Note: Descriptions are shown in the official language in which they were submitted.
CA 02342581 2001-02-21
Reaction chambers coated with nucleic acids, methods for the production and
use
thereof
Description
s
The invention relates to reaction chambers coated with nucleic acids, methods
for the
production thereof by coating of the reaction chambers with standard nucleic
acids, to a test
kit containing a standard strip produced by means of the inventive method, to
a set of at least
two oligonucleotides appropriate for that purpose, to a carrier nucleic acid
as well as to a
io plurality of application possibilities for the quantitative detection of
selected nucleic acids in
biological substances.
Many fields in clinical research and diagnostics, pharmacological drug testing
as well as food
analytics, require a precise cognition of concentrations of determined nucleic
acids
is (deoxyribonucleic acid [DNA] or ribonucleic acid [RNA]) in a sample to be
analyzed. For the
measurement of extremely small analyte concentrations, enzymatic amplification
methods are
frequently used. Hereby, the following methods are inter alia concerned:
polymerase chain
reaction (PCR, US Patents 4,683,195 and 4,683,202, EP 0 201 184; Hoffmann-La
Roche),
ligase chain reactions (LCR, Abbott Diagnostics, North Chicago, IL, USA),
strand
2o displacement amplification (SDA, Walker et al. [1993], PCR Methods Appl..
3: 1-6 Becton-
Dickinson Research Center) and transcription-mediated amplification (TMA, Gen-
Probe Inc.,
San Diego, CA), by means of which analyte nucleic acid concentrations can be
measured with
an extremely high sensitivity. A prerequisite for the quantitative use of all
of the mentioned
technologies is the availability of suitable synthetic or native nucleic acid
standards of a
Zs precisely defined concentration, which either are used as external, i.e.
amplified in parallel
assays, or as internal standards (i.e. so-called competitors amplified
simultaneously in the
same assay). Whereas the preparation of suitable standards is known to the
person skilled in
the art (Zimmermann and Mannhalter 1996, Biotechniques 21: 268-279, Koehler et
al. 1995,
Quantitation of mRNA by polymerase chain reaction - nonradioactive PCR
methods,
3o Heidelberg, Springer-Verlag), hitherto unsolved problems of process
technology exist with
the transfer of these standards into a stable form which is ready for
application, said fact being
a basic prerequisite for the reproducible measurement of unknown nucleic acid
concentrations. Problems exist in particular with the best possible storage of
highly diluted
nucleic acids. These essentially reside in that in practice, extremely low-
concentration
CA 02342581 2001-02-21
standard dilution series are worked with in most cases (approx. 1 - 100000
molecules per
reaction assay), which despite of a storage at temperatures between -
20° C and -80° C, are
often unstable (Koehler et al. 1997, Biotechniques 23: 722-726). This problem
is, inter alia,
so countered that low-concentration nucleic acid dilutions are stabilized in
the form, that a
s defined quantity of a specific carrier nucleic acid is added to the
dilution, said carrier nucleic
acid featuring a sequence homology to the nucleic acid sequence to be detected
which is as
small as possible (DE Kant et. al. 1994, Biotechniques 17: 934-942, Koehler et
al. 1997,
Biotechniques 23: 722-726). However, this does not always lead to success (cf.
Figure 1 ), so
that it is generally recommended to carry out all the required dilution steps
starting every day
io anew from a stock solution with a defined concentration. This, however, is
connected with the
disadvantage that the standards used must be prepared in a labor-intensive
manner, that they
are subjected to a varying pipetting accuracy, and valuable diluted batches
can only be used in
part. Thus, costs and time expenditure automatically increase at a
simultaneously reduced
reproducibility and reliability of the methodology. From the application-
technical point of
is view, the depicted procedure is therewith uneconomical, succumbed to
several disturbance
factors, and hence inappropriate for diagnostic routine laboratories.
It is the object of the present invention to develop vessels and methods by
means of which the
disadvantages of the methods according to the state of the art can be avoided
and which are of
ao simple application and storable for a longer period of time with unchanged
quality, and which
may be components of a test kit.
This task was solved by coating reaction chambers with defined standard
nucleic acid
concentrations, comprising the following substeps:
as ~ preparation and purification of suitable adsorbable nucleic acid
standards
~ determination of the precise concentration of the product by means of high
performance
liquid chromatography (HPLC), called calibration in the following
~ preparation of a dilution series from the calibrated standard with addition
of defined
quantities of a carrier nucleic acid
30 ~ inventive adsorption of the standard / carrier nucleic acid compounds on
reaction
chambers appropriate for amplification reactions, so that these defined coated
chambers
usable as standards could be transformed into a form imperishable for
prolonged periods
of time without impairments of quality.
