Note: Descriptions are shown in the official language in which they were submitted.
WO92/l9775 PCT/EP9~/00929
2la~
ASSAY AND K~.T FOR THE DETECTION OF
CHROMOSOMAL ABNORMALITIES
Field of the Invent on
This invention relates to a process and kit for use in
detecting the nucleic acid sequences that occur in
chromosoma~ abnormalities.
Background of the Invention
Chromosomal abnormalitiPs are the cause of various
undesirable conditions in humans, both inherited and non-
inherited, including neoplastic conditions such asfollicular lymphoma. It is clearly of vital importance to
detect such abnormalities, whether caused by chromosom~l
$ranslocation, transposition, intergene or intragene
recombination, insertion, deletion or point mutation at an
early stage by a simple and r~liable test.
The association between translocations and
~: . pathological states, e.g. neoplastic degeneration, is
: describ~d by Russo et al, in "Recent Advances in
~; ~ Hematology", A.V. Hoffbrand (ed.), 5, 121-130, Churchill
: 20 Livingstone. More particularly, a translocation t(14;18)
that involves portio~s of the qenes c1-2 and JH has been
strongly correlated~ to human follicular lymphoma; see
Tsujimoto et al (:1985j Science 228: 1440-1443, and Science
~; ; 29: 1390-13g3; Stetler-Stevenson et al (1988) Blood ~:
;25 1822-1825; and ;Crescenzi et al (1988) Proc. Natl. Acad.
Sci . U . S . A . ~ 8 5 :~ 4 8 69;-4 8 7 3 .
Follicular~lymphoma (FL) is a B-cell disorder which is
~: ~related to the~presence of a genetic abnormality called the
bc1-2 translocation~ ~ About 90~ of follicular B-cell
:30 lymphomas and 20~ of large diffuse B-cell lymphomas carry
i~the t(14;18~(q32;q213 translocation which directly involves
the IgH locus on chromosome:14 and the c1--2 locus on
chromosome 18. ~ Analogous to the myc translocations in
Burkitt's lymphoma,~ the ttl4;l8)~32;q2~) translocation
occurs 5' or 3~' to~ the:bcl-2 gene, but not withln the
protein coding:portion of the gene. It ap~ears that in FL
the translocation:takes place in pre-B-cells during the
~ ~ ~SUBSTITVTL S~EE~
W092/19775 PCT/EP92/00929
2 :~ , S ~ 1 8 2
recombination of the JH region in the IgH chain locus. The
association of the bc1-2 oncogene with the heavy chain
locus results in high levels of bc1-2 expression.
The FL translocations are structurally uniform. In
about 70% of human FL the breakpoints are clustered within
the 3' untrans~ated region of the gene, de~ignated "Major
Breakpoint Region" (MBR). In another 10-20~ of the cases,
the breakpoints are clustered in a region more than 20 Kb
downstream from bc1-2's second exon, designated "minor
cluster region'l (mcr~. In some cases translocations have
been detected near the 5' exon.
Currently avallable methods for the detection of the
bc1-2 translocation rely on cytogenetic assays (karyotype
anal~sis, which however cannot distinguish between MBR and
mcr) or on DNA digestion with restriction enzymes and
subsequent Southern blotting, usually involving the use of
radioactive probes.
In recent years, many methods for identifying nucleic
acid se~uences have been developed. They are generally
solid-phase methods and relatively rapid and easy to carry
out, but difficult to quantitate and not easily adaptable
~: : for clinical and diagnostic laboratories. If radioactive
labelling is avoided, for easè of operation, it is at the
expense of the sensitivity of the method. This drawbac~
can be overcome~b~ amplifying the sequence to be detected,
e;~g. using the polymerase chain rea~tion (PCR) as disclosed
: ~ ~ in EP-A-02~0362 and EP-A-0258017.
GB-A-2169403~describes a method for the identification
; of nucleic acids, in which two independently-labelled
oligonucleotide probes are reacted in a single solution,
under hybridisihg conditions, with a target analyte. If
the analyte contains a sequence that hybridises to both
probes, this may readily be detected ~y vi~tue of the fact
that one label allows separation of the hybrid and the
3S~ other its detection. The same or similar techni~ues are
described in, for example, EP-A-0128332, EP-A-0145356, EP
~:~ A-01:59719, EP A-0177191, EP-A-01921:68 and EP-A-0198662.
SUE~STITUTE SHEE~
W~)92/1977~ 2 1 ~ ~ 51~ PC~/EP92/00929
. .~
Oligonucleotide probes and their use in detecting
chromosomal abnormalities are described in, for example,
US-A-4701409, US-A-5015568, US-A-5024934, EP A-0181635 and
E:P A-0252685.
S SummarY OI the Invention
According to the present invention, a process of the
general type described in (;B-A-21694Q3 is applied to the
detection of chromosomal abnormalities, e.g.
translocations, using capture and reporter probes that are
respectively complementary to different regions of the
target sequence, e.g. on opposite sides of the
translocation. The re--pective probes, and any other
components used in the procedure, as required, may be
formulated into a novel kit comprising a plurality of
contairlers in which the components are distributed.
