Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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QUANTIFICATION OF NUCLEIC ACID MOLECULES AND THE REAGENT KIT
USED
The invention relates to the quantification of certain
nucleic acid molecules, particularly the degree of
amplification of genes and/or corresponding messenger RNA
molecules using the sandwich or solution hybridization
method, and the reagent kit used.
The number of copies of individual genes in the genome is
usually constant. In some instances there is only one gene
per haploid genome and in others several. Under certain
circumstances the number of copies may change. The
amplification of certain genes has for example been found to
be associated with the development of cancer. It is also
known that external factors such as pharmaceuticals and
metals etc. cause certain genes to be amplified. For the
development of a disease, the faulty or enhanced expression
level of a gene, such as an oncogene, i.e. the quantity of
messenger RNA in the cell, is of major importance. Increased
numbèrs of some chromosomes is the cause of certain
hereditary diseases or other disturbances, whereas some
hereditary di~eases only require duplication of one recessive
gene. In all such instances it is important to determine the
number of chromosomes or genes present. -
The number of certain~DNA molecules, for example the degree
of amplification of given genes, is currently determined by
digesting the extracted DNA to be studied by means of
restrict~ion enzymes and by separating the nucleotide
fragments according to length by agarose gel electrophoresis.
Subsequently the single-stranded DNA is transferred and
affixed to a nitrocellulose filter, where hybridization
takes place using the gene to be studied or part of the gene
as a probe. The results are obtained by autoradiography
(Southern, J. Mol. Biol. 98, pp. 503-517, 1975). In each
parallel~analysis, the quantity of cellular DNA is the same.
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The intensities of the hybridization ba~ds, i.e. the signals a~e
compared and the ratios between the copy numbers of the genes
under study in the test samples are deduced. The method only
yields approximate results. Likewise, RNA is measured using
Northern-blotting or dot-blotting methods. These methods are
quantitatively very inaccurate (Thomas, Methods in Enzymol.,
100, pp. 255-266, 1983).
Known methods, such as the Southern and Northern blotting
methods, are slow and difficult to perform. Since they only
yield approximate results their diagnostic value is doubtful
in cases in which it is important to know the number of
certain nucleic acid molecules per given unit, such as a
cell.
The sandwich or solution hybridization methods, described in
US Patent No. 4,486,539 and in the sri~ish Patent ~pplication
No. GB 2 169 403 are quantitative (Virtanen et al., Lancet 1,
pp. 381-383, 1983). In addition, the method of the present
invention requires a standard nucleic acid, the copy number
of which is constant and enables the determination of the
number of relevant nucleic acid molecules per given unit such
as a cell, nucleus, ribosome or chromosome.
The purpose of this invention is to produce an accurate and
rapid quantitative method of nucleic acid molecule
determination which is also faster and simpler to perform
than those currently used. It can be used for cancer and
prenatal diagnostics, for detecting agents which cause gene
amplification and for demonstrating the development of e.g.
drug resistance as well as for the determination of the
expression level of messenger RNA.
At least two determinations are required in the present
invention. One determines the nucleic acid molecule, which
; may be present in several copies, the test nucleic acid. The -
other determines the constitutive nucleic acid molecule
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advantageously present in constant number, the standard
nucleic acid. In the method according to the invention, a
nucleic acid molecule denotes a certain nucleotide sequence
of 10 - 12 nucleotides or a gene containing several thousand
nucleotides. It can also mean a messenger RNA or a
nucleotide sequence eonsiderably longer than a single gene,
i.e. an amplicone.
The determination of test and standard nucleic acids is done
using an otherwise normal sandwich hybridization method
described, for example, in US Patent No. 4,486,539 or a
solution hybridization method described in British Patent
Application No. GB 2 169 403. The invention also relates to a
reagent kit containing nucleic acid reagents consisting of at
least one test probe pair and at least one standard probe
pair.
The reagents, or probes, used in the method, are prepared,
using recombinant-DNA techniques, from nucleic acids
sufficiently homologous to the test and standard nucleic
acids. Sufficiently homologous nucleic acids can also be
prepared synthetiFally and semisynthetically.
The test and standard nucleic acids may be isolated directly
from ceIls and iden~ified by various hybridization
techniques. Such test and standard nucleic acids are however
also available commercially and from various gene banks.
