Note: Descriptions are shown in the official language in which they were submitted.
.I~m bili~ati~ o~ n~L io aai4~
The invention concerns a method for detecting nucleic
acids, the use of special nucleic acids to immohilize
nucleic acid~ and a system ~or tha detection of nucleic
acids.
The immobili~ation of nucleic a~ids is important in many
areas for example when separating nucleic acids from
other m~tarials, when i~olating nucleic acids with a
special nucleotide sequence from a nucleic acid mixture
and in additiDn for the detaction Gf nucleic acids
~nucleic acid diagnostics). In all these areas
utilization is made of the special affinities o~ nucleic
acids to th~e nucleic acids which have ~ nucleotide
~equence which i8 essentially complementary to them and
can hybridize under certain conditions with the desired
nucleic acids. The complementary nucleic acids which are
used for this which are denoted capture probe in the
following are usllally bound to a solid phase e.g. a
membrane, a gel, a bead or a tube. Recently it has been
established that the immobilization of nucleic acids to
solid phases via covalently bound capture probes ha~
drawbacks in several respects e.g. the rate of
immObiliZatlOn i8 reduced. Therefore it was suggested
that the capture probe be specifically bound to the
solid phase via affinity bindingO Such a binding for
example via antibodies/haptens or biotin/(strept-)
avidin) enables a hybridi~ation of the nucleic acid with
the capture probe in a liquid ancl subsequent
- 2 ~ ~ ~73~Y
immobilization of the hybrid formed. such a procedure is
described for example in EP-A-O 139 489.
The ob;ect of the pre~ent inventlon was to increase the
effectiveness o~ the immobilization in order, in
particular, to obtain more sensitive tests for nucleic
acids.
The inv~ntion conaern~ a method for the detection of an
analyte hucleic acid which comprises the following
steps: `
- Hybridization of a detectahly labelled nucleic
acid or of a detectably labelled nucleic acid
hybrid with an immobilizable nucleic acid probe;
- i~mobilization of the hybrid formed and
- detection of the amount of immobilized hybrid via
the amount of label,
wherein the i~nobilizable nucleic acid prob~
contains two or more nucleotldes modified by
immobilizable groups which are not directly
adjacent in the nucleotide sequence.
The invention als~ concerns the use of the nucleic acids
mentioned in the method as well as a system for
detecting nucleic acids.
An analyte nucleic acid within the sense of the
invention is a nucleic acid which is to be detected~ It
is usually present in a sample which also contains
further components, for example a tissue or a liquid. In
- 3 _ t~r~
particular th~re are ~lso additlonal nucleic acids in
the sampl~ which are not intended to be detected. The
nucleic acid can be any type of nucleic acid r for
example DN~, RN~ or ~ragments thereoP. I~ the analyte
nucleic acid is originally present in the sample in a
double-stranded form it is pre~erably converted into the
single~stranded form. I~ the analyte nucleic acid is
pre~ent bound to a solid phase it is preferably brought
into ~olution.
A detectably labelled nucleic acid is understood as a
nucleic acid whose presence or amount can be determined
with the aid of a detection system. For this purpose a
detectably labelled nucleic acid has modifications as
compared to a normal nu~leotide seguenoes. Suitable
modifications are familiar to one skilled in the art. An
example o~ a modification are chemical groups which are
not normally present in nucleic acids such as enzymes,
coloured or ~luorescent molecules and ligands,
pre~erably residues capable of binding in~unological.ly.
Examples o~ the latter are in particular haptens such as
digoxigenin and also vitamins such as biotin. In the
case of enzymes the detection is usually carried out via
a colour-forming reaction catalyzed by the enzyme.
Coloured or fluorescent molecules can be detected
directly by photometric or fluorometric means. Ligands
are ~sually contacted with a corresponding enzyme-
labelled or dye-labelled receptor which has affinity to
the ligands and are d~tected by this means.
A detectably labelled nucleic acid hybricl i5 understood
as a partially double-stranded hybrid of at least two
mlcleic acids. In this case either both nucleic acids or
only one of them may be detectably labelled as described
above. It is important that the nucleic acid hybrid has
~ ~ r~
a sinyle-stranded region. In a so-called sandwich test
the hybrid preferably consists of the (non l~belled)
analyte nucleic acid and a detectably labelled detector
probe.
An immobilizable nucleic acid probe is understood as a
nucleic acid probe which is modified by a residue which
is not presenk in the usual nucleic acidsu Su~h residues
are residues which have an affinity to another material.