Giverl the importance of assaying ~or chromosomal
abnormalities, the present invent~.on provides a number of
valuable characteristics. Firstly, for example, it is
simple to use, e.g. by relatively unslcilled personnel in
:~ ~ 20 hospitals and less specialised laboratories; it is quick,
:~ : non-radioactive and requires only simple equipment.
Seondly, the absorbance readings allow a quantitative
measurement of the final signal. With other methods, such
as gel: electrophoresis/Southern bl:otting, or dot-blotting,
this quantitatlve de~termination of signal is only possible
wi~h the use of sophisticated instrumentation. These
. ~ traditional methods~ are much more prone to subjectiv~e
interpretation. The:quantitation:of the signal allows much
easier c:omparison of results between e~cperiments, and
between laboratories~
Thirdly, the system will only generate a signal if
both reporter and capture probes (complementary to
::~ sequences on either side of the ~breakpoint3 bind. This
::: provides a very:high degree~ of specificity and helps
35 minimise the risk of false positives~; this is particlllarly
: important as this~techrlology has primarily been desiyned to
-
'5~ TuTE s~ T
WO92/19775 PCT/EP92/00929
~1 ~!Ji1~ 4
detect chromosomal translocations associated with
mallgnancles.
The use of two probes, internally "nested" with
respect to primers used for amplification by PCR, also
reduces the risk of obtaining false pvsitives due to the
detection of PCR artefacts such as truncated elongations,
primer concatenamers and other problems related to the
specificity of the PCR reaction, as well as to the known
imprecision of the Taq I Polymerase enzyme.
Description o~ the Invention
The nucleic acids in the analyte preferably comprise
double-stranded DNA. They may be amplified by the action
of DNA polymerase which is capable of synthesising in the
5'-3' direction a complementary strand from a template, in
the presence of a primer which is complementary to an
extreme portion of the single-stranded analyte sequence.
Preferably, amplification occurs for both strands of the
analyte se~uence, and the DNA polymerase is heat-stable.
The amplified strands may then be denatured.
Advantageously, th~ denaturation occurs by means of
incubation, e.g. at a temperature between 90 and 97C, or
in the presence of NaOH.
Preferably, the capture probe is conjugated to a
hapten such as fluorescein isothiocya~ate (FITC). Then~;'
separation is by means of anti-hapten antibodies, e . g .
anti-FITC, which are immobilised on a ~olid pha~;e,
preferably magnetlsable microparticles which are attracted
or~to magnetic plates. The liquid phase containing free
detection probes may be removed by washing.
The detection probe is preferably conjugated to an
enzyme or biotin. Detection is then condu~ted by means of
incubation with a substrate which is specific for the
enzyme, preferably chromogenic, termination of the
: reaction, e.g. by:adding a stop solution, and colorimetric
reading of the solution itself. Preferably, the enzyme is
an alkaline phosphatase, the specific chromogenic substrate
is phenolphthalein monophosphate, and the colorimetrîc
SU~ ~SHEET
W~92/t9775 ~ l 3 9 i~ pCT/~Pg2/~0929
reading is carried out at a wavelength of 554 nm. A probe
that is conjugated to biotin may be detected by means of
avidin conjugated to an enzyme.
In general, the present invention is particularly
useful for the detection of nucleic acid sequences
comprising con~iguous DNA segments from different
chromosomes, or from different zones of the same
chromosome. This may be the result of any of the foll~wing
biological processes: chromosomal tr~nslocation,
transposition, intergene or intragene recombination,
insertion, deletion or point mutation~
The invention is particularly adapted to the detection
of such biological processes that are correlated to
pathological states of the organism which the analyte
sequence comes from. Thus, the translocation may be
correlated to a neoplastic state, as for instance those
related t~ T and B lymphocytes; for example, the
translocation may be t(l4;18), the analyte sequence bcl-
~iJu~ and the neoplastic state follicular lymphoma. In
this case, the sequence of the analyte DNA contains the
: recombination point of two human chromosomes 14 and 18, and
the probes bind to either side of the tarqet sequence on
the same DNA strand, e.g. the negative strand. It is
important that :the primers be of such length ~ d
2S omposition as not to allow hybridisation to occur with
th mselves or with portions of the analyte DNA segment
: whi~h is complementary to the other primer. Accordingly,
; the extension products are synthesised employing a DNA
polymerase, which is preferably heat-stable, a~d extends
the ter~inal portion to the 3' position of each primer.
The extension products are then separated from their
templates by means of high temperature denaturation (92-
94C). The passage is repeated through a number of cycles
sufficient to increase the amount of the target sequence up
to the concentration at which it can be detected. When the
amplification cycles are completed, a suitable amount of
the analyte sequence is caused to react with a suitable
SlJE3STlTU~E: SHE:T
WO92/19775 PCT/EP92/0~9~9
~1 39~I 8
concentration of NaOH, e.g. 0.08N NaOH, so as to cause
- denaturation of the double-stranded segment.
