DD270383A5 - METHOD FOR THE QUANTITATIVE DETERMINATION OF NUCLEIC ACID MOLECULES AND REAGENT SET HEREFOR - Google Patents

Abstract

Described is a method for the quantitative determination of nucleic acid molecules, in particular of amplified genes and / or corresponding mRNA molecules according to the "sand-wich" or the "solution" hybridization method. Also described is the requisite set of reagents. The method is suitable for both cancer and prenatal diagnosis.

Description

Title of the invention:

Method for the quantitative determination of nucleic acid molecules and reagent set therefor

Field of application of the invention ;

The invention relates to a method for the quantitative determination of nucleic acid molecules, in particular of amplified genes and / or corresponding mRNA molecules according to the "sandwich ¹¹ - or" solution "hybridization principle and the required reagent set.

The copy number of individual genes in the genome is usually constant. In some cases there is only one gene, in other cases several genes per haploid genome. However, under certain circumstances, the copy number may change. It was e.g. found that the amplification of certain genes is linked to the development of cancer. It is also known that external factors, e.g. Drugs and metals that cause amplification of certain genes. For the development of a disease plays the erroneous or increased expression rate of a gene and thus

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the amount of mRNA in the cell plays an important role. An increased number of some chromosomes is the reason for certain hereditary diseases or other disorders, whereas for the development of other hereditary diseases only the doubling

A recessive gene is necessary. In all these cases, it is important to determine the number of chromosomes or genes present.

Characteristic of the known technical solutions :

The number of particular DNA molecules, e.g. the degree of amplification of certain genes is currently determined as follows: The DNA to be tested is extracted, cleaved by restriction enzymes, and the resulting nucleotide fragments are separated according to their length in an agarose gel electrophoresis. Subsequently

¹ ^ the single-stranded DNA is transferred to a nitrocellulose filter, fixed and hybridized against a radiolabelled DNA. The hybridization probe used is the gene to be examined or subsections thereof. The results are shown by autoradiography; see. Southern,

J. Mol. Biol., Vol. 98 (1975), 503-517. Each parallel analysis uses the same amount of cellular DNA. The intensities of the hybridization bands are compared and from this the ratios between the copy numbers of the genes examined in the test samples are deduced.

² * 'The method only leads to approximate values. Similarly, the amount of RNA is estimated by the Northern blotting or dot-blotting method only. All these methods do not allow precise quantitative statements; see. Thomas, Methods in

Enzym., Vol. 100 (1933), 255-266

 30

The known methods, such as the "Southern" and "Northern Blotting" methods, are slow and difficult to perform. Since they provide only approximate values, their diagnostic value is doubtful in those cases in which it is important to estimate the number of particular nucleic acid molecules per given unit, e.g. per cell, to know. The "sandwich" - or

2 703 S3

- 3 • 'solution "-Hybridisierungs ~ _methods, which are described in eggs US Patent 4,486,539 and GB-OS 2,169,403, however, are quantitatively; cf. Virtanen et al, Lancet 1 (1983), 381-.. 383rd

Z iel the invention ; 5

The aim of the invention is to provide an accurate, rapid and quantitative method for the determination of nucleic acid molecules.

Explanation of the essence of the invention ;

The invention has for its object to develop a precise, fast and quantitative method for Bestininung of nucleic acid molecules, which is faster and easier to perform than the known methods. This method should be used for both hrtiss and prenatal diagnostics. It should also be suitable for the detection of substances that cause gene amplification, and for subsequent detection

It indicates the development of drug resistance and the determination of the expression level of mRNA. The method of the invention requires a standard constant copy nucleic acid. It is useful for determining the number of NucJ-acidic molecules per unit, e.g.

a cell, a nucleus, ribosome or chromosome. 20

According to the invention, at least two determinations are required. The first one concerns the nucleic acid molecule, which can be present in multiple copies and is called test nucleic acid. The

another statement concerns the constitutive nucleic acid molecule, the 25th

preferably in constant Kbpienzahl, and is referred to as standard nucleic acid. In the method of the invention, a nucleic acid molecule designates a particular nucleotide sequence of 10 to 12 nucleotides or a gene having several thousands of nucleotides.