CA 02342581 2001-02-21
Another subject matter of the present invention is a set of at least two
oligonucleotides, a test
kit corresponding to the requirements of a routine laboratory, and a plurality
of use
possibilities of said method.
s The subsequently described invention represents a molecular biological
method, being a
foundation for a preferably automated quantitative measurement of smallest
amounts of
analyte nucleic acids in diverse biological materials in conjunction with a
previous enzymatic
amplification. The inventive method consists in transforming nucleic acids
into a form
appropriate for the standardization of quantitative enzymatic amplification
reactions.
io Surprisingly, it was found that the inventive method allows for the
production of nucleic acid-
coated, so-called "ready-to-use" standard reaction chambers, which turned out
to be of simple
use, which are storable without problems for a prolonged period of time with
unchanged
quality, and which may be used as components for test kits, and hence better
comply with the
requirements of routine diagnostic laboratories, in particular in view of
automated analyses.
is
Nucleic acids in the sense of the present invention are single-stranded or
double-stranded
DNA or RNA or synthetic equivalents of DNA and RNA, as well as DNA, the native
deoxythymidine (dT) bases thereof being completely or partially substituted by
deoxyuracil
(dU). The preparation of suitable nucleic acid standards ensues in a form
known to the person
ao skilled in the art, preferably by means of PCR with the use of specific
primer oligonucleotides
(Example 1, item lA-B). As nucleic acid standards, native amplification
products prepared
enzymically, or synthetic nucleic acids are used, the nucleotide sequence of
which is
homologous to a sequence to be determined, or is preferably identical or
characterized by one
or more point mutations, deletions or insertions, which preferably lie outside
of the primer or
is probe bonding points. DNA standards are prepared by means of enzymatic
amplification of
target sequences, preferably by PCR, whereas RNA fragments may be obtained in
a known
manner by means of in vitro RNA synthesis with the use of RNA polymerases (cf.
Example 2,
item 2.1., C). The prepared nucleic acid fragments are subsequently subjected
to a purification
procedure, DNA being preferably cleaned by means of agarose gel
electrophoresis and
so subsequent extraction of the standard nucleic acid from the separating gel
(Example 1, item
1B), whereas in vitro-prepared RNA is extracted from the in vitro synthesis
assay in a manner
known to the person skilled in the art (cf. Example 2, item 2.1., C). The
precise measurement
of the concentration of the purified nucleic acid product preferably ensues by
means of HPLC
(Koehler et al. 1997, Biotechniques 23: 722-726, Example 1, item 2).
Subsequently, a dilution
CA 02342581 2001-02-21
series is prepared of the calibrated standard nucleic acid. For diluting DNA
standards, a DNA
solution is used, which is prepared by preferably transforming the DNA of the
lambda phage
(e.g. strain: lambda cl 857 Sam 7, 48502 bp, lambda DNA) by means of a S x 1-
minute lasting
ultrasonic treatment into fragments of about 1 - 2 kilobases (kb) (Example 1,
item 3; the
s average fragment length was determined by means of agarose gel
electrophoresis). This step
is supposed to entail an improved adsorption during the lyophilization process
and to
contribute to an increased durability of the standard nucleic acid in the
reaction chamber. It is
also possible to use the lambda DNA unmodified, or to use E.coli tRNA instead
of lamda
DNA. For the dilution of RNA standards, a transport RNA (tRNA) solution is
preferably used
io (Koehler et al. 1995, Quantitation of mRNA by polymerase chain reaction -
nonradioactive
PCR methods. Heidelberg, Springer-Verlag, Example 2, item 2.2.).
For the quantification of a measurement parameter, various standard dilutions
are prepared
(Example 1, item 3; Example 2. item 2.2), which preferably allow for the
measurement of the
is entire physiological or technological concentration range of the analyte to
be measured.
Aliquots thereof are used for coating those reaction chambers, wherein the
enzymatic nucleic
acid amplifications necessary for the establishment for example of an
calibration graph, are to
take place. Preferably, eight separate reaction chambers are coated with eight
different
standard dilutions (so-called octet strip). According to the present
invention, the coating is so
Zo carried out that aliquots of the respective standard nucleic acid dilution
supplemented by the
carrier nucleic acid are mildly dried directly in the reaction chambers used
(Example 1, item
4; Example 2, item 2.3.). In a particularly preferred manner, said
lyophilization ensues by
means of a vacuum centrifuge or a freeze-drier. In a further embodiment, the
drying ensues by
means of an equivalent drying method, for example, a method for the
superheating-free
as product drying with the use of microwaves (e.g. distributed via GWE mbH,
Leuna). The
coated reaction chambers produced - such as described - are characterized in
that the adsorbed
nucleic acid standards adhere so fixedly on the inner surface of the reaction
chamber used for
coating, that problem-free shipment via mail may, for example, be guaranteed.