Alternatively, denaturation can be carried out through
exposure of the DNA to a temperature of 94-97C for 5-10
minutes and then cooling suddenly down to 0OC.
Once ~enaturation is completed, a second pair of
oligonucleotides is employed. These are probes which are
~ifferent from the primers employed in the amplification
procedure and which are both complementary to the same
strand of the analyte DNA, in zones which are to those
employed for amplification. The pro~es are added to the
reaction mixture at an excess concentration with respect to
the analyte sequence. The pair of probes consists of a
capture oligonucleotide and of a reporter oligonucleotide.
Each probe is conjugated through its 5' end with a reactive
group, to provide an appropriate label.
Reper molecules include haptens, enzymes and
radioactive labels, or include any substrate thak provides
a chromogenic, fluorescent or chemiluminescent signal. By
way of example, e.g. the report probe is labelled with
alkaline phosphatase and the capture probe with a hapten
such as FITC.
The capture probe is suitably separat~d by linkage to
a solid phase such as plastics beads, microplates, coat~
ubes, latex or, preferably, magnetisable microparticles.
By way of example, a hapten can be linked specifically by
an antibody immobilised on a solid phase, e.g. anti FITC on
magnetisable microparticles.
Next, a neutralising solution, e.g. 0.5 M Tris, pH
7.5, is added to the reaction mixture, in such an amount as
to buffer the NaOH and allow the hybridisation of the
probes to the analyte DNA to occur.
After a suitable incubation period at a constant
temperature, e.g. 30 minutes a~ ~37C, an excess amount of
3~ a solid phase consisting of magnetisable microparticles
coated with an anti-FITC antibody which i5 capable of
binding the whole amount of the FITC-labelled separator
.
` SiL1BSTlTUTE SHEET
W~92/l9775 2 ~ 3 j 1 ~ PcT/EP92/nog29
probe, both the free and that reacted with the DNA
sequence, is added to the reaction mixture, so forming the
analyte sequence-pro~es complex. After a suitable
incubation period at a constant temperature, e.g. 10
minutes at +37C, the reaction tubes are put on a magnetic
plate which, in a short time, e.g. 3 minutes, causes the
magnetisable particles to settle onto the bottom of the
tu~e itself.
The supernatant is then removed by decantation, by
turning the magnetic plate upside down, the magnetised
particles adhering to the bottom of the tube. The tubes
are then removed from the magnetic plate and the solid
phase is resuspended in a suitable washing solution, e.g.
1 ml of 0.075M:Tris-buffered saline, pH = 7.5, allowed to
settle and decanted again. The washing cycle is repeated
as often as is necessary to remove any non~specific binding
of the reag~nts, and in particu~ar of the reporter probe
: which is conjugated to the enzyme, with the solid phase.
During decantation and washing, all those reactants which
are not specifically linked to the magnetic particles are
: removed from the:reaction tube.
Next, a suitable~ amount of a chromogenic substrate
which is enzyme-specific, e.gO 200 ~l of phenolphthalein
monophosphate, is~added~ to the magnetic particles ~d
:25 allowed to react for the time required at a constant
temperature,~e:.g. l hour at 37C. After this period, the
:~ reaction is stopped by adding a stop solution, e.g. 750 ~l
~: o~ a Na2C03 solution, pH 12.
The addition of the stop solution causes th~ formation
and the stabilisation of colour, the absorbance value of
which is measured at a suitable wavelength, for instance
554 nm, on a colorlmeter. A coIour ~evelopment which is
significantly higher than that of blank samples indicates
: that, during~ ampli~fication, ~some extension products were
3~ formed starting from the~ specifie primers and from the
analyte DNA sequence that~has acted~as a template. In the
absence of the analyte sequence, no formation of specific
::
5UE~5TITUTE 5~1EET
WO92~19775 2 1 ~ 9 ~ P~T/EPg2/OOg29
extension products would have occurred, which products are
the only compounds capable of acting as bridges between the
magnetic particles and the reporter probe that bears the
enzyme capable of gener~ting the signal. A standard curve
may be generat0d, employing known concentrations of the
analyte DNA to give a concentration value for each sample
analysed.
The method for conjugating a reactive group to the
oligonucleotide probes obviously depends on the group type
that is to be employed; generally the preferred bond occurs
throu~h the QH group in the 5' position of the
oligonucleotide. During automated synthesis of the
oligonucleotide, employing phosphoroamidite chemistry, it
is possible to introduce an aliphatic amine at the 5' end
employing the Aminolink 2 (ABI) reactant or the
Aminomodifier II (Clontech) reactant; this amino group can
be reacted successively with a specific hapten, for
instance FIT~ or a biotin-hydroxy-succinimide ester, or any
other group containing an ester which is activated and
capable of reacting with a primary amine.