~est and standard nucleic acids may be either DNA or ~NA.
Probe pairs suitable for the sandwich or solution
hybridization method are prepared from nucleic acids
sufficiently homologous to the test and standard nucleic -
acids by recombinant-DNA techniques. The relevant nucleic
acids are~digested by suitable restriction enzymes; at least
two~of the resulting restriction fragments situated
relatively close together are cloned to at least two suitable
vectors. One of thè fragments, the detector probe, is
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labelled with a suitable detectable label and the other, the
capturing probe, is either affixed to a suitable carrier or
an substance is affixed to it, which substance enables
separation of the resulting hybrid from the hybridization
mixture by means cf another substance, such as the
complementary component of an affinity pair.
The test and standard probe pairs can be assembled into
suitable reagent kits wherein the test and standard probe
pairs are both DNA or ~NA, or the test probe pair is DNA and
the standard probe pair RNA or vice versa. The pre- and
further treatment of samples prior to hybridization and the
hybridization conditions should therefore comply with the
probe pairs used in the test.
The method of the present invention is particularly suitable
for determining the number of nucleic acid molecules directly
from cellular homogenates. The method may of course also be
used for the determination of purified or pure nucleic acids.
However, before carrying out the method of the invention, the
most suitable pretreatment of the nucleic acid sample should
be selected.
It is possible to carry out both DNA and RNA determinations
using the method of the invention. Deoxyribonucleic acids
are denatured to obtain single strands if necessary.
Single-stranded messenger RNA molecules potentially
disturbing the hybridization test can be hydrolyzed, for
example by alkaline boiling. The sample is not denatured in
connection with ribonucleic acid determinations since the
double-stranded deoxyribonucleic acid does not interfere with
RNA determination. It is of course possible to disrupt the
DNA with deoxyribonuclease or alter it either chemically
or mechanically so that it cannot participate in the
hybridization reaction. Therefore in connection with DNA and
RNA determinations a suitable method for further treatment of
the sample must be selected or, alternatively, this further
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treatment m~y be omitted. ~he choice of a suitable method
for the further treatment is of course dependent on the
method used for the preliminary treatment of the nucleic acid
sample. Numerous methods of pre- and further treatment of
nucleic acid samples have been described in the literature,
enabling the most suitable method to bs chosen in each case.
Determinations in which both the test and standard nucleic
acids are either DNA or RNA can be performed using an
undivided sample. Determinations in which the test nucleic
acids are DNA and the standard nucleic acids RNA or vice
versa must be performed using a divided sample, as different
methods for further treatment are necessary. The sample may
of course be divided even if the test and standard nucleic
acids are of the same nucleic acid type.
The hybridization test itself is performed by bringing the
undivided sample solution into contact simultaneously with at
least one test probe pair and one standard probe pair. If
the sample solution has been divided it is brought separately
into contact with at least one test probe pair and one
standard probe pair. In such instances, the quantity of test
nucleic acid is determined in one reaction vessel and the
quantity of the standard nucleic acid in the other.
Regardless of whether the sample is divided or not,
hybridization is allowed to take place in the most
advantageous conditions and time in each case. Once the
reaction(s) has/have taken place, the resulting test and
standard hybrids are separated from the hybridization
mixture(s) by~the carrier and washed, or by an isolation
agent such as the complementary member of an affinity pair.
The label attached to the test and standard hybrids is
measured and the result compared with standard curves. In
this way the number of nucleic acid molecules to be studied
can be determined per selected unit.
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The method of the invention is of practical diagnostic value,
particularly in the detection of some types of cancer. In
small cell lung carcinoma, the c-myc gene is often amplified
and its level of expression considerably higher than in
normal t~ssue. In cases of neuroblastoma the N-myc gene is
amplified.
The method of the present invention can also be used for
demonstrating the mutagenic or carcinogenic effects of
certain agents or the development of drug resistance. It is
known that external pressure of selection can result in
enhanced expression of a certain gene. In the treatment of
cancer, cells develop resistance to a given drug by
amplification of the gene, the expression product of which
inactivates the drug. One such case is methotrexate which
induces amplification of the gene for dihydrofola~e reductase
tDHFR). A further example is amplification of the gene for
metallothionine under the influence of cadmium.