In the Figures
1 through 4, it is shown in an exemplary manner, which quality requirements
are fulfilled by
3o the nucleic acid standards prepared according to the inventive method. For
the practice-
relevant test of the products on the basis of the described method, the ABI
PRISMTM 7700
Sequence Detection System (PE Applied Biosystems) has been used, which
presently is the
best, yielding extremely reproducible results. With this detection automate,
and using the
inventive method, as a principle, highly reproducible calibration graphs
comprising
CA 02342581 2001-02-21
correlations > -0.99 may be established (Figure 1). The reaction chambers
coated with DNA
according to the present invention (Example 1), e.g. the so-called "optical"
PCR tubes, are
superior to the actual state of the art, since they have a distinctly higher
stability as compared
to the conventionally used PCR standards (Figure 2), and may be stored even at
room
s temperature for a period longer than one year without loss of quality
(Figure 3). Reaction
chambers coated with in vitro-synthesized RNA, are stable for at least six
months, at 20° C, as
well as at room temperature (Figure 4, Example 2).
Standing upright in a suitable carrier box receiving at least 96 vessels, the
coated reaction
io chambers are closed with a self adhesive film (e.g. plastic film or
aluminum foil, parafilm), so
as to avoid contamination with foreign nucleic acids during storage and
transport. In each case
one octet strip closed with a film / foil, hence a strip containing 8
different concentrations of
the nucleic acids used for coating, is designated as a "ZeptoStrip".
is In said reaction chambers, apart from the calibrated nucleic acids, at
least two specific marked
or unmarked oligonucleotides acting as primers or probes, as well as further
reaction
components required for the enzymatic amplification, may be contained in a
lyophilized form.
Alternatively, reaction chambers used may also solely contain in a lyophilized
form these
specific oligonucleotides acting as primers or probes, the carrier nucleic
acid, and further
2o reaction components required for the enzymatic amplification. The inventive
test kits consist
of at least one "ZeptoStrip", at least two oligonucleotides and one carrier
nucleic acid.
The essence of the invention resides in a combination of known elements and
novel
approaches interacting with one another, and the novel overall effect of which
results in an
Zs advantage of use and the desired success, which consists in that ready-to-
use standard reaction
chambers for the quantitative detection of selected nucleic acids in
biological substances are
now available.
Thus, subsequent to overcoming the above-mentioned disadvantages of prior art,
the inventive
3o method features a series of advantages:
1. A manual or automated transfer of diluted acids used for standardization
into the
reaction compartments used is superfluous, since these are already present in
the
reaction chamber in a lyophilized form. The user-friendliness is therewith
drastically
increased, since the ready-to-use standard strips have only to be taken out
and to be
CA 02342581 2001-02-21
inserted in a 96-well carrier plate. Upon addition of the preferably premixed
reagents
for the subsequent amplification reaction, no further manipulations are still
necessary.
This simplification hence allows a consequent automatization of quantitative
enzymatic reactions.
s 2. Since henceforth, no pipetting of concentrated standards ensues, a
potential
contamination source is eliminated, and thus the danger of incorrectly
positive results
is distinctly diminished.
3. By transferring the standard nucleic acids into a non-aqueous medium, a) a
possible,
undesired microbial decomposition of the nucleic acids used as standards is
restricted
io or prevented, and b) a storability of even extremely low concentrated
nucleic acid for
prolonged periods of time is reached even at room temperature (Figure 2 - 4).
The
advantages facilitate quite drastically the shipment as well as the
application of the test
kits based on this method.
4. The carrier nucleic acids used prevent an unspecific standard adsorption
into the one-
s s way materials used for the preparation of the dilution series, and are -
detectable upon
addition to a PCR assay - at the same time a powerful "enhancer" for the
enzymatic
amplification.
Figure 1 shows a typical calibration graph obtained by means of a real-time
PCR product
zo measurement at the ABI PRISMTM 7700 Sequence Detection System using mdm-2
(murine
double minute-2) nucleic acid standards prepared according to the inventive
method (use of a
standard strip, i.e. eight different lyophilized mdm-2 standard nucleic acid
concentrations)
(Example 1, item S). The calculated correlation coefficient is in this case -
0.996.
zs Figure 2 shows in a graphic form the comparison between the inventive
coating method and
the use of real-time detection alike Figure 1 and the present state of the
art. Conventional, i.e.
mdm-2 standard DNA fragments of various concentrations (S0, 250, 2500, 10000
and 50000
molecules per PCR assay), stored in an aqueous medium and aliquoted the same
test day,
supplemented with carrier DNA, were compared with lyophilized standards of
equal
3o concentration. Whereas conventionally stored standards in particular of a
very low
concentration (50 or 250 molecules per assay) are completely decomposed as
soon as after 14
days of storage and four times freezing / defrosting cycle, (which is
reflected in the
illustration by reaching the threshold cycle 40 corresponding to an
amplification capacity 0),
CA 02342581 2001-02-21
no loss is detectable with the use of lyophilized standards (ZeptoStrips),
even when lowest
nucleic acid concentrations are used.