For conjugation with the enzyme, it is generally
pre~erred to use heterobifunctional reactant~ such as
succini~idyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate
~: (SMCC) and 2-iminothiolane (2-IT), available from Pier~e~
For instance, SMC~ is capable of reacting with the primary
amine in the 5' position of the reporter probe give a
derivative having ~a maleimido group free; the 2-IT is
~ ~apable of reacting~with the NH2 qroups of lysines of the
::: alksline phosph~tase so as ~to give a derivative.having a
free -SH group. The maleimido groups and the -SH group, if
caused to react und~r 5uitable conditions, react
spontaneously so as:to form a very stable carbon-sulphur
covalen* bond. In this way, it is possible to obtain
conjugates in which the reporter probe is linked through
its 5' end to the alkaline phosphata~e through a long and
flexible arbon atom chain, keeping the oligonucleotide
capability of specifically hybridising with a complementary
SUB5TIT~ITE SHEE~
.
WO92/19775 PCT/EP92/00929
2 ~3.~1~
sequence unaltered, and keeping also unaltered the
capability of the enzyme to interact with its specific
substrate, to generate a coloured solution.
Magnetisable particles coated with anti-FITC
antibodies are commercially available (from Ares-Sersno,
Advanced Magnetics) or they can be prepared by well known
procedures. Specific substrates for the phosph~tase and
stop solutions are also commercially available (from
Sigma~.
The extension products can be generated by the
exposure of the primers, hybridised to their templates, to
a DNA polymerase which is preferably heat-s~able, e.g. the
Taq polymerase disclosed in EP-A-0258017. The DNA
polymerase will replicate the sequence of the template, so
synthesising some fresh DNA from the primers in the 5'~3'
dir~ction~ A heat-stable polymerase is preferred, but it
is not indispensable because the simplest way of denaturing
~:: the double-stranded extension product is by exposure to
high temperatures (about 95C) during the cycles of the
PCR/ as disclosed in US-A-4683202. By employing different
: procedures for denaturating the extension products, other
polymerases can be~used, including the Klenow fragment.
Specifically w1th reference to detecting the t(l4;l8)
bcl-2 (JH) translocation, but potentially of more gene~a~
applicability,;~it~ h:as been found that amplification of
either the Major Breakpoint Clus~er Region (MBR~ or minor
: :
clus~er region (mcr~ can be performed at the same time,
~ depending upon wh~ich~target is present, using a mixture of
: primers: (3 ~in total)~. These primers are respectively
: 30 specific for (i) the JH region on chromosome 14; (ii) the
MBR region on chromosome 18 (within the 3' untranslated
region of the; bc1-2 gene); :and (iii) the mcr region on
chromosome 18 in~a:region more than 20 Kb downstream from
the bcl-2 second exon. Preferred primers of these types,
which do not interfere with each other and which yield the
: same efficiency o~ amplification for both the MBR and the
mcr sequences, are the JH primer shown as SEQ ID. No. 3,
: ~ ~
SUBST117~ ~F~
WO92/19775 PCTIEP92/009~9
."1~9518 lo
thP MBR primer shown as SEQ ID No. 1, and the mcr primer
shown as SEQ ID No. 2 (see Sequence Listing, below).
Following amplifica~ion, either the MBR or the mcr-
amplified sequence can be detected using specific
reporters. Further, it is known that 5iX JH regions are
present in the IgH locus. To be able to detect each
individual JH region that may be randomly involved in the
t(14;183 chromosomal translocation, a mixture of six
modified oligonucleotides is preferably used. Each
oligonucleotide is complementary to one of the six specific
JH regions; they have the respective sequences shown as SEQ
ID NOS. 4 9.
Each of these oligonucleotides is modified at both the
3' and 5' end with a NH2 group during the automated
synthesis. Each reporter is thus conjugated at both the
NH2 groups with FITC, and HPLC-purified. The use of 3' and
~; ~5' conjugation increases the system sensitivity.
The FITC-con3ugatéd oligonucleotide acts as a capture
probe, because it reacts with the anti~FITC coated magnetic
~20 particles during the detection assay. The mixture of the
six conjugated oligonucleotides is used in the detection of
both MBR and mcr-amplified sequences. The determination of
;wh~ich breakpoint ls present is~made possible by the use of
specific probes for either~the MBR~or the mcr region of the~
bc1-2 gene. ~ ~
; Both the ~MBR;and mcr repo~rter oligonucleotides are
;modified at both~the~3' and 5'~end with a NH2 group during
the automated synthesis. They are then conjugated to the
enzyme alXaline~ phosphatase and`purified as ~escribed
~; 30 above. The enzyme-conjugated oligonucleotides act as
"signal generatlng"~probes.
The modified oligonucleotides used as reporters (after
:: :
;~ ~ being conjugated to alkaline~phosph~atase) are shown as SEQ
ID~ Nos. 10 and~ (MBR and mcr reporter probes,
respectively)~ The NH2 modification at both ends of the
oligonucleotides increases the amount of enzyme that can be
:: :
,
~JBSTI~UTE St~E~T
WO92/19775 2 i ~ 3 ~ 1 ~ PCT/EPg2/OOg29
linked to the probe and subse~uently the sensitivity of the
detection method.
