,
preferably 25 mM Tris and 150 mM sodium chloride at pH 7,4 and room
temperature (Di Pietro, S.M. et al., Biochemistry 42 (2003) 6218-6227). The
affinity of the protein-DNA binding decreases with increasing salt
concentration
(Record, M.T. et al., J. Mol. Biol. 107 (1976) 145-158).
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Additionally, conditions suitable to bind double-stranded DNA to the different
ds-
DNA binder can be found in the articles as well as in the patents cited in the
chapter "Prior Art Background" and in the examples of this invention.
In a preferred method according to the invention said conditions to bind said
biological molecules potentially present in said reagents to said binding
moieties
coupled to said surface of a solid phase comprise a salt concentration of 10 -
200
mM, most preferably of 10 - 50 mM, a non-denaturing pH, most preferably pH 6 -
8, an incubation time of 5 - 90 minutes, most preferably of 60 minutes, and
room
temperature.
In another preferred method according to the invention said reagents necessary
to
detect said biological molecules in the sample are mixed prior to the adding
step,
whereby said reagents are in physical contact with said binding moieties under
conditions, whereby said biological molecules potentially present in said
reagents
bind to said binding moieties coupled to said surface of a solid phase.
Since in general two or more reagents are necessary in a certain ratio to
perform the
detection of the target molecule, it may be advantageous to mix said reagents
prior
to the addition to the sample, whereby the reagents are in physical contact
with said
binding moieties, preferably at all times. In another embodiment of the
invention,
the reagents are added successively to the sample in appropriate amounts,
whereby
each reagent is in physical contact with said binding moieties, preferably at
all
times.
In a preferred method of the invention said surface of a solid phase is the
inner
surface of a vessel or a pipette tip.
In this embodiment of the invention, the reagents necessary to detect said
target
molecules are provided, maintained and/or mixed in a vessel with binding
moieties
coupled to its surface prior to the addition to the sample. If the reagents
are
CA 02512599 2005-08-17
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transferred from one vessel to another or if the reagents are added finally to
the
sample, in one embodiment of the invention a pipette tip with binding moieties
coupled to its surface is used. The solid phases functionalized with the
binding
moieties according to the present invention are preferably plastic devices,
like e.g.
tubes or pipette tips, or glass devices like e.g. reaction vessels.
In another preferred method of the invention said surface of a solid phase is
the
surface of beads or the surface of a porous material.
In this embodiment of the invention, beads with binding moieties coupled to
the
surfaces of said beads are added to a vessel containing a certain reagent.
Providing
the appropriate conditions for a certain incubation time, the non-target
molecules
and/or the target molecules bind to the binding moieties and the supernatant
comprises the reagent that is preferably free of non-target molecules and of
target
molecules. Most preferably, these beads are magnetic beads, like the
commercial
magnetic Dynabeads0 (Dynal Biotech S.A., Oslo, Norway).
I S In another embodiment of the invention, a porous material with binding
moieties
coupled to the surfaces of said porous material is used to get rid of non-
target
molecules and/or target molecules within the reagents. Preferably, the porous
material is formed as a filter, whereas said filter can be combined with e.g.
a
syringe in order to remove non-target molecules and/or target molecules from
the
reagents during the transfer form one vessel to another or to the sample. The
porous
solid phases possible to couple the binding moieties according to the present
invention are preferably porous inorganic materials, like glass fleeces or
controlled-
pore-glass. An alternative are porous plastics like polyethylene (PE) or
polypropylene (PP) or polyethylenterephthalate (PET), polyacrylnitrile (PAT),
polyvinylidendifluoride (PVDF) or polystyrene. In another alternative
embodiment
of the invention, porous organic materials are used, like e.g. porous polymer
or
copolymer material.
CA 02512599 2005-08-17
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In a preferred embodiment of the method, the binding moieties according to the
invention are coupled to said surface of a solid phase via a covalent bond or
via a
bioaffine bond, preferably a biotin/streptavidin bond.
The coupling of said binding moieties to the surface of said solid phase
includes
covalent bonds like e.g. silane coupling to hydroxy surfaces or amino coupling
to
epoxide, thiol coupling to metals like gold, coordinative bindings like e.g.
between
His-tags and chelators, bioaffine bindings like e.g. a biotin/streptavidin or
biotin/avidin bond. In another embodiment of the invention, the surface of
said
solid phase is covered by a polymer layer comprising the coupling sites for
the
binding moieties.
In a preferred embodiment of the invention, the binding affinity of said
binding
moieties realize a reduction of the biological molecule content in the
reagents by at
least a factor of 10z, preferably by at least a factor of 103.
The biological molecule content in the reagents comprises the content of
target
molecules and/or the content of non-target molecules in the reagents,
depending on
the type of nucleic acid molecules present in said reagents.
The phrase 'binding affinity' is used throughout the invention as a
qualitative
measure for the ability to bind non-target molecules and/or target molecules.
The
ability of a certain binding moiety to reduce the content of non-target
molecules
and/or target molecules may be dependent on the conditions of the environment,
namely e.g. the buffer composition or the temperature. Within the scope of
this
invention, the combination of binding moiety and environment conditions are
adjusted to realize a reduction of the non-target molecule content and/or of
the
target molecule content within the reagents by at least a factor of 102, more
preferably by at least a factor of I 03.
CA 02512599 2005-08-17
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In an also preferred embodiment, the method according to the invention further
comprises the following steps
f) eluting the biological molecules associated with said binding moieties and
g) detecting said eluted biological molecules.
Since the binding of non-target molecules and/or of target molecules to the
binding
moieties according to the invention is reversible, it is possible to detect
the bound
molecules after the decontamination in order to learn about the contaminations
within the used reagents or of the contaminations introduced during the
preparation
steps. Said elution occurs, if the binding condition in terms of buffer
concentration,
temperature, pH, denaturing reagents or mechanical stress are changed. It is
preferred that the release of non-target molecules and/or of target molecules
from
said binding moieties is performed at moderate temperatures and salt
concentrations at alkaline pH. In case of an electrostatic interaction between
the
binding moieties and the non-target molecules and/or target molecules, the
release
can be obtained by increasing the salt concentration and the corresponding
enhancement of screening effects. In general, the coupling of non-target
molecules
and/or of target molecules to the binding moieties can be raptured by
increasing the
temperature.
In yet another preferred method according to the invention said target
molecules
and said non-target molecules are nucleic acid molecules, most preferably said
target molecules and said non-target molecules in the reagents are double-
stranded
nucleic acid molecules.
In a preferred embodiment of the invention, the binding moieties are moieties
capable of binding double-stranded nucleic acid molecules.
CA 02512599 2005-08-17
If the binding moieties bind double-stranded nucleic acid molecules they are
named
double-stranded nucleic acid binding moieties. The phrases double-stranded
nucleic acid binding moiety or 'ds-NA binder' are used as equivalents
throughout
this invention.
In a more preferred embodiment of the invention, the binding moieties are
polycationic entities, minor groove binders, intercalators or anti double-
stranded
nucleic acid antibodies.
A polycationic entity is a large molecule having multiple charges. In case of
a
polymer or a peptide this polycationic entity comprises a certain amount of
monomers, each monomer having a positive charge or a negative charge or is
neutral. Therefore, such a polycationic entity is characterized by its net
charge,
which is the sum of all monomer charges. Examples are peptides or polyamide
derivatives. Minor groove binders (MGBs), like e.g. Distamycin or methyl-
imidazol-polyamide derivatives, are molecules that bind to the minor groove of
a
I S double-stranded nucleic acid. Intercalators, like e.g. Actinomycin D or
ethidium
bromide, are molecules that bind between the stacked base pairs of a double-
stranded nucleic acid. Anti-ds DNA antibodies are known to selectively bind
only
to double-standed DNA and not to single-stranded DNA. These kind of ds-NA
binders are known to someone skilled in the art and additional informations
are
included in the chapter 'prior art background'.
In another embodiment according to the invention, said detection of the target
molecule in the sample is based on a nucleic acid amplification reaction.
Suitable DNA detection methods are known to the expert in the field and are
described in standard textbooks as of Sambrook et al.: Molecular Cloning,
A Laboratory Manual (2nd Addition, Cold Spring Harbour Laboratory Press, Cold
Spring Harbour, NY) or of Ausubel et al.: Current Protocols in Molecular
Biology
(1987) J. Wiley and Sons, NY). The detection methods may include but are not
CA 02512599 2005-08-17
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limited to the binding or intercalating of specific dyes as ethidium bromide
which
intercalates into the double-stranded DNA and changes its fluorescence
thereafter.
The purified DNA may also be separated by electrophoretic methods optionally
after a restriction digest and visualized thereafter. There are also probe-
based
assays which exploit the oligonucleotide hybridisation to specific sequences
and
subsequent detection of the hybrid. It is also possible to sequence the DNA
after
further steps known to the expert in the field. Other methods apply a
diversity of
DNA sequences to a silicon chip to which specific probes are bound and yield a
signal when a complementary sequence binds.
The amplification of a target nucleic acid molecule includes any possibility
to
enlarge the copy number of a nucleic acid molecule within a sample. Examples
are
the PCR or LCR amplification providing an exponential growth of the nucleic
acid
copy number against the cycle number or the replicase amplification providing
a
linear growth of the nucleic acid copy number against the reaction time. Other
possible amplification reactions are already described in the chapter 'Prior
Art
Background'.
All preparation steps prior to said nucleic acid amplification, like e.g.
preparing of
buffer solutions, diluting of stock solutions, providing of enzymes,
nucleotides,
primers and probes, mixing of ingredients and/or pipetting the reagents into
the
reaction vessel for amplification, comprise a certain risk of contaminating
the
sample. Therefore, as many of these preparation steps prior to said nucleic
acid
amplification as possible should be performed with the aid of the binding
moieties
coupled to the surface of a solid phase.