Figure 3 shows PCR results obtained with "mdm-2-DNA" ZeptoStrips, stored
alternatively
s either at -20° C or at room temperature for a period of one year. It
can be recognized that
identical PCR results, i.e. CT-values (i.e. parallel curves), were obtained
independent of the
storage temperature. It may be inferred from these results that the
immobilized mdm-2-DNA
remains stable over the entire test period independent of the assay
concentration and the
storage temperature.
io
Figure 4 shows TaqMan PCR results obtained with "bcl2-cRNA" ZeptoStrips, which
had
been stored for a period of 6 months alternatively at -20° C or at room
temperature. In
analogy to the results for mdm-2-DNA, the immobilized bcl-cRNA as well was
stable over
the entire test period, independent of the assay concentration and the storage
temperature.
IS
The inventive use of the reaction chambers coated with nucleic acids takes
place in test kits
for the detection of selected nucleic acids in biological substances. The test
kits are comprised
of at least one octet strip closed with film / foil, of at least two
oligonucleotides, as well as of
one carrier nucleic acid.
The following Examples serve for the explanation of the invention, without
being limited
thereto.
Exemplary embodiments
2s
Example 1.
Coating of polypropylene reaction chambers ("optical tubes") with defined
concentrations of double-stranded mdm-2 standard DNA
1.1 Preparation of a mdm-2-specific standard DNA fragment by means of PCR
A. cDNA preparation from total RNA, isolated from ADR5000 T-lymphoma cell line
(resistance-selected with 5 ~g of Adriamycin per ml of culture medium)
CA 02342581 2001-02-21
~ 1 pg of RNA purified by means of RNAzoITM "B" (Tel-Test, Friendswood, TX,
USA) in a
20 ~l standard reaction assay consisting of AMV reverse transcriptase buffer
(250 mmol/1
tris/HCI, pH 8.3; 250 mmol/1 KCI, 50 mmol/1 MgCl2, 50 mmol/1 dithiothreitol,
2.5 mmol/1
s spermidine), 5 U AMV reverse transcriptase, 0.5 mmol/1 of each of the dNTPs
(Promega,
Madison, WI, USA), 10 U recombinant RNase inhibitor (AGS, Heidelberg, FRG),
200 ng
Oligo (dT) (Amersham, Pharmacia Biotech, Uppsala, Sweden), to transcribe in
cDNA for
one hour at 42° C.
io B. PCR amplification and purification of the product
PCR
~ 2 ~l each of aliquot of the prepared cDNA are amplified in six identical SO
p,l
standard PCR assays consisting of 100 ng of each 3' or 5' primer,
respectively; 5 pl
is lOx Taq polymerase buffer (100 mmol/1 tris/HCI, pH 8.3; 500 mmol/1 MgCl2,
0.01
[wt/vol] gelatin), 1.5 U AmpliTaq~ polymerase (Norwalk, CT, USA, Perkin-
Elmer)m and 8 ~1 of dNTPs (0.2 mmol/1 of each nucleotide with use of dUTP
instead of dTTP) in GeneAmp~9600 Thermalcycler (Perkin-Elmer).
~ Program: 94° C for 30 s. 55° C for 30 s, 72° C for 1
min
Zo 35 cycles
~ Sequences of the amplification primers used (GenBank Accession Code for mdm-
2: I25341)
MDM2PR1 (1245-1264) 5'-GCC.AAG.AAG.ATG.TGA.AAG.AG-3'
MDM2PR2 (1439-1455) 5'-ACT.GGG.CAG.GGC.TTA.TT-3'
Zs ~ Length of the amplified DNA fragment: 211 by
D Purification of the 211 by fragment by means of agarose gel electrophoresis
~ Preparation of 1.5 % agarose gel (Easy-CastTM Minigel, AGS, Heidelberg), gel
slots with sextuple comb, filling of the chamber with TAE buffer (submarine
gel)
30 ~ Pooling and quantitative application of the prepared PCR assay
~ Carrying out of the electrophoresis at 100V, for 45 min
~ Making ethidium bromide-colored bands visible in UV light, cutting them out
as
precisely and rapidly as possible by means of a scalpel, transferring into 1.5
ml
Eppendorf vessels
CA 02342581 2001-02-21
~ Purification of DNA from gel blocks by means of QIAquick Gel Extraction Kit
(Qiagen, Hilten) according to instruction (elution with H20)
1.2 Calibration of the standard stock solution by means of HPLC
s
HPLC equipment
3-Line Degasser DG-980-50, PU-980 Intelligent HPLC Pump, Low Pressure
Gradient Former, UV-975 UV/VIS Detector, AS-950 Intelligent Sampler,
Column-Thermostat Jetstream 2 (Jasco Labor and Datentechnik GmbH, Gross-
io Umstadt, FRG).