Examples are partners of a biospecific reaction between
a ligand and a receptor for example an immunological
reaction, a reaction between sugar and lectin or between
vitamin and binding protein. Particularly preferred
residues are haptens or biotin. In the method accorcling
to the present invention the residue on the
immobilizable nucleic acid probe is di~fererlt from the
det~ctable residue of the detectably labelled nucleic
acid.
The steps of the method of detection describPd in the
following are known as indlvidual steps or can be
carried out analogou~ to known m~th~ds. Reference ~g in
particular made to Molecular Cloning, Editor Sambrook
et al., CSH 1989. These also include the known methods
for the production of labelled nucleoside triphosphates,
the chemical synthesis of modified ~nd unmodified
oligonucleotides, the choice of hybridization conditions
by which means a specificity can be achieved which i.s
dependent on the extent of homology between the nucleic
acids to be hybridized, their GC content and their
length, as well as the formation of nucleic acids from
nucleoside triphosphates with the aid of polymerases and
if desirsd, using so-called primers. An essential
feature of the present invention is the use of a special
capture probe.
2 ~ 3 ~43
In a first step of the method according to the present
invention a detectably lahelled nucleic acid or a
detectably labelled nucleic acid hybrid is produced
using the analyte nucleic acid. This can for example be
carried out by detectably labelling the analyte nucleic
acid itself. This can be achieved by the enzymatic
incorporation of labelled nucleoside triphosphates or by
elongating the analyte nucleia acid with labelled
nucleoside triphosphates (tailing). A furthex
possibility is to amplify a region of the analyte
nucleic acid whila incorporating detectably labelled
mononucleoside triphosphates or detectably labelled
oligonucleotides. Many methods are known for this from
the state of the art.
One possibility is to carry out an amplification ~y
m~an~ of the pol~mera3e chain reaction aacording to
EP-A-0 200 362 using the analyte nucleic acid as the
template nucleic acid. In this case two primers are used
one of which is complementary to a region of the analyte
nucleic acid and the other is complementary ko a part of
the oppo~ite strand of the analyte nucleic acid to form
a multitude of copies of the strand and opposite strand
of the analyte nucleic acid by elongation of the pri.mer
on the template. In order to produce the detectably
lab~lled nucleicc acid, at least one detectably labelled
mononucleoside triphosphate is used in the elongation of
the primer or a labelled primer is used.
A further possibility ar.ises from the use of the ligase
chain reaction according to W0 90/06376 if at least one
of the oligonucleotides is detectably labelled.
A further possibility is described in EP-A-0 329 822. In
this process a DNA strand is formed which is
- 6 - ~ Q 7 ~
complementary to substantial parts of the analyte
nucleic acid and a primer containing a promoter is
incorporated. After degradation of the analyte nucleic
acid, a transcribable double strand is ~ormed which can
be used to form a multitude of detectably labelled
transcripts while incorporating labelled mononucleoside
triphosphates.
The method according to the present invention has proven
to be particularly efficient when in the form of a
sandwich nucleic acid assay e.g~ according to
EP-A-O 192 168. The method according to the present
invention differs from ~he ~nown sandwich assays in that
a particular type of capture probe is used.
In a ~urther step the detectably labelled nucleic acid
or the detectably labelled nucleic acid hybrid is
brought into contact with an immobllizable nucleic acid
probe under hybridization conditions.
The immobilizable nucleic acid probe according to the
present invention contains two or more nucleotides
modified by immobili~able groups which are not directly
adjacent in the nucleotide sequence. Apart from the
number of ligands (reporter groups) the distance between
the ligands is crucial and should be preferably 10 or
more nucleotides in order to observe this efEect. For
example oligodeoxyribonucleotide probes which contained
5 biotin residues (biotin corresponds to the ligand)
coupled in direct succession and were used as capture
probes to bind an analyte to a streptavidin matrix did
not result in an increase in the sensitivity in the
total assay compared to a reference probe which was only
linked to one biotin residue ~see example 2). The
"capture probe" can ~e an oligonucleotide or a
7 ~ ;3~j~S
polynucleotide (DNA ~r R~) which can hybridlze in a
suitable manner with the analyte nucleic acid to be
detected. It can be single-stranded (e.g.
oligodeoxyribonucleotide, oligoribonucleotide) or
double-stranded ~e.g. plasmid, fragment). In the latter
case the capture probe has to be denatured be~ore
hybridization with the analyte. A particularly preferred
embodiment utilizes nucleic acid probes in which the
terminal nucleotide~ of the nucleic acid probe are
modified in each case.