Particularly preferred detection methods for DNA can be done in so-called
"homogeneous" assays. A "homogeneous" assay system comprises reporter
molecules or labels which generate a signal while the target sequence is
amplified.
An example for a "homogeneous" assay system is the TaqMan~ system ,that has
been detailed in US 5,210,015, US 5,804,375 and US 5,487,972. Briefly, the
CA 02512599 2005-08-17
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method is based on a double-labelled probe and the 5'-3' exonuclease activity
of
Taq DNA polymerase. The probe is complementary to the target sequence to be
amplified by the PCR process and is located between the two PCR primers during
each polymerisation cycle step. The probe has two fluorescent labels attached
to it.
One is a reporter dye, such as 6-carboxyfluorescein (FAM), which has its
emission
spectra quenched by energy transfer due to the spatial proximity of a second
fluorescent dye, 6-carboxy-tetramethyl-rhodamine (TAMRA). In the course of
each
amplification cycle, the Taq DNA polymerase in the process of elongating a
primed DNA strand displaces and degrades the annealed probe, the latter due to
the
intrinsic 5'-3' exonuclease activity of the polymerise. The mechanism also
frees the
reporter dye from the quenching activity of TAMRA. As a consequence, the
fluorescent activity increases with an increase in cleavage of the probe,
which is
proportional to the amount of PCR product formed. Accordingly, the amplified
target sequence is measured by detecting the intensity of released
fluorescence
label.
A similar principle of energy transfer between fluorescent dye molecules
applies to
"homogeneous" assays using so-called "molecular beacons" (US 6,103,476). These
are hairpin-shaped nucleic acid molecules with an internally quenched
fluorophore
whose fluorescence is restored when they bind to a target nucleic acid
(US 6,103,476). They are designed in such a way that the loop portion of the
molecule is a probe sequence complementary to a region within the target
sequence
of the PCR process. The stem is formed by the annealing of complementary arm
sequences on the ends of the probe sequence. A fluorescent moiety is attached
to
the end of one arm and a quenching moiety is attached to the end of the other
arm.
The stem keeps these two moieties in close proximity to each other, causing
the
fluorescence of the fluorophore to be quenched by energy transfer. Since the
quencher moiety is a non-fluorescent chromophore and emits the energy that it
receives from the fluorophore as heat, the probe is unable to fluoresce. When
the
probe encounters a target molecule, it forms a hybrid that is longer and more
stable
than the stem hybrid and its rigidity and length preclude the simultaneous
existence
CA 02512599 2005-08-17
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of the stem hybrid. Thus, the molecular beacon undergoes a spontaneous
conformational reorganisation that forces the stem apart, and causes the
fluorophore and the quencher to move away from each other, leading to the
restoration of fluorescence which can be detected.
More examples for "homogeneous" assay systems are provided by the formats used
in the LightCycler~ instrument (see e.g. US 6,174,670), some of them are
called
"kissing probe" formats sometimes. Again, the principle is based on two
interacting
dyes which, however, are characterised in that the donor-dye excites an
acceptor-
dye by fluorescence resonance energy transfer. An exemplified method uses two
modified oligonucleotides as hybridisation probes, which hybridise to adjacent
internal sequences of the target sequence of the PCR process. The 5'-located
modified oligonucleotide has a donor-dye as a label at its 3' end. The 3'-
located
modified oligonucleotide has an acceptor-dye at its 5' end. Following the head-
to-
tail-oriented annealing of the two modified oligonucleotides to the target
sequence
in the course of an amplification cycle, donor and acceptor dye are brought in
close
proximity. Upon specific excitation of the donor dye by means of a
monochromatic
light pulse, acceptor dye fluorescence is detected providing a measure for the
amount of PCR product formed.
Another assay format is the so-called "array" format. An "array" is an
arrangement
of addressable locations on a device (e.g. US 5,143,854, US 6,022,963, US
6,156,501, WO 90/15070, WO 92/10092). The number of locations can range from
several to at least hundreds of thousands. Most importantly, each location
represents a totally independent reaction site. Each location carries a
nucleic acid as
e.g. an "oligomeric compound", which can serve as a binding partner for a
second
nucleic acid, in particular a target nucleic acid. Methods for the
manufacturing
thereof are described in EP-A-0 476 014, Hoheisel, J. D., TIBTECH 15 (1997)
465-469, WO 89/10977, WO 89/11548, US 5,202,231, US 5,002,867, WO
93/17126. Further developments have provided methods for making very large
arrays of oligonucleotide probes in very small areas (US 5,143,854, WO
90/15070,
CA 02512599 2005-08-17
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WO 92/10092). Microfabricated arrays of large numbers of oligonucleotide
probes,
called "DNA chips" offer great promise for a wide variety of applications
(e.g. US
6,156,501 and US 6,022,963). The basic steps of the method are that nucleic
acid
from control and treatment samples is isolated and labeled with different
fluorescent dyes incorporated during an amplification process. In more detail,
this
is performed according to the method described in US 5,545,522; US 5,716,785;
US 5,891,636 and US 6,291,170, whereby double stranded cDNA is synthesized
with a primer comprising the bacterial T7-Promoter and labeled RNA is
transcribed
in the presence of ribonucleoside triphosphates, whereby labels are attached
to
some of the nucleoside triphosphates. These labeled nucleic acids are then
optionally fragmented, mixed and hybridized to the arrayed oligomeric
compounds.
An optical device is then used to measure the relative intensities of each dye
for
each individual spot. The ratio of fluorescence levels between the two probes
indicates the relative gene expression between the samples. By these processes
researchers can evaluate an entire set of genes simultaneously rather than
looking at
the effects of single genes one at a time. High differential expression of
specific
genes can then be followed up by conventional means such as northern blot or
quantitative real-time PCR. Data from multiple experiments can be combined in
order to assign functional information to genes of otherwise unknown function.
Genes showing similar expression profiles across differing states are likely
to
participate in common physiological or metabolic pathways. Cluster analysis
programs have been developed which allow detection of co-expressed groups of
genes reflecting information on function. In yet another embodiment according
to
the invention, said binding moieties are moieties capable of binding
biological
compartments.
In another embodiment of the invention, the contaminations within the reagents
are
biological compartments and the binding moieties are able to bind these
biological
compartments. Biological compartments are cells or assemblies of cells. Cells
or
assemblies of cells that are potentially present in the reagents and contain
target
CA 02512599 2005-08-17
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and/or non-target nucleic acid molecules are non-target molecules within the
scope
of this invention. Since cells have certain proteins or polymers within their
cell
membrane, the binding moieties of this embodiment of the invention have a
certain
affinity for these molecular structures in order to reversibly bind the cells
or
assemblies of cells. Possible binding moieties for this embodiment of the
invention
comprises antibodies for membrane compounds or cell binding proteins,
preferably
Fibronectin or functional parts of those binding proteins.
In another embodiment, the binding moieties for binding cells or assemblies of
cells are binding moieties that assemble with the membrane structure,
preferably
dye molecules or amphiphilic molecules. Additionally, minor groove binder
(MGB) are applicable as binding moieties for binding cells or assemblies of
cells.
Examples for dyes comprise Crystal violet, methylene blue and Safranin O that
are
functionalized in order to couple to solid surfaces. Examples for amphiphilic
molecules capable of binding to cell membranes comprise e.g sodium dodecyl
sulfate (SDS), cholesterol or lauroyl-lysin, also functionalized in order to
couple to
solid surfaces. Suitable MGBs are e.g. Distamycin or methyl-imidazol-polyamide
derivatives.
In yet another embodiment, the binding moieties for binding cells or
assemblies of
cells are polycationic entities. These polycationic entities, preferably
polycationic
peptides or polymers, bind cells by electrostatic interactions with the
multiple
negative charges of the cell membranes. These polycationic entities are
functionalized in order to couple to solid surfaces, too.
In another preferred embodiment of the invention, said binding moieties are
moieties capable of binding double-stranded nucleic acid molecules and
biological
compartments. A possible variant of this embodiment of the invention is the
combination of binding moieties that are specific for cells or assemblies of
cells
with binding moieties that are specific for double-stranded nucleic acid
molecules.
Therefore, this embodiment of the invention can be realized with two or more
CA 02512599 2005-08-17
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different binding moieties or with one type of binding moiety that is able to
bind
both double-stranded nucleic acid molecules and biological compartments. The
method according to this embodiment of the invention is able to bind
contaminations based on cells or assemblies of cells and on double-stranded
nucleic acid molecules potentially present in said reagents in a single
preparation
step.
Another aspect of the invention concerns a method to amplify a target nucleic
acid
molecule, comprising the steps of
a) providing a sample potentially comprising target nucleic acid
molecules,
b) providing nucleic acid binding moieties coupled to the surface of a
solid phase,
c) providing reagents necessary to amplify said target nucleic acid
molecules,
d) adding said reagents to said sample,
e) amplifying said target nucleic acid molecules in said sample and
wherein in step c) or in step d) or in step c) and d) said reagents are in
physical
contact with said nucleic acid binding moieties under conditions, whereby said
nucleic acid molecules potentially present in said reagents bind to said
nucleic acid
binding moieties coupled to said surface of a solid phase.
Another aspect of the invention concerns a method to amplify a target nucleic
acid
molecule avoiding the amplification of nucleic acid molecules potentially
present
in said reagents necessary to amplify said target nucleic acid molecules in
the
sample, comprising the steps of
CA 02512599 2005-08-17
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a) providing a sample potentially comprising target nucleic acid
molecules,
b) providing nucleic acid binding moieties coupled to the surface of a
solid phase,
c) providing reagents necessary to amplify said target nucleic acid
molecules,
d) adding said reagents to said sample,
e) amplifying said target nucleic acid molecules in said sample and
wherein in step c) or in step d) or in step c) and d) said reagents are in
physical
contact with said nucleic acid binding moieties under conditions, whereby said
nucleic acid molecules potentially present in said reagents bind to said
nucleic acid
binding moieties coupled to said surface of a solid phase.