The expression level of a gene is important from the point of
view of the phenotype and function of the cell. This can be
investigated by measuring the quantity of messenger RNA which
correlates to the quantity of protein coded by it. The
transcriptian product of an oncogene determines the way in
which it will ultimately be expressed.
The express1on levels of an oncogene vary depending on the
cell type, differentiation level and phase of development of
the cell. For example, at a certain stage of fetal
development, the c~myc oncogene is copied rapidly, whereas at
another stage this is very slow. The degree of amplification
often correlates with the level of expression of the gene,
although the latter may significantly increase without the
former.~ In such instances the role of an oncogene is best
determined by measuring its level of expression rather than
the number~of copies. In some instances, quantitative
determination of the messenger RNA may be simpler and handier
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than quantification of the gene product itself. As an
example the c-myc oncogene, a labile protein readily
coagulated by heat, can be mentioned.
The method of the invention can also be used for identifying
numerical chromosomal abnormalities such as Down's syndrome.
In prenatal diagnostics it is also possible to determine
whether the fetus is defective, i.e. homozygous for some
recessive gene.
The method of ~he invention and the nucleic acid reagents
used in the method are described in greater detail below.
EXAMPLE 1
Quantification of an amplified oncogene
a) Nucleic acid rea~ents and_their preparation
STANDARD PROBES
,
Cell standard nucleic acid. The c-Ki-rasI gene is present in
all human cells. The probe pairs for sandwich hybridization
were prepared by subcloning the HindIII fragment of the
c-Ki-rasl gene, measurin~ 3.8 kb in length, the restriction
map of which~has been described by Chang et al., PNAS 79,
pp. 4848-52, 1982. The fragment is available e.g. cloned
into the pBR322 plasmid (ATCC 41032) and can be obtained e.g.
from the ATCC culture collection.
:
Further treatment of the cell standard nucleic acid. The
pBR322 clone described above was treated with sglII and
HindIII restriction enzymes and the resulting fragments were
isolated from the agarose gel; purified fragments located
clo~se ~ogether were su~cloned into two suitable vectors for
p~cparation of the detector and capturing probes.
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~287S58
Standard detector probe. A BglII-BglII fragment measuring
about 1.1 kb in length was subcloned into the ~amHI
restriction enzyme site of the pBR322 plasmid and labelled by
nick-translation with 125I-labelled dCTP.
Standard capturing ~robe. The sglII-HindIII ~ragment of
about 0.5 kb was inserted into the M13 mplO and mpll phage
fvectors between the restriction sites of the BamHI and
HindIII restriction enzymes and affixed to a nitrocellulose
filter (150 ng DNA/dia 1 cm).
TEST PROBES
Test nucleic acid. A probe pair for sandwich hybridization
was prepared from a cloned c-myc gene which can be obtained
for example, from the ATCC culture collection (ATCC 41010).
The restriction map of gene has been described by Watt et
al., PNAS 80, pp. 6307-6311, 1983.
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Further treatment of the test nucleic acid. The c-myc gene
.. . . ..
was treated with HindIII, XbaI and PstI restriction enzymes ;~
and the fragments isolated from the agarose gel, purified and
subcloned into suitable vectors in order to prepare the
detector and capturing probes.
Test detector probe. The single~stranded tails of the
HindIII-XbaI restriction fragment of the c-myc gene,
measuring 3.7 kb in length, were rendered double-stranded by
DNA polymerase. The HindIII linkers were inserted by
T4-DNA-ligase into the resulting blunt-end DNA fragments;
after phenol extraction the DNA was treated with the HindIII
restriction enzyme. The DNA fragment was subsequently cloned
into the pBR322 plasmid at the restriction site of the
HindIII restriction enzyme and labelled by nick-translation
with 125I-labelled dCTP.
Test capturin~ probe. The 1.1 kb XbaI-PstI fragment of the
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c-myc gene was cloned into the M13 mplO and mpll phage
cloning vectors between the restriction sites of the XbaI and
PstI restriction enzymes and affixed to the nitrocellulose
ilter (150 ng DNA/dia 1 cm).
b~ Determination of the standard curve
The sample used for determination of the standard curve
consisted of an alkaline-denatured pBR322 clone of the c-myc
gene. The sandwich hybridization solution to which the above
test probes were added consisted of 4 x SSC, 1 x Denhardt
solution, 200 ~g/ml herring sperm DNA and 0.2 % SDS.