An oligodeoxynuc}eotide is preferred which has a length
between ll and 40 nucleotides whereby the label is
appropriately attached to the 3'- and 5'- end in ordler
to meet the requirement for distance as described above.
The immobilizable residues can in principle be bound to
the base part or to the ~ugar part or to the phosphate
part of the nucleotides. Such immobilizable nucleic acid
probes can be produced acaording to known method~ or
analogous to known methods. The attachment of a ligand
to nucleotides with modi~ied bases is described for
example in Nucleic Acids Res. 15 ~12), p. 4857 to 4876
~1987); Nucleic Acids Res. 16 (9), p. 4077 to 4095
(1988); Nucleic Acids Res. 17, p. 4643 to 7650 (1989) or
can be carried out by introducing a modified nucleotide
which already contains the ligand (e.g. Nucleic Acids
Res. 18 (15), p. 4355 to 4360 (1990). A ~urther suitable
chemical method for incorporating several primary amino
groups in synthetic oligonucleotides which can then be
used for binding to an immobilizable residue is
described in Nucleic Acids Res. 17 (18), p. 7179 to 7194
~1989). The attachment o~ the ligand to a nucleic acid
can also be carried out by a bisulfide-catalyzed
transamination (Nucleic Acids Res. 12 (2), p. 989 to
- 8 - ~f~
1002 (1984)). The attachment of several immobilizable
xesidues can al60 be carried out by enz~matic methods
e.g. by tailing using a nucleoside triphosphate
containing the appropriate residue tDNA 5 (4), p. 333 to
337 (1986~) or by random priming (Analyt. Biochem~ 132,
p. 6 lg~3)~
Chemically synthesized oligonucleotides are particularly
preferred within the scope of the present invention
since they hava the advantage that the distance between
two modified nucleotides can be exactly determined
bef orehand.
The use of multiply labelled nucleic acids as detection
probe~ was known previously. For example in EP-A-0 330
221 an oligonucleotide or a polynucleotide is labelled
with biotin and subse~uently biotin is detected by means
o~ a streptavidin-enzyme complex. The separation of two
~iotin-dUMP residues in the nucleotide sequence by
simultaneous incorporation of dTTP is de~cribed as being
more of a disadvantage. In Nucleic Acids Res. 1~ (15),
p~ 4358 it is shown that by attaching several
immobilizable residues to nucleic acids their binding to
solid phases can be improved.
In Nucleic Acids Res. 18, p. 4345 to 4354 (1990)
multiply labelled nucleic acids are also used as a
detection probe. The production of multiply labelled
nucleic acids in which the label is attached to the
phosphate residue .is described in W0 90/08838.
If the sample contains analyte nucleic acid, a nucleic
acid hybrid forms from the detectably labelled nucleic
acid or the detectably labelled nucleic acid hybrid and
- 9 - l~v ~
the immobilizable nucleic acid probeO In the m~thod
according to the present invention this is bound via the
immobilizable residue to a solid phase. The surface of
the solid phase contains groups which have an affinity
to the immobilizable residue o~ the nucleic acid probe,
for example a receptor. If the immobilizable group is a
hapten, the solid phase preferably contclins an antibody
against this hapten. In the case of biotin the solid
pha~e contains a biotin binding protein such as avidin
or streptavidin.
The amount of immobilizable hybrid is a measure for the
amount or the presence of the analyt~ nucleic acid. This
amount can be detscted via the amount of label which is
immobilized on th~ solid phase. This is preferably
carried out in a known manner and depends on the type of
label used. Before carrying out the detection reaction,
the solid phase is preferably removed from the liquid
phase. This at the same time results in the removal of
the starting material ~or the production o~ the
detectably labelled nucleic acid or for the detectably
labelled nucleic acid hybrid or of an excess detec~ably
labelled nualeic acid probe in the case of a sandwich
hybridization togather with the li~uid. The detection of
a nucleic ac~d by the method according to the present
invention, especially if the test is to be carried out
quantitatively, encompasses comparing the measurement
signal which was obtained with the sample of unknown
analyte nucleic acid content with the measurement signal
or measurement signals which were obtained with one or
several samples with known analyte nucleic acid content.
The use o~ a calibration curve is preferred which can
also be provided in the form of entered data.
1 0 ;~ ?~ (.J ~
The invention in addition concerns the use of a nucle.ic
acid which contains two or more nucleotides modified ~y
immobilizable groups which are not directly adjacent in
the nucleotide sequence ~or immobilizing nucleic acids.