It is preferred that the reaction buffers in which
either the MBR or the mcr detection probes are dissolved
differ slightly from each other, in order to account for
the different lengths of the amplified sequences (200 bp
for the MBR and 400 bp for the ~cr). Also the initial
dilution of the PCR samples may vary for the two targets
(for example, actual dilution for the MBR is 1:10; for the
mcr 1:4), as does the incubation time for the hybridisation
step (for example, 30 minutes at +37C for MBR and 15
minutes at t37C for mcr).
The followi.ng description constitutes specific
embodiments of the present invention. The "Reagents"
illustrate a kit of the invention and the "Recommended
Procedures" illustrate the process of the invention. These
are taken from t~e instructions associated with a kit
~: marketed under the trade name C-TRA~ FL by Raggio-Italgene
:::. S.p.A. This kit is specifically designed fvr in vitro
~: 20 research use, for the detection of the t(14;18) (q32;~21~
:: chromosomal translocation in frozen biopsies, paraffin-
.
: ~ embedded tissues, peripheral blood and bone marrow. Prior
DNA isolation can be conducted by standard methods, as
described: by Maniatis et: al in "Molecular Cloni.ng, A .
2:5 Laboratory Manual", 2nd ed. pub. Cold Spring Harbour.
.
'
~;UB~;TI~UTE SI~EE:T
WO 92/19775 12 PCr/EP92/00929
~3~ 2 J_ J 9 3 1 8
Each kil contains sufficient PCR primers to run 25 amplifications. These amplifications can
be subdivided into a maximum of 5 runs - 3 samples plus 2 PCR con~rols in each run.
There are sufficient detection reagents to assay all the amplified samples for both the MBR
and mcr, as well as for running ~he necessary detection controls.
The following reagents a(e provided:
No.1 PCR Primers lJH; MBR; mcr) 1 vial (Iyophilized)
Contains: 3 nanomoles of each prirner
To be reconstituted with 300 ~LI of distilled water (reagent No. 14). Store at -20 C after
reconstitution .
No.2 Sample Diluent 1 vial (15ml)
Contains: Tris/EDTA (TE) buffer pH 7.5
Ready to use.
No.3 Denaturing Solution 1 vial (3.5ml)
Contains: Diluted NaOH/SDS/EDTA
Ready ~o use. Store at room temperature - DO NOT REFRIGERATE.
; No.4 bc1 2-MBR l: etection probes 1 vi~l (1 5.4ml~
Contains: A set of JH-FiTC reporters IJH1.6 probes conjuga1ed to FITC) and a bc1-2- MBR
probe (conjugated to the enzyme alkaline phosphatase) in reaction buffer.
: ~: Ready to lJse.
No.5 bci-2-mcr Detec1ion probes 1 vial t15.4ml)
Contains: A set of JH-FITC reporters (JH1-6 probes conjugated to FITC) and a bc1-2 mcr _.,
probe (conjugaled with the enzyme alkaline phosphatase) in reaction buffer.
Ready to use. ~ ~
No.6 Separa~ion Reagen~ 1 vial (15.4ml)
Conlains: A suspension of anti-FlTG coated paramagnetic beads in Tris buffered saline.
Ready ~o use BUT FIRST RE-SUSPEND, IMMEDIATELY PRIOR TO USE.
' No.7 Wash Solution ~20x concentrate) 1 vial (13.2ml)
(::ontains: Tris buffered sallne
To be made up wi~h ~50ml of distilled water.
: ~ :
'
~U5$TITUTE SH~El~
W092/19775 ~ 3 1 3 pcr/~P92/~0929
13
No.8 Substrate Solu~ion 2 vials (15.4ml each)
Contains: Phenolph~halein monophospha~e in ~riethanolamine buffer.
Ready to use. DO NOT EXPOSE TO DIRECT SUNLIGHT.
No.3 Stop Soiution 1 bottle (115ml)
Contains: A sodium carbonale/hydroxide solution pH~ 12.
Ready ~o use. CAUTION: (i;AUSTlC MATERIAL
No.10t(14;18) Transtocation Positive PCR Controi 1 vial 160~
Contains: DNA extracted from two cell lines carrying respectively the MBR and the mcr
t~14;18~ translocation. in TE buffer.
Ready ~o use.
No. 1 ~ Negative PCR Control 1 vial (60~
Contains: DNA extracted from a cell line NOT bearing the t(14;18) translocation, in
TE buffer.
Ready to use.
No.12t(14;18) Transloca~ion Positive De~eclion Control 1 vial (480~LI)Contains: Both MBR and mcr arnplified sequences, in TE buffer.
Ready to use
No.13 Negative De~ection Con1rol 1 vial (480~
Contains: DNA exlracted from a ~ cell line NOT bearing the t(14;18) lranslocation
translocation, but subjected to "bc1-2" PCR amplificalion, in TE buffer.
Readytouse.
No.14Dis~illed Water 1 vial (3ml)
Con~ains: HPLC grade, dis~illed water.
Ready to use.
.
SUBSTITUTE 51~EET
WO92~19775 ~ . . . PCT/EP92/00929
1 3 14
Further Reaqents
A. Perkin-Elmer Cetus AmpliTaq DNA Polymerase
B. Perkin-Elmer Cetus lOx Amplification Buffer
C. Per~in-Elmer Cetus MgC12 Solution
D. Deoxynucleotide Triphosphates
Contains: 25mM solutions of dATP, dCTP, dGTP and dTTP
To be diluted 1:20 with distilled water ~reagent E)
,
,.-~
.