Stationary phase:
TSK DEAE-NPR column (4.6 mm ID, length: 35 mm) and DEAE-NPR
precolumn (4.6 mm ID, length: 5 mm) (TosoHaas GmbH, Stuttgart, FRG)
Mobile phase:
is Buffer A: 25 mmol/1 tris/HCI, 1 mol/1 NaCI; pH 9.0
Buffer B: 25 mmol/1 tris/HCI; pH 9.0
HPLC run conditions:
~ Pressure: 80 - 120 bar (maximum 200 bar)
~ Flow rate: 1 ml/min
Zo ~ Temperature: 20° C
~ UV detection at 260 nm
~ Analyte: supplementation of approx. 10 - 200 ng purified PCR fragment
with buffer B on 40 ~l, injection of 20 ~l per run in each case in double
detection
~ Standard (separate run): 36 ~1 of buffer B plus 4 pl of Low Mass DNA
LadderTM
Zs (Life Technologies, Gaithersburg, MD, USA), consisting of 6 smooth-ended
DNA
fragments in the range between 100 and 2000 by (final concentration: 5 to 100
ng
DNA per band), injection of 20 ~l per run (double detection)
~ Carrying out a discontinuous gradient program for 25 min in the manner
described
below:
30 1. Equilibration of the column with 25 % buffer A in buffer B
2. 25 % A in B: sample application up to 0.5 min
3. 25 - 43 % A in B: linear gradient of 0.5 - 4.5 min
4. 43 - 60 % A in B: linear gradient of 4.5 - 20 min
5. 60 - 100 % A in B: linear gradient of 20 - 22 min
CA 02342581 2001-02-21
6. 100 - 25 % A in B: linear gradient of 22 - 24 min
7. 25% A in B: equilibration of 24 - 25 min
The data acquisition ensues by means of integration of the peaks by BorwinTM
s Chromatography Software, version 1.20 (IMBS Developpements, France). For a
precise
measurement of the concentration of the purified DNA standard, the surface
below the
individual peaks is assessed. The unknown concentration of the standard
nucleic acid is
calculated with the calibration graph obtained by means of Low Mass DNA
LadderTM.
io 1.3 Preparation of a standard dilution series with carrier DNA
Carrier nucleic acid:
OD lambda (dam+) DNA (from bacteriophage lambda c1857 Sam7, AGS GmbH,
Heidelberg), dissolved in 0.5 ml 10 mM tris/HCI, pH 8.0; 1 mM EDTA, is
transferred into
is approx. 1 - 2 kb fragments in intervals of S x 1 min and intermediate
cooling on ice by means
of an ultrasonic bath (Transsonic T570, Ultrasonics) at maximum power; a 10
ng/ml dilution
is prepared thereof (1:100), called lambda DNA in the following.
Preparation of a standard dilution series for mdm-2 (20x cone)
StandardDilution SL Final concentrationMolecules per
No. (preparation) [zmol/assay] assay
1 1: 10'' 419.75 252.773
1: 10" -~ 1:5 (20.1[No.l]+80.183.95 50.555
~,)
3 1: 10" ~ 1:10 (10,1 [No.l]+90141.986 25.277
~,)
4 1: 10" -~ 1:25 (lOpl [No.3]+90,116.794 10.111
~,)
1: 10' -~ (201 [No.3]+18014.199 2.528
~,)
1: 10 -~ 1:4 (251 [No.S]+7511.050 632
~,)
7 1: 10 -~ 1:10 (101 [No.S]+9010.420 253
~,)
1: 10 ~ 1:50 (2~1 [No.S]+9810.084 51
~,)
n, = Lambda-1JNA, lU ng/~l
CA 02342581 2001-02-21
For the preparation of the ready-to-use standard solutions, 5 pl each of the
standard dilution
series are pipetted into separate mufti twist top vials (Sorenson Bio Science,
Salt Lake City,
UT, USA; distributor: Carl Roth GmbH: Cat.-No.: 8184.1). For the production of
multi-
functional strips (i.e. strips which are suited for a sequential or
simultaneous quantitative
s measurement of a plurality of different nucleic acid sequences), further 19
standards (5 pl
each per standard nucleic acid) are added and - if required - supplemented up
to a final
volume of 100 pl with lambda DNA.
1.4 Coating of the reaction chambers
io
~ 5 p,l each of the ready-to-use standard solution prepared sub item 3, are to
be
pipetted into 8 different "optical tubes" (Perkin-Eliner, Cat.-No.: N8010935)
(in
decreasing concentration from position Al - A8), carrying out this
aliquotation
preferably with a pipetting robot (e.g. Biomek, company Beckman) for an
is improvement of quality.
~ Insert amplification vessels in black Eppendorf centrifuge adapters (0.2
ml), and
lyophilize the samples for 30 min in a vacuum centrifuge (e.g. Univapo 100 H
with
Unijet Refrigerated Aspirator, company UniEquip) with the rotor counter-
heating
on until complete dryness is achieved.