This aspec~ is based on the ~act that the nucleic acid
probes according to the present invention guarantee a
~urprisingly improved immobilization~ T~he method can for
example be used in the affinity separation of nucleic
acids.
The invention also relates to a system for the detection
of nucleic acids which contains one or several nucleic
acids containing two or more nucleotides modified by
immobilizable groups which are not directly adjacent in
their respective nucleotide sequence and at least one
solid phase which has a specific af f inity to the
lmmobllizable groups of tha nucleic acid. Be~ore
starting a detection method it is pr~ferable that the
nucleic acids and the sclid phase are present separated
~rom one another in the system. ~n addition the system
can also contain further components which are necessary
or helpful for the detection of nucleic acids~ These in
particular include pH buffers and reagents for detecting
the label.
Fig. 1 shows a diagram of the binding of an analyte
nucleic acid A via a capture probe F according to the
present invention to a solid phase coated with
streptavidin S in a sandwich test. The detectably
(represented by a rhombus) labelled detector probe is
denoted D.
Fig. 2 shows the binding to the wall of a labelled
analyte nucleic acid A' via a capture probe according to
the present invention to a solid phase coated with
streptavidin S.
Fig. 3 illustrates the increase of the san~itivity of a
sandwich nucleic acid test in which thQ immobilizable
groups are located on adjacent nucleotide6 by separating
the groups according to the present invention. It is
clear that the increa~e in sensitivity when using only
two biotin residue~ which are each at t:he ends of the
oligonucleotide is unexpectedly even more than when 10
biotin residues are used at one end ~see also
example 2).
Fig. 4 shows the increase in sensitivity in the sandwich
test when using tws biotin residues compared to the use
of one biotin resldue with an oligonucleotide length of
30 nucleotides.
Fig. 5 shows the comparison of sensitivity of Figure 4,
but with oligonucleotides having a length o~ 20 nt.
Fig. 6 shows the compari~on of sen~itivity for a method
in which an analyte nucleic acid wa5 amplified and at
the same time labelled by PCR and in which nucleic acid
probes with a length of 15 nucleotides and having one or
two biotin residues were used as the capture probe.
12 - ~ Q 7 3 9 ~ ~
The curves shown in figures 4 to 6 can also be used as
calibration curves for determining an unknown a~ount of
an analyte nucleic acid.
Fig. 7 shows the formula for the aminomodifier II.
The invention is elucidated in more detail by the
following example~.
- 13 ~ ,~J,~
PrQduction of oli~onucleot~des
A: Explanatory notes
All oligonu~leotides were produced with the aid of
a DNA synthesizer 8700 from the Biosearch Company
using the "phosphoramidite method" published by
Caruther~ et alO Methods Enzymol. 154, 287, 1987.
The ~-cyanoethyl~nu~leoside phosphoramidites used
were obtained ~rom Roth ~Karlsruhe, GFR), D
biotino~l-aminocaproic acid-N-hydroxysuccinimide
eq~ter was from Boehringer Mannheim ~Mannheim, GFR),
aminomodifier 2 (AM II) from Beckmann Instrumen~s
GmbH ~Fullerton, CA, USA). All other reagents and
solvents were used in the best ~uality available.
The purification of the biotlnylated
oligonucleotide~ was carried out by HPLC (Spectra
Physics, Darmstadt, GFR) using a Mono Q HR 5/5 ion-
exchange column from Pharmacia (Freiburg, GFR) or a
Cl~ column (LiChrosorb RP 18-5, CS-Chromatographie
Service, Langerwehe, GFR). Spectra/Por lO00
membranes (Roth, Karlsruhe, GFR) were used for the
dialysis.
B) Synthesis ~ad ~ri~ication of t e biotlnvlated
oli~onucleotides
The biotinylated oligonucleotides were all prepared
in the Biosearch synthesizer on a 1 ~mol scale
using the trityl-off as well as cleave-off
programme. Since there was no carrier material
available with aminomodifier 2, a th~midine carrier
was used and subsequently aminomodifier 2 (see Fig.
7) was condensed to this according to the standard
coupling program ~0.1 M in acetonitrile).