Hybridization took place at 6SC for 17 - l9 hours,
whereafter the filters were washed in the wash solution (0.1
x SSC O . 2 % SDS ) at 50C. The label attached to the sandwich
hybrids was then counted in the gamma counter.
Table 1
Sample cpm
molecules/test c-myc-filter
0 40
1o6 75
5 x 106 190
107 3~0
8 2200
c) Determination of the number of genes
The samples comprised 1) cells from a human placenta and 2)
Colo 320 cells, which can be obtained e.g. from the ATCC
culture collection (ATCC-CCC220). DNA was isolated from both
samples, and the same quantity of cell DNA, denatured by
alkaline boiling, was added to both tests. Alkaline
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denaturation hydrolyzed any RNA present in the sample.
The test was performed by adding to each sample both the
c-myc and c-Ki~rasI filters and the two labelled reagents,
enabling both the standard and test DNA to be measured for
each sample. On the basis of c-Ki-ras~ determinations, each
test was found to contain the same quantity of DNA and it can
be deduced that the c-myc gene in Colo 320 cells is present
in about 16 - 20 higher copy number than in the normal
situation. The results are shown in Table 2.
Table 2
~ Sample c-Ki-rasI filter c-myc filter
- cpm* cpm* number
Human placental cells 486 340 107
Colo 320 cells 432 3205 1.6 x 108
-
*the reading obtained from the blank filter has been
subtracted from the readings.
EXAMPLE 2
Quantification of amplified gene
a) Nucleic acid reagents and_their preparation
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STANDARD PROBES
Cell standard nucleic acid. The control nucleic acid was
taken from the promoter area of the metallothionine gene in
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the mouse, i.e. the MT gene, and the DNA immediately upstream
of it. The structure of the MT gene has been described by
Pavlakis and Hamer, PN~S 80, pp. 3g7-401, 1983. The
reference nucleic acid fragment is available e.g. cloned into
the p~PV-MMTheo(432-12) vector ~ATCC 37224) and can be
obtained, for example, from the ATCC culture collection.
Further treatment of cell standard nucleic acid. The MT gene
described above was treated with KpnI, BglII and EcoRI
restriction enzymes for subcloning into the pAT153 plasmid.
The KpnI tail was converted into a HindIII tail with a
linker.
Standard detector probe. The EcoRI-KpnI-(HindIII)
fragment measuring about 1.2 kb and located upstream of the
promoter area of metallothionine gene was cloned to the
pAT153 plasmid between the restriction sites of the EcoRI
and HindIII restriction enzymes and labelled by
nick-translation with 32P-labelled nucleoside triphosphates.
Standard capturing probe. The 0.8 kb KpnI-~glII fragment
comprising the promoter area of the metallothionine gene and
the area upstream of it was cloned into the M13 mpl8 and M13
mpl9 phage vectors between the restriction sites of the RpnI
and BamHI restriction enzymes and affixed to the
nitrocellulose ilter.
TEST PROBES
Test nucleic acid. The probe pair for the sandwich
hybridization test was prepared using the commercially
available pMTVdhfr plasmid (Bethesda Research Laboratories,
product~No. 5369SS), the structure of which is described by
Lee et al., Nature 294, pp. 228-232, 1981.
Further treatment of test nucleic acid. The pMTVdhfr plasmid
containing cDNA of the dihydrofolate reductase (DHFR) gene
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was treated with HindIII and 8glII restriction enzymes.
Test detector probe. The Hind~ sglII fragment, measuring
0.75 kb and corresponding to the area coding for the DHFR
gene of the pMTVdhfr plasmid, was inserted into the plasmid
pAT153 vector between the restriction sites of the HindIII
and ~amHI restriction enzymes and labelled by nick-
translation with 32P-labelled nucleoside triphosphates.
Test capturing probe. A HindIII fragment measuring 1.4 kb
taken from the MMTV gene area of the pMTVdhfr plasmid was
cloned into the M13 mpl8 and M13 mpl9 phage vectors.
b) Determination of the standard curve
. . . _ . . . _ _
The sample used for the test was purified DNA from the
pMTVdhfr plasmid. The test itself was carried out as
described in Example lb except that a liquid scintillation
counter was used for counting. The resulting standard curve
is shown in Table 3.