Yet another aspect of the invention concerns a method to amplify a target
nucleic
acid molecule in a sample comprising the steps of
a) providing nucleic acid binding moieties coupled to the surface of a
solid phase,
b) providing reagents necessary to amplify said target nucleic acid
molecules,
c) adding said reagents to the sample potentially comprising target nucleic
acid molecules,
d) amplifying said target nucleic acid molecules in said sample and
CA 02512599 2005-08-17
-2g-
wherein in step b) or in step c) or in step b) and c) said reagents are in
physical
contact with said nucleic acid binding moieties under conditions, whereby said
nucleic acid molecules potentially present in said reagents bind to said
nucleic acid
binding moieties coupled to said surface of a solid phase.
Yet another aspect of the invention concerns a method to amplify a target
nucleic
acid molecule in a sample avoiding the amplification of nucleic acid molecules
potentially present in said reagents necessary to amplify said target nucleic
acid
molecules in the sample, comprising the steps of
a) providing nucleic acid binding moieties coupled to the surface of a
solid phase,
b) providing reagents necessary to amplify said target nucleic acid
molecules,
c) adding said reagents to the sample potentially comprising target nucleic
acid molecules,
d) amplifying said target nucleic acid molecules in said sample and
wherein in step b) or in step c) or in step b) and c) said reagents are in
physical
contact with said nucleic acid binding moieties under conditions, whereby said
nucleic acid molecules potentially present in said reagents bind to said
nucleic acid
binding moieties coupled to said surface of a solid phase.
To amplify a nucleic acid molecule several reagents are necessary, comprising
buffer solutions, enzyms, primers, probes, labels and/or nucleotides. Since
these
reagents are added to the sample, it is important to assure that the used
reagents are
free of contaminations with non-target nucleic acid molecules as well as
nucleic
acid target molecules in order to avoid false positive results detecting the
target
molecule within the sample.
CA 02512599 2005-08-17
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The nucleic acid molecules potentially present in said reagents are selected
from
the group consisting of only target nucleic acid molecules, only non-target
nucleic
acid molecules and target and non-target nucleic acid molecules together.
The amplification methods mentioned above for a target nucleic acid
amplification
in a sample that avoids the amplification of non-target nucleic acid molecules
and/or target nucleic acid molecules potentially present in said reagents,
reduce the
risk of false positive results due to contaminations.
It is preferable that the binding of non-target nucleic acid molecules and/or
target
nucleic acid molecules occurs at conditions comprising room temperature and
moderate salt concentrations as described before.
In another preferred method to amplify a target nucleic acid molecule
according to
the invention, said reagents necessary to amplify said target nucleic acid
molecules
are mixed prior to the adding step d), whereby said reagents are in physical
contact
with said nucleic acid binding moieties under conditions, whereby nucleic acid
molecules potentially present in said reagents bind to said nucleic acid
binding
moieties coupled to said surface of a solid phase.
As described before regarding the detection of target molecules, more than one
reagent is necessary to amplify the target nucleic acid and therefore, it may
be
advantageous to mix said reagents prior to the addition to the sample, whereby
the
reagents are in physical contact with said binding moieties, preferably at all
times.
The detection of the amplified target nucleic acid can be performed during the
amplification, e.g. in real-time PCR, or after the amplification.
In a preferred method to amplify a target nucleic acid molecule according to
the
invention, said surface of a solid phase is the inner surface of a vessel or a
pipette
tip.
CA 02512599 2005-08-17
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In another preferred method to amplify a target nucleic acid molecule
according to
the invention, said surface of said solid phase is the surface of beads or the
surface
of a porous material.
As described before regarding the detection of target molecules the reagents
necessary to amplify said target nucleic acid molecules are preferably
provided,
maintained and/or mixed in a vessel with binding moieties coupled to its
surface
prior to the addition to the sample. If the reagents are transferred from one
vessel to
another or if the reagents are added finally to the sample, in one embodiment
of the
invention a pipette tip with binding moieties coupled to its surface is used.
The beads with binding moieties coupled to the surfaces of said beads can be
added
e.g. to a vessel containing a certain reagent to bind non-target nucleic acid
molecules and/or target nucleic acid molecules and to obtain a supernatant
that is
preferably free of contaminations. The porous material with binding moieties
coupled to the surfaces of said porous material can be used to get rid of
contaminations within the reagents, too. Preferably, the porous material is
formed
as a filter, wheras said filter can be combined with a syringe in order to
remove
non-target nucleic acid molecules and/or target nucleic acid molecules from
the
reagents during the transfer form one vessel to another or finally to the
sample.
In a preferred method to amplify a target nucleic acid molecule according to
the
invention, said non-target nucleic acid molecules and said target nucleic acid
molecules in the reagents are double-stranded nucleic acid molecules,
preferably
double-stranded DNA molecules.
In another preferred method to amplify a target nucleic acid molecule
according to
the invention, said nucleic acid binding moieties are double-stranded nucleic
acid
binding moieties.
Possible double-stranded nucleic acid binding moieties are already described
before regarding the detection of target molecules. In order to enable the
binding of
CA 02512599 2005-08-17
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the double-stranded nucleic acid molecules to the ds-NA binders, the
temperature
has to be kept below the melting temperature of the hybrids.
In a more preferred method to amplify a target nucleic acid molecule according
to
the invention, said binding affinity of said double-stranded nucleic acid
binding
moieties to single-stranded nucleic acid molecules is lower than to double-
stranded
nucleic acid molecules.
As mentioned before, the binding affinity is a qualitative measure for the
ability of
a binding moiety to bind non-target molecules and/or target molecules
throughout
this invention. Note, that the binding affinity may be dependent on the
conditions
of the environment, namely e.g. the buffer composition or the temperature. In
case
of the ds-NA binder that should distinguish between double-stranded nucleic
acid
and single-stranded nucleic acids, the binding affinity towards these two
molecule
types must be different. Therefore, within the scope of this invention, the
combination of ds-NA binder and the conditions of the environment is adjusted
to
realize such an affinity difference.
A method to determine this affinity difference of a ds-DNA binder in a
quantitative
way is to provide two samples comprising the same amount of single-stranded
and
double-stranded DNA molecules, respectively. After a certain incubation time
of
both samples with the same number of ds-DNA binder under equal reaction
conditions, the remaining amount of both the single-stranded and the double-
stranded DNA molecules in the samples is quantified. The ratio of the
remaining
amounts in the samples is the affinity difference of a ds-DNA binder to single-
stranded and double-stranded DNA.
More general, the affinity difference of the ds-DNA binder must be large
enough to
provide the possibility to bind a small number of long, double-stranded
nucleic acid
molecules present in a reagent containing a large number of short, single-
stranded
primer molecules in an efficient manner. If the affinity difference of the ds-
DNA
CA 02512599 2005-08-17
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binder is not sufficient, the short single-stranded primer molecules will
occupy all
binding moieties and the long, double-stranded nucleic acid molecules will
remain
in the reagent (see Example 8).
In another preferred method to amplify a target nucleic acid molecule
according to
the invention, said binding affinity of said double-stranded nucleic acid
binding
moieties realize a reduction of the double-stranded nucleic acid content
within the
reagents by at least a factor of 10z, more preferably by at least a factor of
103.
The double-stranded nucleic acid content comprises the content of double-
stranded
target nucleic acid molecules and/or the content of double-stranded non-target
nucleic acid molecules in the reagents, depending on the type of nucleic acid
molecules present in said reagents.
According to another embodiment of the method to amplify a target nucleic acid
molecule, said double-stranded nucleic acid binding moieties are polycationic
entities, minor groove binders, intercalators or anti double-stranded nucleic
acid
antibodies.
According to a preferred embodiment of the method to amplify a target nucleic
acid molecule, said polycationic entities are nucleic acid binding polyamide
derivatives.
According to a more preferred embodiment of the method to amplify a target
nucleic acid molecule, said nucleic acid binding polyamide derivatives have a
positive net charge, preferably at least with 0.1 positive net charges per
monomer,
more preferably at least with 0.2 positive net charges per monomer.
As defined before, a polycationic entity is a large molecule having multiple
charges. In case of a polymer or a peptide this polycationic entity comprises
a
certain amount of monomers, each monomer having a positive charge, a negative
CA 02512599 2005-08-17
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charge or is neutral. Therefore, such a polycationic entity is characterized
by its net
charge, which is the sum of all monomer charges. In order to compare
polycationic
entities with a different amount of monomeric units, the net charge is
normalized
on this number of monomeric units, providing the quantity 'positive net
charges per
monomer'. These polycationic entity provide a certain selectivity between
double-
stranded and single-stranded nucleic acids, namely the polycationic entities
have a
higher affinity towards double-stranded nucleic acids compared to single-
stranded
nucleic acids. Without being bound to theory, this effect may originate from
the
difference in molecular charge of double-stranded and single-stranded nucleic
acids. In a preferred embodiment of the invention, where long double-stranded
nucleic acids (e.g. bacterial DNA) are present in a reagent containing short
single-
stranded nucleic acids (e.g. primers), the selectivity based on charge
differences
between the double-stranded and the single-stranded nucleic acids in the
reagents is
even more pronounced.
According to a preferred embodiment of the method to amplify a target nucleic
acid molecule, said minor groove binders are Distamycin, Netropsin, methyl-
imidazol-polyamide derivatives or 4',6-diamidino-2-phenylindole (DAPI)
derivatives.
According to another preferred embodiment of the method to amplify a target
nucleic acid molecule, said intercalators are acridine derivatives, preferably
acriflavine derivatives or phenanthridinium compounds, preferably ethidium
bromide derivatives.