1.5 Testing of the produced strips by means of ABI PRISMTM 7700. Sequence
Detection System
Optimized mdm-2 TaqManTM method:
2s
Program: 2-step-PCR 95° C 110:0 min, subsequently
40 cycles 95° C 00:1 S min
60° C 01:00 min
3o Reaction conditions: 60 mM MgCl2
10 pM primer mdm-2Pr1 l and mdm-2Pr21
2 pmol mdm-2Probe
50 ng lambda DNA (Spl)
2.5 U AmpliTaqGoldTM
CA 02342581 2001-02-21
dNTPs, buffer from TaqManTM PCR Core Reagents Kit with AmpliTaqTMGold
assay volume: 50 pl
Primer and probe sequences used (GenBank Accession Code fiir mdm-2: I25341)
s mdm2Pr11 (1295-1318) 5'-GAG.AGT.GTG.GAA.TCT.AGT.TTG.CCC-3'
mdm2Pr21 (1352-1373) 5'-TGC.AAC.CAT.TTT.TAG.GTC.GAC.C-3'
mdm2Probe (1320-1350) 5'-FAM-TTA.ATG.CCA.TTG.AAC.CTT.GTG.
TGA. TTT. GTC.A-XT-3' -TAMRA
io
Example 2.
Coating of polypropylene reaction chambers ("optical tubes") with defined
concentrations of bcl2 standard copy RNA (cRNA)
is
2.1 Preparation of bcl2 standard cRNA, which is sequence-homologous to native
bcl2
mRNA
(according to a modified method by: Grassi G., Zentilin L., Tafuro S.,
Diviacco S., Ventura
2o A., Falaschi A., Giacca M., A rapid procedure for the quantitation of low
abundance RNAs
by competitive reverse transcription-polymerase chain reaction. Nucleic Acids
Res 1994;
22:4547-4549).
A. Preparation of bcl2 template DNA with incorporated T7 promoter
D Preparation of a bcl2 target fragment with PCRl
~ 100 ng of CCRF ADR5000 cDNA (cf. Example 1.1, A) were amplified such as in
Example 1.1, B, in a TRIO-ThermoblockTM 48 Thermal Cycler (Biometra,
so Gottingen).
Temperature profile PCRI: 94° C for 30 s, 55° C for 30 s,
72° C for 1 min
40 cycles
CA 02342581 2001-02-21
~ Sequences of the amplification primers used (GenBank Accession Code fur
bcl2:
M 14747)
BCL2PR1 (3498-3516): 5'-CTT.TTG.CTG.TGG.GGT.TTT.G-3'
BGL2PR2 (3896-3915): 5'-CTT.CTC.CTT.TTG.GGG.CTT.TT-3'
s
~ Theoretical length of the amplified DNA fragment: 418 by
Incorporation of the T7 promoter sequence into the synthesized bcl2 target
fragment by means of PCR2
io
Table 2/l : Pipetting scheme for PCR2 (preparation of 6 identical assays)
Reaction components Assay volume
pl
HZO (HPLC - pure) 21.5
lOx PCR-buffer with 1.5 mM MgCl2 (PE Applied5
Biosystems)
DNTPs (1.25 mM each, Promega/Boehringer 8
Mannheim)
Primer bcl2-1T7 (SO ng/~.1), diluted with 6
HZO
Primer bc12Pr2 (50 ng/~l), diluted H20 2
bcl2 target product (cf. 2.1. A, 1:1000 5
diluted with H20)
AmpliTaq Gold (0,5 U/~1, PE Applied Biosystems)2.5
~ The amplification ensued according to the following temperature profile:
is once through 95° C for 10 min, 40 cycles 95° C for 30 s,
SS° C for 30 sec, 72° C for 1
min, one through 72° C, S min, 4° C o0
~ Sequences of the amplification primers used:
BGL2PR2 (3896-3915) : (see above)
Zo bcl2-1T7: (underlined sequence: T7 promoter)
5' -cgg. gat. ccg. gat. cct. aat. acg. act. cac.tat. agg. gag. aCT. TTT. GCT.
GTG. GGG.TTT. TG-3'
~ Theoretical length of the amplified DNA fragment: 455 by
CA 02342581 2001-02-21
B. Purification and calibration of the bc12T7-DNA fragment
such as described in Example 1.1. and 1.2
s C. In vitro cRNA synthesis (preparation of 3 identical assays)
Table 2/2: Pipetting scheme for the preparation of bcl2-cRNA synthesis assays
Reaction components Assay volume
~ul
RNase-free H20 (treated with DEPC) 1
l Ox transcription buffer (Boehringer Mannheim)2
ATP (20 mM, Amersham Pharmacia) 1
UTP (20 mM, Amersham Pharmacia) 1
GTP (20 mM, Amersham Pharmacia) 1
CTP (20 mM, Amersham Pharmacia) 1
Purified bcl2-T7 dsDNA fragment (6 ng/pl) 10
T7-RNA polymerase (Boehringer Mannheim, 2
20U/~l)
RNase inhibitor (Amersham Pharmacia,l0 1
U/~1)
to ~ The incubation of the assays was carried out in a thermoshaker for 1 h at
37° C.
~ For increasing the cRNA yield, further 20 U T7 polymerase were added per
assay after
termination of the incubation, thereafter ensued another incubation for 1 h at
37° C
(thermoshaker).