~ ~ '
,
: :
SlJBSTlTUTE SI~EET
... . ... .. .... . . . . . . . . .
WO 92/1 977S ~ 1 C 3 J 1 8 PCI /EP92/00929
fica~ion of the bc!-2,translocated ~I\IA sequence by the Polymera~e Chain
Rea~ion (P~R).
1. Reconstitute Ihe PCR primers (reagent No. 1) with 300~1 of distilled water
(reagent No. 14), mix vor!ex for several minutes a,nd spin in a microfuge.
Store at -20 C after reconstitu~ion.
2. DiJute dNTPs (reagenl D) 1:20 with distilled water (reagent E) by transferring the
contents of the tube (110~11) inlo a vial in which 2.09ml of distilled wa~er have
been pipetted. Aliquot and store at -20 C.
3. Into an autoclaved tube suitable for a PCR thermai cycler, pipette:
-46.5,ul of distilled water (reagsnt No. 14)
-10.0111 of PCR primers (reagent No. 1)
-10.0111 of 10x Amplification buffer ~reagen~ B)
-7.0',11 of MgCI2 solution (reagent C)
-16.O~LI of dNTPs solution (reagent D)
-0.5111 of Amplitaq (reagent A) - equivalent to 2.5 units of enzyme
-10.0~,11 of sample; 1 llg of total DNA
100.0 ~11 final volume
Layer on the top of the solution 50~L1 of mineral oil, spin in a microfuge and start the
thermal cycles.
We suggest the following procedure:
i) prepare a 'master mix' (at 0-4 C in a 'protected' environment) of all the
reagents necessary for the PCR reaction excepl the DNA sample. Sufficien~ 'master
mix' should be prepare~ for each of the samples plus the positive and negative PCR
controls (plus 5% excess).
ii) pipette the 'master mix' into the PCR tubes at 4 C.
iii) pipette the DNA samples into their respective PC~ tubes.
iv) add the mineral oil.
v) spin in a microfuge.
vi) quickly start 1he thermal cycles. f~,
~he recommended pr~otocol for the Perkin-Elmer Ihermocycler 9600 includes the
use of:
0.5mm thin-walled vials
1 OO,ul reaction volume/ 50111 minerai oil
time constant ot 12.5 (100~1)
and the following instrumerlt set-up parameters:
No.of cycles Denaturation Annealing Extension
T(C) tirre(s) T(C~ time(s) T (C) time(s)
95.0 120 56.5 3 0 72 .0 3 0
6 95.0 30 ~6.0~ 30 72 . 0 20
3 0 94.0 30 53.0 3 0 72.0 60~
94.0 30 53.0 30 72.0 300
stop and hold at 4 C
set lempera1ure de~r~a~ = 0.6 degreeslcycle
set time ln~ - 1 second/cycle
- ~3UB~iTlTUTE SH~:ET
WO 9~/19775 2 1 ~ ~ 5 1 8 16 pcr/Eps2/oo9~9
For other PCR inslrumen!s which make use of different vials and different
hea~ing/cooling devices, we sugges~ the foilowing set-up parameters. (NB Each user
should optimize these parameters for the instrument they are using).
No. of cy~les Denaturation Annealing Extension
T(C) time(min) T(~C) time(min) T(C) time(min)
94.0 5 53.0 2 7Z.0 3
9~.0 1 53.0 ~ 72.0 3
3 0 ~ 92.0 1 53.0 2 72.0 3
92.0 1 53.0 2 72.0 10
stop and hold at ~ C
Detection ~f the am~lifiedDN~ se~uence
Before use, bring all the reagents to room temperature, gently but thoroughly mixing thern
using a rolling or orbital mixer, or equivalent device. (Take them out af the refrigerator at
least half an hour before use.)
Do no1 expose to direct sunlight.
I~o not expose to direct heat sources.
NB The detection of the MBR and mcr must be carried out in two separate experiments.
1. Add the entire conlents of the Wash Solution - 20x concentrate (reagent No.7) to
250ml of dislilled water and mix well.
2. Make sure the PCR sample is clear, if not then vortex mix and spin in a microfuge.
3 a. To detect the MBR, dilute sach ~PCR reaction mixture 1:10 wilh Sample Diluenl
(reagent No.2), using at least 20~1 of sampte, vortex mix and spin in a microfuge.
3b. To detect the mcr, dilut~ ~ach PCR reaclion mixtur~ 1:4 with Sample Diluent
reagent No.2), using a~ least 20~11 of sample, vo~ex rnix and spin in a rnicrofuge.
4. Take the t~be rack out of the magnetic separator. Place in 1he rack Iwo r~action tubes
for each of the diluled PCR samples and for both the Posilive Detection Control
(System reagent No. 12) and the Negative Detection Control (reagenl No.13). Label
the tubes appropria1ely.