The minor groove binders and intercalators mentioned above as well as other
molecules that can be used for the present invention, too are e.g. described
in the
text book of Demeunynck et al (Eds.): DNA and RNA binders (Vol. 1 & 2, 2003,
Wiley-VCH, Weinheim).
CA 02512599 2005-08-17
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In another preferred method to amplify a target nucleic acid molecule
according to
the invention, said double-stranded nucleic acid binding moieties are coupled
to
said surface of the solid phase via a covalent bond or via a bioaffine bond,
preferably a biotin/streptavidin bond.
In another preferred method to amplify a target nucleic acid molecule
according to
the invention, biotinylated double-stranded nucleic acid binding moieties are
bound
to the streptavidin coated surface of the solid phase.
Using this standard system of a bioaffine bond has the advantage that several
solid
phase materials already functionalized with Streptavidin are commercially
available. For example Streptavidin coated Dynabeads~ M-280 (Dynal Biotech
S.A., Prod. No. 112.06, Oslo, Norway) or Streptavidin-coated PCR tubes (Roche
Cat. No. 1741772, Roche Diagnostics GmbH, Mannheim). In addition, the
modification of different organic molecules with a biotin group is widely used
in
the art (see text book of Kessler (Ed.): Nonradioactive labeling and detection
of
biomolecules, 1992, Springer Verlag, Heidelberg).
In another preferred method to amplify a target nucleic acid molecule
according to
the invention, the method further comprises the steps
f) eluting the nucleic acid molecules associated with said nucleic acid
binding moieties and
g) detecting said eluted nucleic acid molecules.
This preferred method according to the invention provides the opportunity to
detect
the bound non-target nucleic acid molecules and/or target nucleic acid
molecules
after the decontamination in order to learn about the contamination within the
reagents used or of the contamination introduced during the detection
procedures.
The detection of the eluted nucleic acid molecules can be performed with
CA 02512599 2005-08-17
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techniques known to someone skilled in the art, e.g. PCR amplification or
certain
array technologies.
In a more preferred method to amplify a target nucleic acid molecule according
to
the invention, the elution in step f) comprises temperature treatment, change
of
ionic strength, pH modification or denaturing reagents.
Said elution of bound non-target nucleic acid molecules and/or target nucleic
acid
molecules can be performed by temperature treatment, change of ionic strength,
pH
modification and/or denaturing reagents. In case of a ds-NA binder as binding
moiety and consequently double-stranded DNA as bound molecules, the elution is
equivalent to the denaturing of the DNA hybrid, whereas increasing the
temperature above the melting temperature or alkaline pH conditions are
preferred.
In yet another preferred method to amplify a target nucleic acid molecule
according
to the invention, the amplification of said target nucleic acid is a PCR
amplification.
In an also preferred method to amplify a target nucleic acid molecule
according to
the invention, said reagents comprise oligonucleotides, nucleotides, enzymes
and
buffer solutions.
In more preferred method to amplify a target nucleic acid molecule according
to
the invention, said oligonucleotides comprise primers and probes and said
enzymes
comprise DNA-polymerases and uracil-N-glycosylase (UNG).
In another embodiment of the method to amplify a target nucleic acid molecule
according to the invention, the amplification of said target nucleic acid
molecule is
a nucleic acid amplification in a test for microbiological infection of a
sample.
Examples for a microbiological infection test of a sample are sepsis tests for
detecting Gram-positive bacteria, like e.g. staphylococcus aureus or
streptococcus
CA 02512599 2005-08-17
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pneumoniae, and Gram-negative bacteria, like e.g E.choli or Enterobacter
aerogenes. Such a test is known to someone skilled in the art and comprises
several
steps starting from obtaining the biological samples potentially containing
the
bacterial cells, lysis of the cells and finally isolating the genetic material
for
amplification.
An other aspect of the invention concerns a solid phase material, whereas
double-
stranded nucleic acid binding moieties are coupled to the surface of said
solid
phase material.
The complex out of the solid phase material and a certain number of ds-NA
binders
that are coupled to its surface can be any combination of a solid phase
material and
binding moieties according to the invention. The surface of this solid phase
may be
of arbitrary shape and therefore, includes planar surfaces of e.g. a cover
slide, the
curved surface of e.g. a test tube, a vessel or a pipette tip, the surface of
small
particles like e.g. magnetic beads or the surface of porous materials like
e.g. glass
fleeces. Throughout this invention a vessel is a single reaction vessel, like
e.g. a
Eppendorf cap or a centrifugation tube. As solid phase material all materials
are
possible within the scope of this invention, as far as the surface of this
solid phase
material comprises coupling groups for said binding moieties or as far as the
surface of this solid phase material may be functionalized with coupling
groups for
said binding moieties. In some cases it is necessary to provide said surface
with a
functional coating, before the binding moieties may be coupled. Such a
functional
coating is e.g. a polymer layer.
Yet another aspect of the invention concerns a solid phase material, whereas
double-stranded nucleic acid binding moieties are coupled to the surface of
said
solid phase material and whereas said solid phase material is a vessel, a bead
or a
pipette tip.
CA 02512599 2005-08-17
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According to another preferred embodiment of the invention, said solid phase
material is a bead or a porous material.
In a preferred embodiment of the solid phase material according to the
invention,
the binding affinity of said double-stranded nucleic acid binding moieties to
single-
stranded nucleic acid molecules is lower compared to double-stranded nucleic
acid
molecules.
In another preferred embodiment of the solid phase material according to the
invention, the binding affinity of said double-stranded nucleic acid binding
moieties realize a reduction of the double-stranded nucleic acid content
within the
reagents by at least a factor of 102, preferably at least a factor of 103.
According to another preferred embodiment of the invention, said double-
stranded
nucleic acid binding moieties are polycationic entities, minor groove binders,
intercalators or anti double-stranded nucleic acid antibodies.
According to a more preferred embodiment of the solid phase material according
to
the invention, said polycationic entities are nucleic acid binding polyamide
derivatives.
According to a even more preferred embodiment of the solid phase material
according to the invention, said nucleic acid binding polyamide derivatives
have a
positive net charge, preferably at least with 0.1 positive net charges per
monomer,
more preferably at least with 0.2 positive net charges per monomer.
According to yet another preferred embodiment of the invention, said minor
groove
binders are Distamycin, Netropsin, methyl-imidazol-polyamide derivatives or
4',6-
diamidino-2-phenylindole (DAPI) derivatives.
CA 02512599 2005-08-17
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According to yet another preferred embodiment of the invention, said
intercalators
are acridine derivatives, preferably acriflavine derivatives or
phenanthridinium
compounds, preferably ethidium bromide derivatives.
In another preferred embodiment of the solid phase material according to the
invention, said double-stranded nucleic acid binding moieties are coupled to
said
surface of a solid phase via a covalent bond or via a bioaffine bond,
preferably a
biotin/streptavidin bond.
In a more preferred embodiment of the solid phase material according to the
invention, biotinylated double-stranded nucleic acid binding moieties are
bound to
the streptavidin coated surface of the solid phase.
An other aspect of the invention is a solid phase material, whereas
polycationic
entities or intercalators are coupled to the surface of said solid phase
material as
double-stranded nucleic acid binding moieties.
In a preferred embodiment of the solid phase material according to the
invention,
biotinylated polycationic entities or biotinylated intercalators are bound to
the
streptavidin coated surface of the solid phase as double-stranded nucleic acid
binding moieties.
Yet another aspect of the invention is a solid phase material, whereas binding
moieties are coupled to the surface of said solid phase material that are able
to bind
biological compartments and whereas said solid phase material is a vessel or a
pipette tip.
As mentioned before, biological compartments are cells or assemblies of cells.
Since cells have certain proteins or polymers within their cell membrane, the
binding moieties of this embodiment of the invention have a certain affinity
for
CA 02512599 2005-08-17
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these molecular structures in order to reversibly bind the cells or assemblies
of
cells.
In a preferred embodiment of the solid phase material to bind biological
compartments according to the invention, said binding moieties are minor
groove
binders, antibodies, dyes, amphiphilic entities or polycationic entities.
Possible binding moieties for this embodiment of the invention comprises
antibodies for membrane compounds or cell binding proteins, preferably
Fibronectin or functional parts of those binding proteins. Also possible are
binding
moieties that assemble with the membrane structure, preferably dye molecules
or
amphiphilic molecules. Additionally, minor groove binder (MGB) are applicable
as
binding moieties for binding cells or assemblies of cells. Examples for dyes
comprise Crystal violet, methylene blue and Safranin O that are functionalized
in
order to couple to solid surfaces. Examples for amphiphilic molecules capable
of
binding to cell membranes comprise e.g sodium dodecyl sulfate (SDS),
cholesterol
or lauroyl-lysin, also functionalized in order to couple to solid surfaces.
Suitable
MGBs are e.g. Distamycin or methyl-imidazol-polyamide derivatives. Moreover,
polycationic entities can be used as binding moieties for binding cells or
assemblies
of cells. These polycationic entities, preferably polycationic peptides or
polymers,
bind cells by electrostatic interactions with the multiple negative charges of
the cell
membranes. These polycationic entities are functionalized in order to couple
to
solid surfaces, too.
In another preferred embodiment of the method and of the solid phase material
to
bind biological compartments according to the invention, biotinylated binding
moieties are bound to the streptavidin coated surface of the solid phase.
A further aspect of the invention is a solid phase material, whereas dyes,
amphiphilic entities, polycationic entities or minor groove binders are
coupled to
the surface of said solid phase material as binding moieties for biological
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compartments. It is preferred that said binding moieties are biotinylated and
that
said biotinylated binding moieties are bound to the streptavidin coated
surface of
the solid phase.