A~er the amlnomodifier, a further th~midine was
coupled to the 5' end in order to protect the
primary hydroxyl group of the aminomodi.~ier ~rom
attack during the ammonia treatment and the
glyceryl residue from being again partially cleaved
o~f. A~ter completion of the .~ynthesis and cleavage
of the protecting groups with concentrated NH3
solution at 55C/5h, all solvents were removed in a
vacuum, the residue was taken up in water and the
product was purified by ion-exchange chromatography
(Mono ~ HR 5~5; A~ 0.25 mM Tris/HCl, 0.3 M NaCl; ~:
0.25 Tris/HCl, 1 M MaCl; in 60 min from 0 to 100
B, flow 1 ml/min). Subsequently the
oligodeoxynucleotide was desalted by dialysis
(Spectra/Por 1000, Roth). Afterwards the
oligonuclotides were biotinylated by taking up 5-10
O.D.260nm of the oligodeoxynucleotides in 0.5 ml
0.05 M K2HP04/KH2P0~ buffer and adding a solution
of 5 mg D-biotinoyl-aminocapxoic acid-N-
hydroxysuccinimide ester in 0.5 ml DMF. The
reaction mixture was incubated overnight at 37C,
subsequently the solvent was removed in a vacuum,
the residue was taken up in redistilled water and
- 15 ~ a~
excess biotin was removed by filtration. The
further purification was carried out by means of
reversed phase ~PLC with a gradient o~ ~: 0.1 M
triathylammonium acetate p~I 7; 5 % acetonitrile, B:
O.1 M triethylammonium acetate pH 7; 40 %
acetonitrile. The gradient was run ~rom 20 % B to
80 % B within 40 min at a flow rate of 2 ml/min.
The dif~erence in the ret~ntion time between the
twice biotinylated oligonucleotides and the
corresponding aminomodi~ier-modi~ied starting
oligonucleotides was between 2 and 4 minutes
depending on the length of the sequence. The
biotinylated oligodeoxynucleotid2s could bs
obtained in 1.5 to 3.5 O~D.2~0 after renewed
dialysis.
~etaction o~ ~v analyte D~A in a sandwich form taklnq
into account_the site o~. modification
The test is carried out in a sandwich with binding of
the analyte DNA to a solid phase. A partial sequence of
the hepatitis B virus DNA which is present clon~d in a
plasmid is used as the analyte DNA. The analyte DNA is
bound to a streptavidin solid phase by means of a
biotin-labelled oligonucleotide whose sequence is
complementary to a region of the HBV DNA. The detection
of the bound analyte DNA is then carried out by means o~
a diyoxigenin-labelled oligonucleotide which in turn is
complementary to another region of the analyte DNA. This
can be recognized by anti-digoxigenin antibodies which
are conjugated with horseradish peroxidase and the
hybrid is subsequently detected by an enzyme-catalyzed
colour-forming reaction.
- 16 -
A dilution series of the plasmid in a range of
4~4 ~g/ml, 2.2 ~g/ml, 1.1 ~g/ml, 0.55 ~g/ml in TE buffer
~10 mM Tris-HCl, 1 mM EDTA, pH 7.5~ is prepared. In
order to denature khe double-strande~ DNA, 10 ~1 ~ M
NaOH 16 added to 90 ~1 of the plasmid solution and
i~cubated for 10 min at room temperatur~. 20 ~1 of the
denaturation prepaxation i~ subse~uently pipetted into a
well o~ a microtitre plate ~oated with streptavidin and
immediately neutralized by addition of 180 ~1
hybridization solution ~50 mM Na phosphate buffer,
0.75 M NaCl, 0.075 ~ Na citrate, 0.05 % bovine ~erum
albumin, pH 5.4). This results in the following
concentration~ of the plasmid in the testo 5~, 100, ~00,
400 ng/ml. The hy~ridi~ation solution in addition
contains 200 ng/ml of a digoxigenin-labelled
oligonucleotide (labelled once with digoxigenin at the
5' end, position 287c-24~c in the HBV genome) and of a
biot~n-labelled oligon~cleotide (position 2456c-2417c in
the HBV genome). The te~t is carried out with
oligonucleotldes havi~g different degrees of bio~in
labelling:
HBV-Oli 7-4 (1 biotin, 40mer), SEQ ID NO 1:
5'-T(bio-AMII)-CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-
3'
HBV-Oli 7-5 ~5 biotin, 40mer), SEQ ID NO 2:
5'-T-(bio-AMII)5-
CATTGAGATTCCCGAGATTCAGATCTTCTGCGACGCGGCG-3'
HBV-Oli 7-6 (10 biotin, 40 mer), SEQ ID NO 3:
5'T-(bio-AMII)10-
CATTGAGATTCCCGAGATTGAGATCTTCTGCGACGCGGCG-3'
17 ~ /7 ;~ ~3 f~ J
HBV-Oli 7-7 (2 biotin, 40mer~, SEQ ID N0 4~
5'T-(bio MII)-cATTGAGATrrcccGAGATTcAGATcTTcTGcGAcGcGGcG
(AMII-bio)-T-3'
The hybridization preparation i~ incubated for 3 h at
37C in the microtitre plate while shaking. A~t~r
aspirating the solution it is washed 2 x 10 min with
0.3 M NaCl, 0.03 M Na citrate, 002 % Na dodecylsulate
at 370c and ~ubsequently once ~or a short time at room
temperature with 0.9 % NaCl in order to remove non-bound
reaction partners from the test. 20 mU/ml o~ an anti-
digoxigenin antibody-horseradish peroxidase conjugate in
103 mM Tris-HCl (p~ 7.5), o.s % NaCl, 1 ~ bovin~ serum
albumin is added and incubated ~or 30 min at 37C while
shaking. Non-bound con~uyate is removed by briefly
washing three times with 0.9 % NaCl at room temperat.ure.