However, it also denotes an mRNA or a nucleotide sequence which is 30

is considerably longer than a single gene.

The determination of the test and standard nucleic acids is performed either by the usual "sandwich" hybridization method, as described e.g. in the

U.S. Patent 4,486,539, or after the solution hybridization-35

Method described in GB-OS 2,169,403. The invention further relates to a kit containing the nucleic acid samples. The nucleic acid samples consist of at least one pair of test nucleic acids and one pair of standard nucleic acids.

The nucleic acid probes used in the method of the invention are prepared by recombinant DNA technology from nucleic acids having sufficient homologous regions to the test and standard nucleic acids. Sufficient homologous nucleic acids can be produced both synthetically and semi-synthetically.

The test and standard nucleic acids can be isolated directly from cells and identified by different hybridization techniques. However, such test and standard nucleic acids are also commercially available from various gene libraries. Test and standard nucleic acids can be either DNA or RNA molecules.

Sample pairs suitable for the "sandwich" or "solution" hybridization method are prepared by the ΓΝΑ-recombinant technique from nucleic acids having sufficient homologous regions to the test and standard nucleic acids. The nucleic acids are cleaved to this end with suitable restriction enzymes. Of the restriction fragments obtained, at least two, which are relatively close together in the uncleaved nucleic acid molecule, are cloned into at least two suitable vectors. One fragment, the "detector" sample, is coupled to a suitable recognizable marker, while the other fragment, the "capturing" sample, is either coupled to a suitable carrier or to eii: ⁱ ? Tussende substance bound v / ird. For example, this substance is complementary to another substance so that affinity pairs form, and with the aid of these pairs, the resulting hybrids can be separated from the hybridization mixture.

The test and standard sample pairs may coexist in a suitable reagent composition in which both the test and standard sample pairs consist of either DNA or RNA only. It is also possible that the test sample pair as DNA

uml the standard sample pair as RNA or vice versa. The advantages and further treatment of the samples prior to hybridization and the hybridization conditions themselves are therefore selected such that they correspond to the employed in the test sample • ^ft pairs.

The method according to the invention is particularly suitable for determining the number of nucleic acid molecules from homogenized cell material. Of course, the method can also be used to determine purified or pure nucleic acids. However, prior to carrying out the process of the invention, the most suitable pretreatment of the nucleic acid sample should be selected.

It is possible to carry out both DN / 2 and RNA determinations with the method according to the invention. Desoxyribonucleic acids are optionally denatured to obtain single strands. Single-stranded mRNA molecules that may interfere with the hybridization assay may be hydrolyzed, e.g. by cooking under alkaline conditions. The sample is not denatured for ribonucleic acid determinations because dope-stranded deoxyribonucleic acid does not interfere with the determination of RNA. Of course, it is also possible to destroy the DNA with deoxyribonucleases or to chemically or mechanically alter the DNA so that it can no longer interfere with the hybridization reaction. Therefore, in DNA and RNA determinations, a suitable method for the further processing of the sample must be chosen, or alternatively, this further treatment must be omitted. The 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 are described in the literature for the pre- and post-treatment of the nucleic acid samples, so that in each case the most suitable method can be selected. Provisions in which both the test and the

Standard nucleic acids, either as DNA or RNA, can be made in a non-separated sample. Provisions in which the test nucleic acids are present as DNA and the standard nucleic acids as RNA or vice versa must be carried out in a separate sample, since different methods are necessary for the further treatment. Of course, the sample can be separated even if the test and stagnant nucleic acids are of the same nucleic acid type

 10

The hybridization test itself is performed by simultaneously contacting the non-separated sample solution with at least one test sample pair and one standard sample pair. However, if the sample solution has been separated, it is separately contacted with at least one test sample pair and a stanoard pair of samples. In these cases, the quantification of the test nucleic acids in one reaction vessel and the quantification of the standard nucleic acid in another reaction vessel are performed ,