Another aspect of the invention concerns a solid phase material, whereas
binding
moieties are coupled to the surface of said solid phase material that are able
to bind
double-stranded nucleic acid molecules and biological compartments.
In this embodiment of the invention, said binding moieties are moieties
capable of
binding double-stranded nucleic acid molecules and biological compartments. A
possible variant of this embodiment of the invention is the combination of
binding
moieties that are specific for cells or assemblies of cells with binding
moieties that
are specific for double-stranded nucleic acid molecules. Therefore, this
embodiment of the invention can be realized with two or more different binding
moieties or with one type of binding moiety that is able to bind both double-
stranded nucleic acid molecules and biological compartments. The solid phase
material according to this embodiment of the invention is able to bind
contaminations based on cells or assemblies of cells and on double-stranded
nucleic acid molecules potentially present in said reagents in a single
preparation
step.
In a preferred embodiment of the solid phase material to bind double-stranded
nucleic acid molecules and biological compartments, said solid phase material
is a
bead, a porous material, a vessel or a pipette tip.
In another preferred embodiment of the solid phase material to bind double-
stranded nucleic acid molecules and biological compartments, two or more kinds
of
binding moieties are coupled to the surface of said solid phase material.
In a more preferred embodiment of the solid phase material to bind double-
stranded
nucleic acid molecules and biological compartments, said two or more binding
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moieties are choosen from the group consisting of minor groove binders,
intercalators, antibodies, dyes, amphiphilic entities or polycationic
entities.
In yet another preferred embodiment of the method and of the solid phase
material
to bind double-stranded nucleic acid molecules and biological compartments,
biotinylated binding moieties are bound to the streptavidin coated surface of
the
solid phase.
Yet an other aspect of the invention concerns the use of a solid phase
material
according to the invention for a target nucleic acid amplification in a sample
avoiding the amplification of nucleic acid molecules potentially present in
the
reagents necessary to amplify said target nucleic acid molecules in said
sample.
Thus, by using a solid phase material according to the invention for a target
nucleic
acid amplification in a sample that avoids the amplification of non-target
and/or
target nucleic acid molecules potentially present in said reagents, the risk
of false
positive results due to contaminations can be reduced.
In a preferred use according to the invention, said target nucleic acid
amplification
is a PCR amplification.
In another preferred use according to the invention, said target nucleic acid
amplification is part of a test for microbiological infection of a sample.
Yet an other aspect of the invention concerns a kit comprising the reagents
necessary to perform a target nucleic acid amplification and a solid phase
material
according to the invention.
In a preferred kit according to the invention, said reagents comprise
oligonucleotides, nucleotides, enzymes and buffer solutions.
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In more preferred kit according to the invention, said oligonucleotides
comprise
primers and probes and said enzymes comprise DNA-polymerases and uracil-N-
glycosylase (UNG).
In another preferred kit according to the invention, said solid phase material
comprises pipette tips and vessels.
Example 1:
Synthesis of Minor Groove Binder (MGB), peptides and intercalator
Materials
The rink amide resin and Fmoc-Glu(biotinyl-PEG)-OH were purchased from
Novabiochem (Merck Biosciences AG, Laufelfingen, Switzerland). 4-(Fmoc-
amino)-1-methylpyrrole-2-carboxylic acid (Fmoc-Amp-OH) and 4-(Fmoc-amino)-
1-methyl-1H-imidazole-2-carboxylic acid (Fmoc-Im-OH) were purchased from
Fluka and Fmoc-Orn-OH and Fmoc-Nva-OH were purchased from Bachem
(Bubendorf, Switzerland). All other amino acids were obtained from Applied
Biosystems (Foster City, USA). HBTU and HOBt were from Iris Biotech
(Marktredwitz, Germany). Peptide synthesis solvents (DMF, NMP) were obtained
from Merck AG (Darmstadt, Germany) and like all the other solvents were of the
highest biochemical grade commercially available.
General synthesis procedure
All the compounds were assembled either manually or on an Applied Biosystems
Inc. 433 peptide synthesizer using FastMoc Chemistry. Normal coupling
reactions
were performed using Fmoc amino acids ( 1 mmol) activated with HOBt/HBTU
and DIPEA for 1 h unless otherwise noted. Fmoc removal was effected by
treating
the resin for 2x5 min. with 20 % piperidine in DMF. The side chains of
trifunctional amino acids were protected as follows: Arg(Pmc), Asn(Trt),
Asp(OtBu), Gln(Trt), Glu(OtBu), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc),
Tyr(tBu).
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1. Minor Groove Binder:
MGB 1
Bi-PEG-Distamycin
H Glu(biotiuyl-PEG)-Nva-Orn-Nva Amp Amp Amp Arg-NH2
After conventional anchoring of the first arginine to 0.25 mmol of rink amide
resin
(0.43 mmol/g) and subsequent removal of its Fmoc group, the resin was
successively washed with NMP, isopropanol and NMP. DIPEA (2M in DMF) was
added to a solution of Fmoc-Amp-OH (2 mmol) and HOBt/HBTU (1:1; 2mmol).
After 2 min. of activation, this mixture was added to the resin. The
suspension was.
shaken for three hours, and the resin was washed thoroughly with DMF and
isopropanol. This procedure was repeated three times until all three Amp-
residues
were incorporated into the sequence. The Fmoc-group was cleaved as described
above and the peptide chain was elongated by Nva, Orn, Nva and Glu(biotinyl-
PEG) under normal coupling conditions employing double couplings. After
cleaving the N-terminal Fmoc-group and washing the resin with DMF, the resin
was cleaved with 2.8 ml (per 100 mg of resin) of a mixture containing TFA,
triethylsilane and ethandithiol (0.8/0.5/1.5). The resin was filtered off and
the
peptide was precipitated by 300 ml of cold diisopropylether to the eluate. The
precipitate was washed with ether, dried in vacuo and dissolved in acetic
acid/water
(1/5) for preparative HPLC purification on vydac polygosil.
LC-ESI-MS: m/z = 1410.12 [M+H]+; M,.= 1409.59 Da calcd. for C6qH104N200145~
MGB 2
Bi-PEG-Distamycin (without Arg)
H-Glu(biotinyl-PEG)-Nva-Orn-Nva-Amp-Amp-Amp-NHZ
This substance was synthesized according to the procedure as described for MGB
1, except that Fmoc-Arg (Pmc) was not coupled as first amino acid to the
resin.
LC-ESI-MS: m/z = 1253,74 [M+H]+; Mr = 1253,51 Da calcd. for CSgH92N~6O~3S
CA 02512599 2005-08-17
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MGB 3
Bi-PEG-Imidazol-derivative
H-Glu(biotinyl-PEG)-Nva-Orn-Nva-Im-Im-Im Arg-NHZ
This substance was synthesized according to the procedure as described for Bi-
PEG-Distamycin, except that Fmoc-Amp-OH was replaced by 4-(Fmoc-amino)-1-
methyl-1H-imidazole-2-carboxylic acid (Fmoc-Im-OH).
Cleavage from the resin was achieved with 1.2 ml (per 100 mg of resin) of a
mixture containing TFA, water, thioanisol, ethandithiol (1/0.05/0.05/0.1).
Workup
and purification was done as described above.
LC-ESI-MS: m/z = 1412.86 [M+H]+; M,. = 1411.9 Da calcd. for C6iH,piN23O~4S.
MGB 4
Bi-PEG-lmidazol- derivative (short)
H-Glu(biotinyl-PEG)-Im-Im-Im Arg-NH2
This substance was synthesized according to the procedure as described for MGB
3, except that Fmoc-Glu(biotinyl-PEG)-OH was directly coupled to Im.
LC-ESI-MS: m/z = 1100,5 [M+H]+; M,. = 1100.20 Da calcd. for Cq6H~3N~9O"S
2. Peptides:
Synthesis was carried out according to the general procedure described above.
Cleavage from the resin was achieved as described for the imidazole distamycin
compound and the substances were purified by preparative HPLC.
Peptide 1
H-Glu(biotinyl-PEG)-QGRVEVLYRGS WGTVA-NHZ
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- 45 -
LC-ESI-MS: m/z - 1168.06 [M+2H]2+; Mr - 2334.1 Da. calcd. for
C I04H I 6gN3o~29S
Peptide 2
H-Glu(biotinyl-PEG)-IGAVLKVLTTGLPALISWIKRKRQQ-NH2
LC-ESI-MS: m/z - 1116.58 [M+3H]3+; M,. - 3346.7 Da. calcd. for
C I 54H269N43~37s.
Peptide 3
Bi-ZU-KKNKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRA-NH2
LC-ESI-MS: m/z = 696.6 [M+7H]7+; M,. = 4869.2 Da.
Peptide 4
Bi-XUZU-
KKNKRNTNRRPQDVKFPGGGQIVGGVYLLPRRGPRLGVRATRKTS-NH2
M~ = 5639.8 Da
Peptide 5
Bi-XUZU-PWPLYGNEGLGWAGWLLSPRGSRPSWGPTDPRRRSR-NH2
Mr = 4728.8 Da
Peptide 6
Bi-XUUUU-QPGPPSEKAWQPGWT-NH2
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M~ = 2303.6 Da
Peptide 7
BXUZU-GGGGDDLGANDELISFKDEGEQEEK(Amid)
M,. = 3219,44 g/mol
Peptide 8
H Glu(biotinyl-PEG)-(Arg-Gly)5-NHZ
LC-ESI-MS: m/z = 411.39 [M+4H]4+; M,.= 1640.9 Da calcd. for C6sH,2,N3~O~~S.
Peptide 9
H Glu(biotinyl-PEG)-(Arg-Gly)~5-NH2
LC-ESI-MS: m/z = 755.8 [M+SH]s+; Mr= 3773.3 Da calcd. for C~45H2~~NgiO3~S.