Table 3
Sample cpm
molecules/test DHFH filter
0 17
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3 x 106 79
107 210
c) Determination of the number of genes
Cell lines derived from the mouse fibroblast cell NIH 3T3, available
from the ATCC culture collection under the number CRL 1658, were
transfected with different quantities of cDNA corresponding to the mRNA
of the DHFR gene, and
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13
were cultured on cell culture plates and used as the sample.
The cells were lysed using sodium dodecyl sulphate and their
DNA was sheared by squeezing through a fine hypodermic needle
from a syringe. A 250 ~l sample corresponding to about 10
cells was taken from the homogenate and 50 ~1 NaOH added.
The sample was boiled and neutralized with acetic acid and
the hybridization mixture. The total volume was 0.5 ml. All
the probes described above were added simultaneously and a
so-called blank filter was added as a background control.
Hybridization, washing and label counting were done as in
Example lb except that a liquid scintillation counter was
used for counting. The results are shown in Table 4.
Table 4
Cell MT No.of cells DHFR
cpm* in the sample cpm* No. of molecules No. of
in the sample copies
Control cell
(No DHFR-cDNA) 1821.05 x 106 21 < 106
Line I 138 0.9 x 106 803 x 106 3
Line II 210 1.25 x 106 7325 x 107 40
* cpm: the reading given by the blank filter has been subtracted
The MT gene is an internal marker which measures the number
of cells present in a sample. The results show that in this
test 106 cells gavean MT-specific signal of 165 + 20 cpm. The
DHFR reagents measure the quantity of DHFR-CDN~. The number
of celIs was deduced from the MT-specific signal. It was thus
possible to determine the number of DHFR-cDNA copies in
different cell lines as shown in Table 4.
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EXAMPLE 3
Quantification of messenger RNA
a) Nucleic acid reagents and their preparation
Using the test probes described in Example 2 it is also
possible to measure the quantity of mRNA derived from
DHFR-cDNA. The structure of the pMTVdhfr plasmid is such
that transcription of the DHFR gene begins at the MMTV
promoter. The resulting messengers are about 1.0 kb in
length. Of this, about 0.25 kb are derived ~rom the M~TV
promoter area and the rest from DHFR-cDNA (Lee et al., Nature
294, pp. 228-232, 1981).
:
STANDARD PROBES
The cell standard nucleic acid, standard detector and
standard capturing probe were as described in Example 2.
TEST PROBES `
The test nucleic acid, test detector and test capturing probe
were as described in Example 2.
~ -
b) Determination of the s~andard curve
The sample used for standard curve determination consisted of
messenger RNA~corresponding to the dihydrofolate reductase
gene produced by in vitro transcription. The DNA needed for
transcription was~prepared by subcloning the 1.4 kb HindIII
fragment of the MMTV~promoter of the pMTVdhfr plasmid and the
0,75~kb HindIII-BglII fragment (D~IFR-cDNA) next to each other ;
into the pSP64 plasmid (Promega Biotec) between the
restriction sites of the HindIII and BamHI restriction
enzymes. The sample RNA was stored in 0~2 % SDS aque~ous
solution.
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~he sandwich hybridization test was carried out as described
in Examples lb and 2b but denaturation was omitted.
Table 5
Sample cpm
molecules/test DHFR filter
0 20
5 x 106 ~5
107 130
5 x 107 390
lo8 653
-----
c) Determination of the number of messenger RNA molecules
The number of messen~er RNA molecules corresponding to the
DHFR gene was determined from the cell lines described in
Example 2.
The cells were lysed using sodium dodecyl sulphate and their
DN~ was sheared slightly by squeezing through a fine
hypodermic~needle from a syringe. A 250 ~l sample of the
homogenate was taken corresponding to about 5 x 106 cells
The~homogenate was then added to the sandwich hybridization
test without denaturation. Sandwich hybridization took place
as desc~ibed in Examples 2c and lb, except that only the DHFR
probes were added ~o the hybridization solution. In a
parallel sample of 250 ~l of homogenate, the cell number was
determined usinq~the MT probe as described in Example 2c.