5~ Pipettè in duplicate 20~11 of each sample and control into their respective tubes,
making sure tha~ you pipetlc into the bottom of the tube.
6. Using ~ multipipetle, dispense into each tube 20 111 of Denaturing Solution
(reagent No, 3).
~Yol~e: The addition of the l:)enaturlng Solution as all tubes should ~e completed within
3 minutes.
7 . ~hake the rack manuaily,~using a side-to-side motion for some seconds, making sure
the samples come into contact with the Denaturing Solution.
8~ !ncubate the rack of tubes in a waterbath at 37 C for 10 minutes.
9. Usin~ ~ multipette, dispense 200111 of either MB:R Detection Probes
(rea~ent No.4) or mcr l:)eteclion Probes (reagent No. 5), into each tub~. -
10. ~ Shake 1he rack manually, using a side-to-side motion for some seconds.
11. Inoubate the rack of tubes in a waterbath at 37 C for eil~er 3V minu~es when
delecting the MBR Qr for 15 minules when detec1ing lhe mcr.
-
~;UBSTITIllTE S~ T
WO 92/19775 17 2 1 0 c~ 8 PCI/EP92/00929
12. Disp~nse 0.1 ml of thoroughly mixed Separation Reagent (reagent No. 6) intoeach tube.
Notes:
- do not use a magnetic stirrer to mix the Separation Reagent.
- the magnetic antibody suspension must be thoroughly mixed before use to ensure a
uniform suspension of magnetic particles. After pipetting into 10 to 20 tubes swirl
the viai.
- the addition of the Separation Reagent to all tl~bes should be completed within 3
minutes.
13. Cover the tubes. Gently vortex mix the rack using a rnulti-vortex. Alternatively,
shake the entire rack using a side-~o-side mo~ion.
Note: gentle but complete and sirnultaneous mixing is critical to assure good assay
performance.
14. Incubate the rack of tubes in a waterbath at 37 C for 10 minules.
15. Slide lhe rack of tubes into 1he magnetic separator and allow magnetic sedimentation
to occur for 4 minutes, making sure all the tubes are in contact with the surface of
the separator.
16. Decant Ihe supernatant from all the tubes by inverting ~he separator in one large,
slow, circular movement. Place Ihe inverted separator on absorbent paper in a tray
and hit the base of the separator firmly several times to dislodge any droplets of
liquid adhering to the sides of the tubes.
Notes:
- a loss of magnelic black particles indicates incorr~ct decanging teohnique.
- try to avoid excessive splashing in order to minimiza "amplicon~ aerosol
formation.
- ~lean up the area thoroughly immediateiy after use with 0.5% bleach.
- discard lhe absorbent paper in a sealed bag.
- do not touch the rim of the tubes with hands/pipettes.
- be aware that in lhis phase of the procedure large amounts of amplified sequences
may be present in the reaction tubes which may give rise to serious oontamination
problems if adequate precautions are not taken.
17. Place the separator upright and add O.5ml of already diluted Wash Solution
(reagent No.7) to each tube.
18. Remove the rack from the separator. Place in a multi-vortex mixer. Vortex
~igorously - thorough mixing is essential to ensure good assay per~rmance.
19. Slide the rack of tubes into the magne~ic separator. Check lo see that all lubes are in
cQnlact with the surface of the separator. Wait ~or 3 minutes 10.allow particles to
sediment magnetically.
20. Decanf ~he supernatant from all 1he tubes as in Step t~.
21. Repeat Steps 16 to 19 twice (three washing steps in total).
Note: at the end of the magnetic separation step, complete draining of all the tubes is
vital to avoid an increase in background signal.
22. Label two lubes for ~'blankingU the spectrophotometer and place them in the rack.
23. Remove the rack frsm the separalor and pipette 0.2ml of Substrate Solution
(reagent No. 8) in10 each tube, including the blank tubes.
Note: the addition of the Subslrate Solution ~o all tubes should be compleled within 5
minutes.
24. CoYer the rack with plastic filrr. Thoroughly mix all the tubes using a side-to-side
motion.(Note: discard the plastic film with grea! care as it will be heavily
contaminated with amplicons.)
25. Inc~bate the raek in a waterbath at 37 C for 60 minutes.
51JBSTITUTE SHEET
WO 92/1977S PCl~/EP92/OOg29
~1~3~18 1~
26. Pipe~te 0.75ml of Slop Solution (reagent No.9) inlo each tube, including the blank tubes.
Note: it is critical tc add StQP Solution at approximately the same rate and in the
same sequence, as when adding the Substrate SoluRon.
27. Slide the tack into 1he magnetic separalor and allow the particles to sediment
magnetically for al least 5 minutes.
28. Blank the spectrophotometer at 550nm using Ihe blank tubes and th0n measure the
absorbances (A) for samples and controls.
Note: Samples fa,r which the absorbance exceeds the upper limit of the
spectrophotometer should be read at 492nm. A550 is approximately equal to 5 x A492,
though the precise relationship should be determined for each instrument.
:
::: : :
:::: :
,
`:
:
SlJE~STITlJTE SHEET
;~ ::.