Peptide 10
H Glu(biotinyl PEG)-(Lys-Gly)5-NHZ
LC-ESI-MS: m/z = 501.51 [M+3H]3+; Mr= 1501.4 Da calcd. for C6sHi2iNaW7S.
Peptide 11
I S H Glu(biotinyl PEG)-(Lys-Gly)15-NHZ
LC-ESI-MS: m/z = 839.43 [M+4H]4+; Mr = 3354.6 Da calcd. for ClasH2nNs~437S.
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Peptide 12
H Glu(biotinyl-PEG)-(Arg)ZO-NHZ
LC-ESI-MS: m!z = 61?.61 [M+6H]6+; Mr= 3699.5 Da calcd. for C~45H286N860275
Abbreviations
The nomenclature of amino acids and peptides is in accordance with the
proposals
of the IUPAC-IBU Commission on Biochemical Nomenclature (Europ. J.
Biochem. 138 (1984) 9-37)
ABI Applied Biosystems
Amp Aminomethylpyrrole-2-carboxylic acid
Boc tert.-Butyloxycarbonyl
Da Dalton
DIPEA Diisopropylethylamine
DMF Dimethylformamide
ESI-MS Electrospray Ionisation Mass Spectrometry
Fmoe Fluorenyl-9-methyloxycarbonyl
HBTU 2(1H-Benzotriazol-lyl)-1,1,3,3-tertramethyluroniumhexafluorophosphate
HOBt 1-Hydroxybenzotriazole
HPLC High Performance Liquid Chromatography
CA 02512599 2005-08-17
_ 4g _
Im Aminomethyl-1 H-imidazole-2-carboxylic acid
MS mass spectrometry
NMP N-Methylpyrrolidin
OtBu O-tert.-Butyl
PEG Polyethylenglycol
Pmc 2,2,5,7,8-Pentylmethylchroman-6-sulfonyl
TFA Trifluoroacetic acid
Trt Triphenylmethyl (Trityl)
3. Intercalator:
Acriflavine-Biotin Conjugate: 2, 7-Bis(amino)-9-(biotinylamidoethylamino)
acridine
Toronto Reasearch Chemicals, North York, Canada; Cat.Nr.: A191100; M,. _
493.64 Da (CZSH3~N~02S)
CA 02512599 2005-08-17
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Example 2:
Several biotinylated ds DNA-binder (Example 1) were immobilized onto
Streptavidin coated Dynabeads~ M-280 (Dynal Biotech S.A., Prod. No. 112.06,
Oslo, Norway ; binding capacity for biotin: 650 pmol/mg ; SA-magnetic beads
throughout these examples) according to the following procedure:
2001 (2mg) of resuspended Dynabeads~ M-280 were transferred to a PP tube.
The tube was placed on a magnet for 1-2 min. After removing the supernatant,
the
beads were washed twice with 500 ~l of washing&binding buffer (5 mM Tris-HCl
pH 7.5; 0,5 mM EDTA; 1.0 M NaCI). Thereafter, 200 ~l of washing&binding
buffer and 2~1 (2000pmol) of biotinylated ds DNA-binder (c = 1000pmo1/~1) were
added and incubated for 60 min at room temperature on an Eppendorf thermomixer
comfort. The supernatant was removed from the derivatized beads by supplying a
magnet, then the beads were washed 5 times with 500 ~l of washing&binding
buffer. The derivatized beads were stored in washing&binding buffer until
usage
(final concentration: lmg derivatized beads / 1001 washing&binding buffer).
Example 3:
The minor groove binder MGB l, MGB3 and the peptide 3 (Example 1 ) were
immobilized onto Streptavidin coated Dynabeads~ M-280 (Dynal Prod. No.
112.06, Oslo, Norway; binding capacity for biotin: 650 pmol/mg; SA-magnetic
beads throughout these examples) according to the following procedure:
200 ~1 of resuspended Dynabeads~ M-280 were transferred to a PP tube. The tube
was placed on a magnet for 1-2 min. After removing the supernatant, the beads
were washed twice with 200 ~l of washing&binding buffer (5 mM Tris-HCl pH
7.5; 0,5 mM EDTA; 1.0 M NaCI). Thereafter, 200 ~l of washing&binding buffer
and 20 ~l (20 ~mol) of biotinylated ds DNA-binder were added and incubated for
60 min at room temperature on a Eppendorf thermomixer. The supernatant was
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removed from the derivatized beads by supplying a magnet, then the beads were
washed 3-4 times with 200 ~l of washing&binding buffer. The derivatized beads
were stored in washing&binding buffer until usage (final concentration: 1 mg
derivatized beads / 100 ~l washing&binding buffer).
Example 4:
The minor groove binder MGBl, MGB3 and the peptide 3 (Figure 1 and Figure 2)
were immobilized onto Streptavidin-coated PCR tubes (Roche Cat. No. 1741772,
Roche Diagnostics GmbH, Mannheim; binding capacity for biotin: 61,5 pmol/200
~l tube) according to the following procedure:
The tubes were washed twice with buffer (50 mM Tris-HCl pH 8.1; LC Sepsis Kit,
Id.nr.04493613, Roche Diagnostics GmbH, Germany), then 195 ~1 of buffer and 5
~l of biotinylated ds-NA binder (in 10 mM Tris, pH 8.0 ; c = 1000 pmol/~1)
were
added. After incubating for 20 min at 37 °C (water bath) the
supernatant was
carefully removed with a pipet, and the tubes were washed with buffer 3-4
times.
Example 5:
In this experiment the decontamination from bacterial DNA (Staphylococcus
epidermidis) with several minor groove binder/SA-magnetic bead complexes was
performed.
For each experiment 501 (0,5 mg) of MGB/SA-magnetic bead complex (see
example 2) were transferred in a sterile and siliconized tube, washed with
2501
buffer ( 10 mM Tris-HCl pH 8.0) about four times and the supernatant was
removed
completely. Afterwards, 40 ~l of a Staphylococcus epidermidis DNA dilution
(DNA standard from LC Sepsis Test, Roche Identnr.: 04493613) were added to
each tube. During incubation at room temperature the tubes were mixed for 60
min.
The supernatants were analyzed by PCR on a Light Cycler 1.2. The standard
curve
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was obtained by a 1:10 dilution series of the bacterial DNA (Staphyloccocus
epidermidis stock solution ; c = 105 cp/10~1) in 50 mM Tris-HCl buffer pH 8.1.
The quantification reactions were run on the Light Cycler Version 1.2 using
the
Light Cycler Staphylococcus Kit Mg'~de (Roche Cat.No. 3 376 419, Roche
Diagnostics GmbH, Mannheim) with the following cycling conditions:
denaturation at 95 °C for 10 min, then 45 cycles comprising of a
profile of 50 °C
for 15 s with fluorescence measurement, 72 °C for 10 s and 95 °C
for 10 s.
Following cycling a melt analysis was done immediately by rapid cooling to 40
°C
and then increasing the temperature to 95 °C with a ramp rate of 0,1
°C/min using
continous fluorescence measurement.
The results of this experiment are summarized in Figure 3. In the experiment
1,7 x
104 cp were decontaminated. As a conclusion the decontamination efficiency is
in
the range of 103-T04 for all binders except peptides 6 and 7.
Examnle 6:
The decontamination efficiency of several ds DNA-binder molecules was
estimated
using the Light Cycler Control Kit DNA (Roche Cat.No. 2158833, Roche
Diagnostics GmbH, Mannheim), Light Cyler DNA Master Hybridization Probes
(Roche Cat.No. 2 015 102, Roche Diagnostics GmbH, Mannheim) and the
LightCycler 1.2 apparatus (Roche Cat.No 2 011 468; Software Version LC 3.5).
For each decontamination experiment 601 of a hg DNA (template for control
reaction from Light Cycler Control Kit DNA (110bp fragment of 13-globin gene))
containing solution was decontaminated with O,Smg of ds DNA binder/SA
magnetic bead complex (same preparation as in example 2). The standard curve
was obtained by a 1:10 dilution series of the hg DNA template from kit in IOmM
Tris pH 8Ø
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After incubation for 60 min at room temperature on an Eppendorf thermomixer
comfort the supernatant was analyzed by PCR according to the kit instruction
manual.
Quantifications were run on the Light Cycler apparatus and the assay was
performed according to the procedure of the kit instruction manual and using
the
following cycling conditions: denaturation at 95 °C for 30s, then 45
cycles
comprising a profile of 55 °C for 10 s with fluorescence measurement,
72 °C for 5
s and 95 °C for 0 s and final cooling to 40 °C with a ramp rate
of 20 °C/s. The
standard curve was evaluated with a dilution series of hg DNA from the kit.
The results of this experiment are summarized in Figure 4. In the experiment
1,2 x
104 cp were decontaminated. As a conclusion the decontamination efficiency is
in
the range of 102-104 for all binders except peptides 6 and 7.
Example 7:
The decontamination efficiency of the three ds-NA (MGBl, MGB3 and the peptide
3) binders was estimated using the Parvovirus B19 Quantification Kit (Roche
Cat.No. 3 246 809, Roche Diagnostics GmbH, Germany) and the LightCycler 1.2
apparatus (Roche Cat.No. 2011468, Roche Diagnostics GmbH, Germany; Software
Version LC 3.5).
For each decontamination experiment 10 ~l (0.1 mg) of ds-NA binder / SA-
magnetic bead complex from example 3 were transferred to a PP tube and washed
twice with 10 mM Tris, pH 8Ø After removal of supernatant, 20 ~l of a
plasmid
DNA (Parvovirus DNA standard from the Parvovirus B19 Quantification Kit
Roche Cat.No. 3 246 809, Roche Diagnostics GmbH, Germany) containing
solution were added. After incubation for 60 min at room temperature on a
mixer
(Thermomixer Comfort, Cat.no. 5350000.013, Eppendorf, Germany) the
supernatant was analyzed by PCR according to the kit instruction manual.