~ Subsequently, all three assays were mixed and digested with 60 U DNase I
(RNase-free,
is Boehringer Mannheim) for 50 min at 37° C.
~ The purification of the cRNA ensued by means of conventional phenol
chloroform
isoamyl alcohol (25:24:1) extraction such as described in: Koehler T., Lassner
D., Rost
A.-K., Thamm B., Pustowoit B., Remke H., Eds.: Quantitation of mRNA by
polymerase
chain reaction - nonradioactive PCR methods, Springer-Verlag Heidelberg, pages
36 - 37.
Zo ~ The precipitation of the cRNA was carried out over approx. 3 h at -
20° C, thereafter, it
was washed three times with 300 ~.1 of 75 % ethanol, then centrifuged, and the
pellet was
stored over night at -20° C under 100p1 of 96 % ethanol.
CA 02342581 2001-02-21
~ The next day, drying of the pellet was carried out in a vacuum centrifuge
(Univapo 100 H,
UniEquip), the dried pellet was solved in 25 ~l DEPC H20; the concentration
detection
ensued in 500 ~1 quartz cells after an UV 260/280 measurement of 2x 2 ~l
aliquots.
~ The total yield of all three assays amounted to 23 ~g cRNA at a ratio of
260/280 > 1.8.
s
D. Characterization of the synthesized cRNA by means of non-denatured agarose
gel
electrophoresis
(according to a modified method by: Collins M.L., Zayati C., Detmer J.J., Daly
B., Kolberg
J.A., Cha T.A., Irvine B.D., Tucker J., Urdea M.S., Preparation and
characterization of RNA
io standards for use in quantitative branched DNA hybridization assays. Anal.
Biochem. 1995;
226:120-129).
~ A minigel chamber (Easy-CastTM, AGS Heidelberg) was freed from RNase over
night by
means of 2% Absolve NEF-9716 cleaning solution (DuPont), and was subsequently
is rinsed twice with DEPC H20.
Preparation of the gel: Prepare 1 % agarose gel (Qualex-Gold-Agarose, AGS)
with 1 x
TAE buffer (from SOx stock solution with DEPC H20) (ethidium bromide (1.6 ~1
10
EtBr] poured in), decuple comb; run buffer: 1 x TAE (4 ~l EtBr/ 1 OOmI).
4 ~1 of formamide were added to the purified and re-suspended cRNA (approx. 1
pl per 4
zo ~l of volume), as well as 4 pl of a 0.16 - 1.77 Kb RNA ladder (Gibco BRL),
and
incubated subsequently at 65° C for 5 min.
~ Thereupon ensued a rapid cooling down on ice and a subsequent addition of 1
~l of
(RNase-free) gelling buffer per tube; from the finished assay, 4 ~l aliquots
are pipetted
into the test gel slots (marker into the outer slots, cRNA sample into the
inner slots).
Zs ~ The electrophoretic separation was carried out for about 2 h at 80 V in
the RNase-free
minigel chamber cooled in an ice bath. A buffer circulation was achieved by
intermittently
moving the run buffer.
~ The RNase-free documentation of the results ensued by means of a GelPrint
Video
Documentation Workstation (MWG-Biotech, Ebersberg) with the use of ONE-DscanTM
so Software (Scanalytics, Billerica, MA, USA).
~ Opposite the theoretical molecular weight of the synthesized bcl2 cRNA of
418 b stood an
experimentally determined molecular weight of approx. 400 b.
2.2. Preparation of a bcl2 cRNA dilution series
CA 02342581 2001-02-21
The dilution of the purified bcl2 cRNA ensued with E. coli tRNA solution (100
ng/pl DEPC
HZO, Boehringer Mannheim).
s Table 2/3: Preparation of a bcl2 cRNA dilution series according to the
following scheme:
Standard cRNA dilution factor RNA concentration (indicated
No. in
zmol per 5 pl of diluted
solution)
1 1:10' 1610
2 1:10" 161.05
3 1:10' 16.105
4 1:10'-~ 1:5 3.22
1:10 1.6105
6 1:10~ 1:5 0.322
7 1:10' 0.16105
8 1:10 0.016105
2.3 Coating of 96 "optical tubes" (1 plate) with bcl2 standard cRNA-defined
io concentration
~ From each of the bcl2 cRNA dilutions (see Table 2/3), 150 ~l were prepared
and
positioned in a prepared coolable rack of a Biomek~2000 pipetting work station
(Beckman Instruments Inc. Fullerton, CA, USA).
Is ~ A 96-well carrier plate was charged with 96 "optical tubes" and was
equally inserted in
the therefor intended position in the work station.
~ By means of the robot and using suitable plugged one-way pipetting nozzles,
5 pl each of
standard " 1 " was pipetted into the "optical tubes" of position A 1 - A 12,
of standard "2"
into the tubes B 1 - B 12, of standard "3" into the tubes C 1 -C 12, etc.