:
WO 92/19775 2 1 a ~ Pcr/EP92/oo929
lnterpretatiQn of results
The resulls of the assay are indicated by the absorbance values. Samples which yield an A550
which is significanlly higher than the PCR Negative Control should be scored as positive.
ie. (AX550 - 3 S.D.~ > (AC560 + 3 S-D-)
where:
x = Sample
c= Negative PCR control
S.D.= Standard Deviation
Expected CV (co-efficient of varia~ion~ value for the Negative PCR Controls is
approximately 15%, where CV= S.D./As5O
As a check on the crucial issue of PCR carry-over contamination and false posi~ive tesults,
the A5so of the Negative PCR Control should not be significantly different from ~he A550 of
the Negative Detection Control. tf Ihe A550 of the Negative PCR Control does significantly
exceed the As5o of the Negative Detection Control ~i.e. (AC550 3 S.D.) > ~Ad550 + 3 S.Z:1.),
where d denotes the Negative Detection Con~rol3, Ihen the results of the whole t~s~ run
should be disregarded and actions implemented to avoid further PCR carry-over.
To confirm that both Ihe PCR amplificalion and the detec~ion procedure have been performed
correctly, both the PCR PosiliYe control and lhe Deteclion Positive Control must yield A550
values withiri the range indicaled in ~he ht-specific data sheet provided with each kit.
: :
Sensitivity
In our laboratories we hav~ been able to detect the presence of 1 translocated cell in 50l000
cells. A negative result in the translocation assay could occur simply as a r~sult of yery low
concentrations of translocated cells in the sample. Additionally, when investigating
potentiaily low level occlJrrence of the t(14;18) translocation, statistical sampling methods
should be emp!oyed.
` ~ ~ Precision
.
Intra-assay precision sf the detr~t~ s~ep was determined by measuring A~so replicates of
the same PGR amplified sampies and resulted in an~average C\/ of 8-1Q%.
~ ~ : : . :
Inter-assay precision of the delecliQn step (delermined as above~ gave a CV of 1Q-1~%.
; ~:
:
~:
: :
~ : :
WO92/1g775 PCTJEP92/009~9
SE~UENCE LISTING
EQ ID No. 1
Sequence Type: Oligonucleotide
Sequence Length: 24 bases
Strandedness: Single
Topology: Linear
TGA CCT TTA GAG AGT TGC TTT ACG
~Q ID No. 2
Sequence Type: Oligonucleotide
Sequence Length: 21 bases
Strandedness: Single
Topology: Linear
GAT GGC TTT GCT GAG AGG TAT
S~Q ID No. 3
Sequence Type: Olig~nucleotide
Sequence Length: 2 a bases ~-~
Strandedness: Single
Topology: Linear
ACC l'GA GGA GAC GGT GAC CA
S~Q ID No. 4
5equence Type: Oligonucleotide
Sequence Length: 27:bases
Strandedness: Single
Topology: Linear
NH2-AAT ACT TCC AGC ACT GGG GCC AGG GCA-NH2
::
- SllBSTJT~ SH~:E'r
~ r..~t~
W092/t9775 ~ 1 ~}~ ¦ ~ PCT/EP92/00929
21
SEQ I~ NoO 5
Sequence Type: Oligonucleotide
Sequence Length: 27 bases
Strandedness: Single
Topology: Linear
NH2-GGT ACT TCG ATC TCT GGG GCC GTG GCA-NH2
SEQ ID No. 6
Sequence Type: Oligonucleotide
Sequence Length: 27 bases
Strandedness: Single
Topology: Linear
NH2-ATG CTT TTG ATG TCT GGG GCC AAG GGA-NH2
SEQ ID ~o. 7
Sequence Type: Oligonucleotide
Sequence Length: 27 bases
Strandedness: Single ~.
Topology: Linear
NH2-ACT ACT TTG ACT ACT GGG GCC AAG GAA-NH2
8EQ ID No. 8
Sequence Type: Oligonucleo~ide
Sequence Length: 27 bases
Strandedness: Single
: Topology: ~inear
NH2 ACT GGT TCG ACT CCT GGG GCC AAG GAA-~H2
SU 135TITUTE SHE~:T
.
WO92/19775 PCTJEP92/009~9
2 1r~9 ~ 1 ~ 22
SEQ ID No. 9
Sequence Type: Oligonucleotide
Sequence Length: 27 bases
Strandedness: Single
Topology: ~inear
NH2-ACG GTA TGG ACG TCT GGG GGC AAG GGA-NH2
~EQ ID NoO lO
Sequence Type: Oligonucleotide
Sequence Length: 27 bases
Strandedness: Single
Topology: Linear
NH2-TTT CAA CAC AGA CCC ACC CAG AGC CCT-NH2
~EQ ID No~ ll
Sequence Type: Oligonucleotide
Sequence Length: 25 bases
Strandedness: Single ~.,
Topology: Linear
NH~-CGC TCr TGT TGA CTG GCT GGC TTA G-NH~
SU13ST~TlJTE SHEE:T