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Quantifications were run on the Light Cycler apparatus and the assay was
performed according to the procedure of the kit instruction manual without the
internal control and using the following cycling conditions: denaturation at
95 °C
for 10 min, then 45 cycles comprising a profile of 60 °C for 15 s with
fluorescence
measurement, 72 °C for 10 s and 95 °C for 10 s and final cooling
to 40 °C with a
ramp rate of 20 °C/s. The standard curve was evalutated with DNA
standards from
the kit.
The amplification curves of these decontamination experiments are summarized
in
Figure 5. As a conclusion the following decontamination efficiencies were
obtained: 4 x 10z (for MGB 1), 2,6 x 102 (for MGB 3) and 1,6 x 103 (for
peptide 3).
Table:
Results of Example 7
supernatantcomplete solution
[cp/5~1] [cp/20p1]
Starting concentration52920 2,1 x 105 100
of
contaminated solution
DNA solution after supernatantcomplete solution% left
decontamination with:[cp/Spl] [cp/20p1]
1. MGB1 128 5,1 x 102 0,242
Z. MGB3 199 7,9 x lOz 0,376
3. Peptide 3 32 1,2 x 102 0,060
Example 8:
The following experiment was performed in order to evaluate the selectivity of
the
three different ds-NA binders (MGBl, MGB3 and the peptide 3) with respect to
double-stranded and single-stranded oligonucleotides in a time dependent
manner.
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Two single-stranded oligonucleotide solutions (5'-CCC ATC CCC AAA AAC
ACA AAC CAC A P04-3 ' ; c = 14,189 OD/ml [49,9pM] in 10 mM Tris ph 8.0),
one with and one without plasmid DNA (Parvovirus DNA standard from the Light
Cycler Parvovirus B19 Quantification Kit, Roche Cat.No. 3 246 809, Roche
Diagnostics GmbH, Mannheim) were both decontaminated with ds-NA binder/SA-
magnetic bead complexes (from example 3) in a time dependent manner. The
oligonucleotide concentration in the supernatant was determined after 1, 6 and
48 h
by UV-adsorption (UVIKON 931 spectrophotometer, Kontron, Germany) and
additionally, in case of the solution containing plasmid DNA, the
decontamination
efficiency was evaluated by PCR as described in the preceding examples. For
sake
of comparison, a solution of just the plasmid DNA in buffer (TBE buffer: 10 mM
Tris-HCl pH 8.0) was decontaminated and quantified by PCR as well.
The Parvovirus B19 plasmid standard DNA (c = 5 x 105 cp/~l) was added to one
of
the single stranded oligonucleotide solutions (ratio: 1: 4 (~/,,)). For each
experiment
of Figure 6 and 7, 100 ~1 (1 mg) of Bi-PEG-Distamycin/SA-magnetic bead
complex in washing&binding buffer were transferred into a siliconized and
sterile
PP tube, washed twice with 10 mM Tris, pH 8.0 and the supernatant was removed
completely. Thereafter, 200 ~1 of both oligonucleotide solutions were added to
two
different tubes containing the beads and the absorbance of the oligo solutions
was
determined (7~ = 260 nm) after l, 6 and 48 h in order to determine the
concentration
in the supernatant. Additionally, the plasmid DNA containing solutions with
and
without single stranded oligonucleotides were analyzed by PCR on a Light
Cycler
1.2 as described in the preceding experiments using all three ds-NA binders
(Figure
7).
The results of this experiment are summarized in Figure 6 and 7. From the
adsorbance experiment (Figure 6) it can be seen that the ds DNA binding
selectivity of the Bi-PEG-Distamycin/SA-magnetic bead complex in the presence
of ss oligonucleotide is provided. Even in the presence of a large amount of
single-
stranded oligonucleotides, the ds DNA molecules are efficiently removed from
the
CA 02512599 2005-08-17
-55-
sample. The LightCycler quantification (Figure 7) demonstrates that the time-
dependent selective binding of ds DNA in the presence of ss oligonucleotide is
best, if the biotinylated peptide 3 is used as ds-NA binder.
Example 9:
In this experiment the decontamination from bacterial DNA (streptoccocus
pneumonia DNA from LC Sepsis Kit, ld.nr.04493613, Roche Diagnostics GmbH,
Germany) with the Bi-PEG-Distamycin/SA-magnetic bead complex was performed
and subsequently, the bound DNA was eluted from the beads.
For each experiment 10 ~l (0.1 mg) of Bi-PEG-Distamycin/SA-magnetic bead
complex (from example 3) were transferred in a sterile and siliconized tube,
washed with buffer (50 mM Tris-HCl pH 8.1) twice and resuspended in 20 ~l of
the same buffer. The standard curve was obtained by a 1:10 dilution series of
the
bacterial DNA (Streptoccocus pneumonia stock solution ; c = 106 cp/gl) in 50
mM
Tris-HCl buffer pH 8.1.
Afterwards, 20 ql of streptoccocus pneumonia DNA dilution were added to each
tube. During incubation at room temperature the tubes were mixed for 60 min.
The
supernatants were analyzed by PCR on a Light Cycler 1.2. The quantification
reactions were run on the Light Cycler Version 1.2 (Cat.No. 2011468, Roche
Diagnostics GmbH, Germany; Software Version LC 3.5) using the Light Cycler
Sepsis Kit (Id.nr.04493613, Roche Diagnostics GmbH, Germany) with the
following cycling conditions: denaturation at 95 °C for 10 min, then 45
cycles
comprising of a profile of 60 °C for 25 s with fluorescence
measurement, 72 °C for
40 s and 95 °C for 20 s. Following cycling a melt analysis was done
immediately
by rapid cooling to 40 °C and then increasing the temperature to 95
°C with a ramp
rate of 0,1 °C/min using continous fluorescence measurement.
CA 02512599 2005-08-17
-56-
After removal of the supernatant, each tube (containing Bi-PEG-Distamycin/SA-
magnetic bead complex) was washed twice with PCR-water (from Sepsis Kit).
Then 40 ~l of 10 mM NaOH were added and the tubes were incubated for 60 min
at room temperature on a mixer (Thermomixer Comfort, Cat.no. 5350000.013,
Eppendorf, Germany). The supernatants containing the DNA eluted from the beads
were analyzed by PCR.
The amplification curves of this experiment are summarized in Figure 8,
demonstrating the decontamination efficiency towards bacterial DNA. After
decontamination, the supernatant contained 17 cp/~1, whereas after subsequent
elution of bound molecules from the beads the supernatant contained as much as
340 cp/pl.
Table:
Results of Example 9
Supernatant
[cp/lOpl]
Starting concentration of DNA solution372
after decontamination (aver. out of 17
3 det.)
after NaOH (lOmM) treatment (aver. 340
out of 3
det.)
Example 10:
In this experiment the decontamination of bacterial DNA (streptoccocus
pneumonia DNA from LC Sepsis Kit, Id.nr.04493613, Roche Diagnostics GmbH,
Germany) in a Bi-PEG-Distamycin/SA-coated tube was performed and
subsequently, the bound DNA was eluted from the tubes.
A solution of streptoccocus pneumonia DNA (Streptoccocus pneumonia stock
solution; c = 106 cp/~1) in buffer (TBE buffer: 50 mM Tris-HCl pH 8.1 ) was
CA 02512599 2005-08-17
-57-
prepared (ratio 1:1 (~/,,)). The standard curve was obtained by a 1:10
dilution series
of the bacterial DNA in 50 mM Tris-HCl buffer pH 8.1.
40 ~1 of the prepared solution were added to each tube (Bi-PEG-Distamycin/SA-
coated tube complex from example 4) and incubated at room temperature for 60
min on a mixer (Thermomixer Comfort, Cat.no. 5350000.013, Eppendorf,
Germany). The supernatants were analyzed by PCR on a Light Cycler 1.2 (Cat.No.
2011468, Roche Diagnostics GmbH, Germany; Software Version LC 3.5) using the
Light Cycler Sepsis Kit, whereas the quantification reactions were performed
with
the following cycling conditions: denaturation at 95 °C for 10 min,
then 45 cycles
comprising of a profile of 60 °C for 25 s with fluorescence
measurement, 72 °C for
40 s and 95 °C for 20 s. Following the cycling a melt analysis was
performed
immediately by rapid cooling to 40 °C and then increasing the
temperature to 95 °C
with a ramp rate of 0,1 °C/min using continous fluorescence
measurement.
After removal of the supernatant, each tube was washed twice with PCR-water
(from Sepsis Kit). Then 40 ~l of 10 mM NaOH were added and the tubes were
incubated for 60 min at room temperature on a mixer. The supernatants
containing
the DNA eluted from the tubes were analyzed by PCR accordingly.
The amplification curves of this experiment are summarized in Figure 9,
demonstrating the decontamination efficiency of the tubes towards bacterial
DNA.
After decontamination, the supernatant contained no detectable molecules at
all,
whereas after subsequent elution of bound molecules from the tubes the
supernatant contained as much as 38 cp/pl.
CA 02512599 2005-08-17
-58
Table:
Results of Example 10
Supernatant
[cp/lOpl]
Starting concentration of DNA solution 510
after decontamination (aver. out of 0
3 det.)
after NaOH (lOmM) treatment (aver. out 38
of 3 det.)
Example 11:
In this experiment the decontamination from bacteria (Staphylococcus
epidermidis)
with several bacteria binder/SA-magnetic bead complexes was performed.