:
The results are shown in Table 6. ;
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Table 6
Cell MT Cell number DHFR
cpm* in the sample cpm* No of molecules No. per
in the sample cell
Line I 380 3.5 x 106 1465 3.45 x 108 100
Line II 430 4.2 x 10 4800 2 x 10 500
*cmp: The reading given by the blank filter has been
subtracted.
The results showed that cell line I produced per cell about
100 messenger RNA molecules from the DHF~ genes and cell line
II produced about SOO messenger RNA molecules from the DHFR
genes.
EXAMPLE 4
Quantiflcation of amplified gene by solution hybridization
,,
; a)~Nucle~lc_a-cid reagents and their preparation
STANDARD PROBES
The cell 6t6ndard~nucleic~acld,~ standard detector and
standard~capturing probe were as described in Example 2. The
1.~2~kb EcoRI-KpnI-(HindrII) fragment in pAT153 was labelled
by nick-translation with 125I-labelled deoxycytidine. The 0.8
kb-KpnI-BglII fragments in Ml3 mpl8~and M13 mpl9 were
~modified~;-with~blotln~u6ing the PhotoprobeTM reagent ~Vector
Labo~ratorle~6, CA~, USA, product No SP-1000).
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TEST PROBES
The test nucleic acid, test detector and test capturing probe
were as described in Example 2. The 0.75 kb HindlII-BgllX
fragment in pAT153 was labelled with 125I-labelled deoxy-
cytidine. The 1.4 kb HindIII fragments in M13 mpl8 and M13
mpl9 were biotinylated using PhotoprobeTM as above.
b) Determination of the standard curves
A cell standard curve was prepared using a known amount of
cells, from which the hybridization signal was measured using
the standad probes recognizing the MT-gene. A test nucleic
acid standard curve was prepared with the pMTVdhfr plasmid
and the test probes recognizing this plasmid. Hybridizations
were carried out in 200 ~l of a solution consi~ting of 0.6 M
NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, 4 %
polyethylene glycol (PEG 6000) for 1.5 hours at 70C. After
the reaction 50 ~l of streptavidin-agarose (Betheseda
Research Laboratories, Maryland, USA, product No. 5942SAJ,
and 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA was
added~to a final volume of 500 ~l. The hybrids were collected
on the streptavidin-agarose at 37C for 15 min. The agarose
was washed once for 5 min. with the buffered 1 M NaCl
solution at 37C and twice for 2 min. with 15 mM NaCl, 1.5 mM
sodium~citrate at 55C. The amount of bound hybrids was
determined by measuring the agarose in a gamma counter.
(Syvanen~et aI., Nucleic ~cids Res. 14, 5037-5048, 1986).
The results are shown In table 7 and 8.
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Table 7
Sample cpm
cells/test MT probes
0,8 x 106 162
1,6 x 106 216
3 x 106 298
.. . _ :
Table 8
Sample cpm
molecules/test DHFR probes
106 - 148
5 x 106 394
5 x 107 2240
c? Determination o the number of genes ~~
, - . .
Samples of the cell lines described in ExampIe 2 were treated
in a similar way:, except that the volume per sample
correspondinq to approximatedly 2 x 106 cells was 125 ~l.
The~determinations of number of cells and number of test
nucleic acid molecules were carried out in separate vials by
~adding the cell sample, the appropriate detector and
capturing prabes, and the components of the hybridization
mixture to a final~volume of 200 ~1. Control assays without
cell~standard or test DNA were included.:Hybridization,
collection::of:hybrids, washing and measurement was done as
desc:ribed~in Example 4b. The results were read from standard
curves prepared in parallel as described in Example 4b. The
:results;~are~shown in Table 9. :
- 1~87~;~;8
-- 19 --
Table 9
Cell MT DHFR
. . . _ _ . . . _ _ . . . _
cpm No of cells cpm* No. of No. of
in the sample molecules copies
in the sample
Control cell 253 2.3 x 106 73 < 105
Line I 210 1.5 x 106 2333.8 x 106 3
Line II 237 2.1 x 106 30598.8 x 107 42
-- -
* cpm: values from control assays without cell standard or
test nucleic acid have been subtracted.
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