The deteotion r~action is started by addition of thQ
substrate solution ABTS~ ~.9 mM, 2,2'azino.di~[3-
ethylbenzthiazoline sulfonic acid (6)3-dia~m~nium salt).
The incubation is carried out ~or 30 min at 37C while
shaklng. The absorbance i8 subsequently measur~d at
405 nm by means of an E~ISA reader.
The results ar~ ~hown in Fig. 3.
~amp~ 3
Detection of HBV-analYte DNA in a sandwich format takinq
into account the chain_len~th and the site oE
modification
A d.ilution series of the plasmid which contains HBV-
specific sequences is prepared in a range from 62.5 to
250 ng/ml in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5. 10 ~1
5 M NaOH is added to 90 ~1 of the plasmid solution and
incuhated for 10 min at room temperature~ 20 ~1 of this
denaturation preparation is pipetted into a well of a
microtitre plate coated with streptavidin and 180 ~1
hybridization solution (50 mM phosphate buffer, 0.75 M
NaGl, 0.075 M Na citrate, 0.05 ~ bovine serum albumin
(~SA), pH 5.4) is added. 200 ng/ml o~ the biotin-
labell~d captux~ oligonucleotide and of th~ digoxigenln-
labelled oligonucleotides ~pos. 405-444 in the HBV
genome, 40mer, labelled twice with digoxigenin at the 3'
and 5' end) are added to the hybridization solution.
Thus the analyte DNA concentration in the test is 6.25,
12.5, 25 and 50 ng/ml.
HBV-Oli 25-3 ~pos. 2327-2356, 30mer, 1 biotin),
SEQ ID NO 5:
5'-T-(bio-AMII)-CACTTCCGGAAACTACTGTTGTTAGACGAC-3'
H~V-Oli 25-4 (pos. 2327-2356, 30mer, 2 biotin),
SEQ ID NO 6:
5'-T-(blo-AMII~-CACTTCCGGAAACTACTGTTGTTAGACGAC-~MII-
bio-~-T-3'
HBV-Oli 33-1 (pos. 2332-2351, 20mer, 1 biotin).
SEQ ID NO 7:
5'-T-(bio-AMII)-CCGG~AACTACTGTTGTTAG-3'
HBV-Oli 33-2 (pos. 2332-2351, 20mer, 2 biotin),
SEQ ID NO 8:
5'-T-(bio-AMII)-CCGGAAACTACTGTTGTrrAG-(AMII-Bio)-T-3'
~he stated positions relate to the HBV genome.
- 19 - ~?~ ~ 3 ~
The hybridi.zation preparation is shaken for 3 h at 370C.
Subsequently it is washed 2 x lo min with 0.3 M NaCl,
0.03 M Na citrate, o.~ % Na dodecylsulfate at 37OC and
then once briefly wi~h o.s % NaCl at room temperature.
Su~sequently 20~ ~l of the anti-digoxigenin antibody-
horseradish peroxidase conjugate (200 mU/ml in 100 mM
Tris-HCl, pH 7.5 9 0 . 9 % NaCl, 1 % BSA) is added and
incubated for 30 min at 370C whil2 shaking. After
washing again three times with O . 9 % NaCl, the substrate
solution ~1.9 mM ABTSR) is pipetted shaken again for 30
min at 370C and th~n the absorbance is measured at 405
nm by mean6 of an ELISA reader.
The result~ ~or the 30 nt oligonucleotid~s are shown in
Fig. 4, those for the ~o nt oligonucleotides are shown
in Fig. 5.