For each experiment 50 pl (0,5 mg) of bacteria binder/SA-magnetic bead complex
(see example 2 for the immobilization of biotinylated binder on Streptavidin
coated
Dynabeads~) were transferred in a sterile and siliconized tube, washed with
250 pl
buffer (10 mM Tris-HCl pH 8.0) about four times and the supernatant was
removed
completely. Afterwards, 40 pl of a Staphylococcus epidermidis bacteria
dilution
were added to each tube. During incubation at room temperature the tubes were
mixed for 60 min. The supernatants were analyzed by PCR on a Light Cycler 1.2
(Roche Diagnostics GmbH, Germany; Software Version LC 3.5). The standard
curve was obtained by a 1:10 dilution series of the bacterial stock solution
(Staphyloccocus epidermidis stock solution ; c = 9103 DNA equivalents
copies/10
pl) in 10 mM Tris-HCl buffer pH 8Ø
The quantification reactions were run on the Light Cycler Version 1.2 using
the
Light Cycler Staphylococcus Kit Mg'~de (Roche Cat.No. 3 376 419, Roche
Diagnostics GmbH, Germany) with the following conditions: denaturation at 95
°C
for 10 min, then 45 amplification cycles each with the following profil: 50
°C for
15 s with a fluorescence measurement, 72 °C for 10 s and 95 °C
for 10 s. After the
amplification reaction an immediate melt analysis was performed by rapid
cooling
CA 02512599 2005-08-17
-59-
to 40 °C and then increasing the temperature to 95 °C with a
ramp rate of 0,1
°C/min using continous fluorescence measurements.
The results of this experiment are summarized in Figure 10. In the experiments
about 3,4M03 and 4,SM04 copies of bacteria were removed. As a conclusion,
intact
bacteria can be depleted with decontamination efficiencies up to the range of
103,
whereas minor groove binders and positively charged peptides worked best, and
negatively charged peptides did not function at all.
CA 02512599 2005-08-17
-60-
List of References
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Abravaya, K. et al, Nucleic Acid Ampl. Technol. Chapter 9 (1997) 125-133
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Barany, F., Proc. Natl. Acad. Sci. USA 88 (1991) 189-193
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Di Pietro, S.M. et al., Biochemistry 42 (2003) 6218-6227
Dore, K. et al., JACS 126 (2004) 4240-4244
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Hilali, F. et al, Mol. Biotechnol. 7 (1997) 207-216
Hoheisel, J. D., TIBTECH 15 (1997) 465-469
IUPAC-IBU Commission on Biochemical Nomenclature, Europ. J. Biochem. 138
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Kessler (Ed.): Nonradioactive labeling and detection of biomolecules, 1992,
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Klaschik, S. et al, Mol. Biotechnol. 22 (2002) 231-242
Koepsel, R.R. and Russell, A.J., Biomacromolecules 4 (2003) 850-855
Kwoh, D.Y. et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177
Li, M. et al Bioorg. Med. Chem. Letters 12 (2003) 4351-4354
CA 02512599 2005-08-17
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Mantero, G. et al, Clin. Chem. 37 (1991) 422-429
Pauluhn, J. et al., Ber. Bunsenges. Phys. Chem. 82 (1978) 1265-1278
Prince, A.M. and Andrus, L., Biotechniques 12 (1992) 358-360
Record, M.T. et al., J. Mol. Biol. 107 (1976) 145-158
Sambrook, J. et al.: Molecular Cloning: A Laboratory Manual (2nd ed., Cold
Spring Harbour Laboratory Press, Cold Spring Harbour, NY
Stewart, K.D. et al., J. Phys. Org. Chem. 5 (1992) 461-466
Stryer, Biochemistry, 4th edition (1995) W H Freeman & Co, NV, USA
Tijssen, P.: Practice and theory of enzyme immunoassays (1985, Elsevier,
Amsterdam, Netherlands
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Vogelstein, B., and Gillespie, D., Proc. Natl. Acad. Sci. USA 76 (1979) 615-
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Whelen, A.C. and Persing, D.H., Annu. Rev. Microbiol. 50 (1996) 349-373
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WO 02/06117
WO 02/48164
WO 03/33698
WO 04/01418
WO 89/10977
WO 89/11548
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WO 90/15070
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WO 2002/82078
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Zimmer, G. et al., Prog. Biophys. Moles. Biol. 47 (1986) 37-112
CA 02512599 2005-08-17
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: F. HOFFMANN-LA ROCHE AG
ROCHE DIAGNOSTICS GmbH
(ii) TITLE OF INVENTION: A METHOD TO REDUCE FALSE POSITIVE
RESULTS
(iii) NUMBER OF SEQUENCES: 16
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: OGILVY RENAULT
(B) STREET: 1981 McGill College Avenue - Suite 1600
(C) CITY: MONTREAL
(D) STATE: QC
(E) COUNTRY: Canada
(F) ZIP: H3A 2Y3
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: ep 04019636
(B) FILING DATE: 2004-08-19
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Christian Cawthorn
(B) REGISTRATION NUMBER: 11,005
(C) REFERENCE/DOCKET NUMBER: 3580-975CA CC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 514-847-4256
(B) TELEFAX: 514-288-8389
(C) TELEX:
(2) INFORMATION FOR SEQ ID N0: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: nucleic acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: Artifical sequence for example
CA 02512599 2005-08-17
-64-
(ix) FEATURE:
(A) NAME/KEY: misc-structure
(B) LOCATION: (25)...(0)
(D) OTHER INFORMATION: 3'-phosphate
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
cccatcccca aaaacacaaa ccaca 25
(2) INFORMATION FOR SEQ ID N0: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: Minor groove Binder
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (2)...(0)
(D) OTHER INFORMATION: Nva
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (3)...(0)
(D) OTHER INFORMATION: Orn
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (4)...(0)
(D) OTHER INFORMATION: Nva
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (5)...(7)
(D) OTHER INFORMATION: Xaa = amino-1-methylpyrrole-2-carboxylic
acid
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (8)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 2:
Glu Xaa Xaa Xaa Xaa Xaa Xaa Arg
1 5
CA 02512599 2005-08-17
(2) INFORMATION FOR SEQ ID N0: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: Minor Groove Binder
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (2)...(0)
(D) OTHER INFORMATION: Nva
(1x) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (3)...(0)
(D) OTHER INFORMATION: Orn
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (4)...(0)
(D) OTHER INFORMATION: Nva
(ix) FEATURE:
(A) NAME/KEY: VARIANT
(B) LOCATION: (5)...(7)
(D) OTHER INFORMATION: amino-1-methyl-1H-imidazole-2-carboxylic
acid
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (8)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 3:
Glu Xaa Xaa Xaa Xaa Xaa Xaa Arg
1 5
(2) INFORMATION FOR SEQ ID N0: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
CA 02512599 2005-08-17
-66-
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: Minor Groove Binder
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: VARIANT
(B) LOCATION: (2)...(4)
(D) OTHER INFORMATION: amino-1-methyl-1H-imidazole-2-carboxylic
acid
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (5)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 4:
Glu Xaa Xaa Xaa Arg
1 5
(2) INFORMATION FOR SEQ ID N0: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 1
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (17)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 5:
Glu Gln Gly Arg Val Glu Val Leu Tyr Arg Gly Ser Trp Gly Thr Val
1 5 10 15
Ala
(2) INFORMATION FOR SEQ ID N0: 6:
CA 02512599 2005-08-17
_67_
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 2
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (26)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 6:
Glu Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
(2) INFORMATION FOR SEQ ID N0: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 3
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Bi-ZU attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (40)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 7:
Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
1 5 10 15
Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
20 25 30
Gly Pro Arg Leu Gly Val Arg Ala
CA 02512599 2005-08-17
-68-
35 40
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 4
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Bi-XUZU attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (45)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Lys Lys Asn Lys Arg Asn Thr Asn Arg Arg Pro Gln Asp Val Lys Phe
1 5 10 15
Pro Gly Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg
20 25 30
Gly Pro Arg Leu Gly Val Arg Ala Thr Arg Lys Thr Ser
35 40 45
(2) INFORMATION FOR SEQ ID N0: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 5
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Bi-XUZU attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (36)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
CA 02512599 2005-08-17
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Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp Leu
1 5 10 15
Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro Arg
20 25 30
Arg Arg Ser Arg
(2) INFORMATION FOR SEQ ID N0: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 6
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Bi-XUUUU attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (15)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr
1 5 10 15
(2) INFORMATION FOR SEQ ID N0: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 7
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Bi-BXUZU attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (25)...(0)
CA 02512599 2005-08-17
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 11:
Gly Gly Gly Gly Asp Asp Leu Gly Ala Asn Asp Glu Leu Ile Ser Phe
1 5 10 15
Lys Asp Glu Gly Glu Gln Glu Glu Lys
20 25
(2) INFORMATION FOR SEQ ID N0: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 8
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (11)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 12:
Glu Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 9
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (31)...(0)
CA 02512599 2005-08-17
-71 -
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 13:
Glu Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg
1 5 10 15
Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly Arg Gly
20 25 30
(2) INFORMATION FOR SEQ ID N0: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 10
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (11)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 14:
Glu Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly
1 5 10
(2) INFORMATION FOR SEQ ID N0: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 11
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (31)...(0)
CA 02512599 2005-08-17
-72-
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 15:
Glu Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys
1 5 10 15
Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly Lys Gly
20 25 30
(2) INFORMATION FOR SEQ ID N0: 16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21
(B) TYPE: amino acid
(C) STRANDEDNESS: unknown
(D) TOPOLOGY: unknown
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Artificial Sequence
(ix) FEATURE:
(D) OTHER INFORMATION: peptide 12
(ix) FEATURE:
(A) NAME/KEY: MOD RES
(B) LOCATION: (1)...(0)
(D) OTHER INFORMATION: Biotinyl-PEG attached
(ix) FEATURE:
(A) NAME/KEY: AMIDATION
(B) LOCATION: (21)...(0)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:
Glu Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10 15
Arg Arg Arg Arg Arg