20 ~ The chambers containing standard cRNA were all shock-frozen for 30 min at
-80° C, and
thereupon, avoiding an intermediate defrosting, immediately lyophilized in a
pre-cooled
Lyovac GT2 (AMSCO Finn-Aqua GmbH, Huerth) for 1 h.
~ The tubes were subsequently manually closed with a self adhesive foil (e.g.
BiomekTM
Seal & Sample aluminum foils, Beckman Instruments). The "RNA" ZeptoStrips then
were
CA 02342581 2001-02-21
so manufactured that the foil adhering to the tubes was vertically cut, so
that continuous
strips of the positions A 1 - H 1, A2 - H2, A3 - H3, etc., were formed, in
each case
containing one concentration of each of the bcl2 cRNA dilutions.
~ From the so prepared 12 strips, 6 were in each case stored over the test
period either at -
s 20° C or at room temperature.
2.4. Analysis of the prepared bcl2 cRNA strips by means of ABI PRISMTM 7700
Sequence Detection System
io A. Materials:
Sx TaqMan EZ buffer: 250 mM of bicine, 575 mM of K-acetate, 0.05 mM of EDTA,
300
nM of ROX (6-carboxytetramethyl rhodamine), 40 % of (w/v) glycerol; pH 8.2.
~ 25 mM Mn(OAc)z
i s ~ dNTP s: dATP ( 10 mM), dCTP ( 10 mM), dGTP ( 10 mM) and dUTP (20 mM)
mixed in a
volume ratio of 1:1:1:1
B. Sequences of the oligonucleotides used
zo bc12Pr1 (3498-3516) see above
bc12Pr21 (3572-3591) 5'-GCA.AGT.GCA.GCC.ACA.ATA.CT-3'
bcl2Probe (3547-3568) 5'-FAM-CAG.TTC.TGG.GGC.CAA.GAG.GCT.GTXT-3'-
TAMRA
(FAM = 6-carboxyfluorescein, TAMRA = 6-carboxytetramethyl rhodamine)
zs
C. Combined reverse transcription and PCR (RT-PCR) with use of the enzyme rTth
polymerase (PE Applied Biosystems)
~ On the respective test day, into each of the bcl2 cRNA strips stored
alternatively at -20° C
30 or at room temperature, the following reaction components were pipetted
(Table 2/4, S = standard No., assay volume: 50 ~1)
CA 02342581 2001-02-21
Table 2/4: PCR3
Reaction component S1 S2 S3 S4 SS S6 S7 S8
DEPC-H20 10.510.5 10.5 10.5 10.5 10.5 10.5 10.5
Sx EZ buffer with ROX 10 10 10 10 10 10 10 10
DNTPs for TaqMan 6 6 6 6 6 6 6 6
bc12Pr1 (50 ng/pl) 1 1 1 1 1 1 1 1
bc12Pr21 (50 ng/pl) 1 1 1 1 1 1 1 1
bcl2 probe (0,79 pmol/pl)2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Mn(OAc)2, 25 mM 5 5 5 5 5 5 5 5
RTth-polymerase (PE Applied2 2 2 2 2 2 2 2
Biosystems, 2.5 U/pl)
s Temperature profile at the ABI PRISMT"" 7700 Sequence Detection System:
59° C, 30 min (reverse transcription)
95° C, 5 min
40 cycles 95° C, 15 sec
59° C, 1 min
Io
~ By means of PCR3 (Table 2/4), the stability of the inventive bcl2 cRNA
strips was
analyzed in each case after 7, 14, 28, 90 and 180 days of storage (at -
20° C, as well as at
room temperature). The results are summarized in Figure 4.
is
Legend to the figures
Figure l:
Characteristic calibration graph established by means of a PCR product
measurement at the
Zo ABI PRISMTM 7700 Sequence Detection System with use of a "ZeptoStrip"
coated with
mdm-2 DNA according to the inventive method (cf. Example l, item 5), r = -
0.996.
Figure 2:
CA 02342581 2001-02-21
Advantages of ZeptoStrips (ZSs) as compared to standard nucleic acids, which
had been
conventionally stored in an aqueous medium according to the present state of
the art. Dashed
lines: conventional storage; continuous lines: ZeptoStrip. Legend: Initial
number of nucleic
acid molecules per PCR assay.
s
Figure 3:
Storability of mdm-2 DNA ZeptoStrips over a test period of 1 year without loss
of quality.
Legend: Initial number of nucleic acid molecules per PCR assay. Continuous
lines: storage at
+25° C, dashed lines: storage at -20° C.
io
Figure 4:
Stability of "bcl2 cRNA" ZeptoStrips, which had been alternatively stored over
a period of 6
is months either at -20° C or at room temperature. Represented are the
regression lines
calculated for each standard concentration with consideration of all
measurement values
obtained in each case at a storage at -20° C and +25° C. Legend:
Concentration of the bcl2
cRNA immobilized in the reaction chamber in zeptomol per assay.