B~mpla
Detection of_~Ç~ Products wit~ nqlY=_and_~ e-
lahelled oli~onucleotides
For the DNA test for the hepatitis B viruses, the viral
DNA is firstly amplified by means of the polymerase
chain reaction (EP~A-0 200 362). During this
amplification digoxigenin is incorporated into the DNA
in the form of Dig~ dUTP (EP-A-32~474). Subsequently
this hapten allows a sensitive detection of the PCR
product in a heterogeneous immunoassay. The binding of
the digoxigenin-labelled DNA to the walls of the
streptavidin-coated tubes is carried out by means of a
biotin-labelled oligonucleotide who~e region is
complementary to a region of the PCR product. The test
is again carried out using an anti-digoxigenin antibody-
~ ~ ~ 3 ~ . ~
~ 20 ~
horseradi~h peroxidase conjugate and a sub~e~uent colourreaction.
HBe-positive human plasma with a virus titre of 1 x 101
hepatitis B viruses per ml is diluted in normal serum so
that the virus content in the dilutions is 1 x 107
viruses/ml. For the lysi~, 10 ~l 0.2 M NaOH is added to
10 ~l of the virus dilution and incubatisd for 1 h at
37C. The lysis preparation i6 neutrali2ed by addition
of 30 ~l neutrali~ation mixture (100 mM KCl, 50 mM Tris-
HCl, pH 6.5, 3 mM MgCl2). 20 ~l of this solution is used
in the subsequent amplification reaction (amplification
conditions: 200 nM of each of the PCR prim~rs, 200 ~M
each of dATP, drTp/ dGTP, 175 ~M dTTP, 25 ~M
digoxigenin~ 2'-dUTP, 2.5 U Thermus aquQticus DNA
polymerase in 50 mM KCl, 10 mM Tris-HCl, pH 8.9, 1.5 mM
MgCl2, 0.01 % gelatin; total volume 100 ~l). The
preparation is covered with a layer of 100 ~l mineral
oil and incubated ~or 30 cycles in a thermo-cycler
(Perkin-Elm~r):
30 sec 92C, 30 sec 50C, 60 sec 70C.
Due to the position of the PCR primers (PCR primer 1:
position 1937-1~60; PCR primar 2: position 2434c-2460c
in the HBV genome) a 500 bp long DNA fragment is
produced which is subsequently detected in a two-
component test system in streptavidin-coated microtitre
plates.
The PCR preparation is diluted in H20 in such a way that
in relation to the original virus concentration of 1 x
107 viruses per ml serum, dilutions ar~ made which
correspond to a range of 1 x 105 to 1 x 104 viruses/ml.
- 21
20 ~1 of the diluted PCR preparatlon is incubated in a
~inal concentration of 0.1 N NaO~ in a ~olume of 50 ~1
for 10 min at room temperature for the denaturation.
20 ~1 of the denaturation solution are pipetted together
with 180 ~1 hybridization solution (1 M NaCl, 0.~ M Na
citrate, 67.5 mM Na phosphate, 0.05 % BSA, p~ 6.7) into
a ~treptavidin-coated well of a microtitre plate. The
biotin-labelled oligonuoleotides are added at a
concentration of 100 ng/ml to the hybridization buffer:
HBV-Oli 34-1 (pos. 2299-2313, 15mer, 1 biotin) r
SEQ ID NO 9:
5'-T-(~MII-bio)-AGACCACCAAATGCc~3'
~BV-Oli 34-2 (pos. ~299-2313, 15mer, 2 biotin)
SEQ ID NO 10~
5' T-(AMII-bio)-AGACCACCAAATGCC-(bio-AMIIH)-T-3'
The hybridization preparation i5 incubated for 3 h at
37C while skaking. Subsequently the ~olution is
aspirated and the well is washed three time~ with 0.9 %
NaCl. 200 ~1 of a solution of 200 mU/ml anti-digoxigenin
antibody-hor~eradlsh peroxidase con~ugate in 100 mM
Tri~-HCl, pH 7.5, 0.9 % NaCl, 1 % BSA is added and
incubated for 30 min at 37C. After washing again with
0.9 % NaCl the substrate reaction is started by addition
of 200 ~1 ABTSR. The photometric measurement is carried
out after 30 min at 405 nm.
The results are shown in E'ig. 6.
~o~uerlce Protocol
SEO ID ~0 1
~ength of sequence: 41 bases
Type of sequence: nucleotide sequence
Type of strand: single strand
Topology: linear
Type of molecule: part of genome DNA with
modifications N
Position- HBV genome 2456c-2417c (Hpbadw-data
bank~
Antisense
Modification N: T on this is bound to
aminomodifier II via 3'-0 biotin
5'-N CAT TGA GAT TCC CGA GA~ TGA GAT CTT CTG CGA CGC GGC
G-3'
SEQ I~ NO_2
Length of sequence: 41 base~
Type o~ sequence: nucleotide sequence
Type of strand: single strand
Topology: linear
Type of molecule: part of genome DNA with
modifications N
Position: HBV genome 2~56c-2417c
Antisense
Modification N: T on this is bound to 5
aminomodifiers II via 3'-0 5 biotin
5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G-3'
- 23 -
2 ~
5EQ ID NO 3
Length of sequence: ~1 bases
Typ~ of saquence: nucleotide se~uence
Type of strand: single strand
Topology: linear
Type of moleculeo part of genome DNA with
modifi~ations N
Position: HBV genome 2456r-2417c
Antise~se
Modification N: T on this is bound to 10
aminomodifiers II via 3'-0 10 biotin
5l-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
G~3'
SE Q ID NO 4
Length of se~uence- 42 base~
Type of sequence: nucleotide sequence
Type of st~and: single strand
Topology: linear
T~pe of molecule: part of genome DNA with
modifications N
Position: HBV genome 2456c-2417c
Antisense
Modification N at T on this is bound to
the 5' end: aminomodi~ier II via 3l_0 biotin
Modification N at T on this is bound to
the 3' end: aminomodifier II via 5' 0 biotin
5'-N CAT TGA GAT TCC CGA GAT TGA GAT CTT CTG CGA CGC GGC
N-3'
- 2~ -
. f
SE QID NO_5
Length of sequence: 31 basQs
Type of se~uence: nucleotide sequence
Type of strand: single strand
Topology: linear
Type of molecule: part of genome DNA with
modifications N
Position: HBV genome 2327-2356
Modification N: T on this is bound to
aminomodifier II via 3'-0 biotin
5'-N CAC TTC CGG AAA CTA CTG TTG TTA ~AC GAC 3'
SE ~ID NO 6
Length o~ ~equence: 32 bases
Type of sequence: nucleotide sequence
Type of strand: single ~trand
Topology: linear
Type of molecule: part of genome DNA with
modifications N
Position: HBV genome 2327-2356
Modi~ication N at T on this is bound to
the 5~end: aminomodifier II via 3'-0 biotin
Modification N at T on this i.s bound to
the 3'end aminomodifier II via 5'-0 biotin
5'-N CAC TTC CGG AAA CTA CTG TTG TTA GAC GAC N-3'
-- 25
SEQ I D NO 7
Len~th of sequence: 2 O bases
Type of sequenceO nucleotide sequence
Type of strand- ~;ingle strand
Topology: linear
Type of molecule: part Q~ genome DNA with
modi~ications N
Position: H~Y genoma 2332-2351
Modi~ication N: T on this i~3 bound to
aminomodif ier II via 3 ' -O biotin
5 '-NC CG(; A~A CTA CTG TTG TTA G-3 1
- 26 -
~ ;.3
SEO ID NO 8
Length o~ sequence: 21 basPS
Type of sequence: nucleotide sequence
Type o~ ~trand: single strand
Topology: linear
YPQ of molecule: part of genome DNA with
modi~ication~ N
Position~ HBV genome 2332-2351
Modification N at T on this is bound to
the 5' end: aminomodifier II via 3'-0 biotin
Modification N at T cn this is bound to
the 3' end: aminomodifier II via 5'-0 biotin
5'-NC CGG AAA CTA CTG ~TG TTA GN-3
SEQ ID NO ~
Length o~ sequence: 16 bases
Type of sequence: nucleotide sequence
Type of strand: single strand
Topology: linear
Type of molecule: part of genome DNA with
modifications N
Position: HBV genome 2299-2313
Modification N: T on this is bound to
aminomodifier II via 3'-0 biotin
5'-N AGA CCA CCA AAT GCC-3'
- 27 -
SE~ ID NO 10 !.~
Length of sequence: 17 bases
Type of sequence: nucleotide sequenc~
Type of strand: single strand
Topology: linear
Type of molecule: part of genome DNA with
modi~ication6 N
Position: HBV genome 2299-2313
Modification N at T on this is bound to
the 5' end aminomodifier II via 3'-0 biotin
Modification N at T on this is bound to
the 3' end: aminomodifier II via 5'-0 biotin
5'-N AGA CCA CCA AAT GCC N-3'