Regardless of whether the sample is divided or undivided, hybridization can take place under the most favorable conditions each time. At the end of the hybridization reaction, the resulting test and standard hybrids are separated from the hybridization mixture. Then the test and standard hybrids are bound or coupled to a carrier or to a suitable substance. This substance is complementary to another substance, so that affinity pairs form, which can then be isolated. The marker bound to the test and standard hybrids is then measured and the results compared to those of the standard curves. In this way, the number of nucleic acid molecules to be tested per unit selected can be determined. The method of the invention has practical diagnostic value, especially in the recognition of some

Types of cancer. In small lung cell carcinoma that is

c-myc gene is often amplified and its expression level significantly higher than in normal tissue. In the case of tumors of the nervous tissue one finds an amplified N-myc gene. 5

The method of the invention can also be used to demonstrate the mutagenic or carcinogenic effects of certain substances or the development of drug resistance. It is known that an external selection pressure can lead to an increased expression of a particular gene. In the treatment of cancer, cells develop resistance to the drug employed by amplifying that gene whose expression product inactivates the drug. One such case is with methotrexate, which induces the amplification of the dihydrofolate reductase gene (DHFR). Another example is the amplification of the metallothionein gene under the influence of cadmium.

The expression level of a gene is important in terms of the phenotype and function of the cell. This can be examined by quantifying the mRNA, since the amount of mRNA correlates with the amount of translation product. The transcriptional product of an oncogene determines the extent to which it is ultimately expressed.

The level of expression of an oncogene varies depending on cell type, cell differentiation and developmental phase of the cell. For example, at some stage of fetal development, the c-myc oncogene is rapidly depreciated, while in another phase it is very slow. The degree of amplification often correlates with the expression level of the gene, although the level of expression may also increase significantly without amplification of the gene. In such cases, the role of an oncogene is best determined by measuring not the gene copy number but the expression level. In some cases, the

Quantifying the mRNA can be simpler and more useful than quantifying the gene itself. As an example, one may cite the c-myc oncogene, which is a labile protein and which rapidly clumps on heat.

The method of the invention may also be used to detect numerical abnormalities of the chromosomes, such as the TDown Syndrome. "Also, the present invention's method of aural diagnosis may be used to determine if the fetus is abnormal, whether homozygous for example recessive gene.

Exemplary embodiments :

example 1

Quantitative determination of an amplified oncogene

a) Nucic acid samples and their preparation Standard samples

Cell standard nucleic acid

The c-ki-ras I gene is present in all human cells.

The sample pairs for the "sandwich" hybridization were.

prepared by subcloning the 3 »8 kb Hind III fragment of the c-Ki-ras I gene. The restriction map of the Hind III fragment was published by Chang et al., PNAS 79 (1982), 484B-4852. The fragment is cloned in pBR 322 (ATCC 41 032). It can be obtained from the ATCC Collection.

Further Treatment of Cell Standard Nucleic Acid The pBR 322 clone described above was cleaved with the restriction enzymes Bgl II and Hind III and the resulting fragments were isolated from the agarose gel.

Purified fragments that are relatively close together in the uncleaved DNA molecule were transformed into two appropriate ones

Vectors subcloned to produce "detector" and "capturing" samples.

Standard "detector" sample

A 1.1 kb Bgl II Bgl II fragment was subcloned into the Bam HI restriction site of pBR 322. The resulting plasmid was radiolabeled by ¹² 5j-dCTP in a nick translation.

Standard "capturing" sample

The approximately 0.5 kb Bgl II-Hind III fragment was ligated into the restriction sites Bam HI and Hind III of the phage vectors M 13 nip 10 and mp 11. The resulting DNA molecules were bound to a nitrocellulose filter

1 & (150 ng DNA / 1 cm).

Test samples Test nucleic acid

From a cloned c-myc gene, which can be obtained, for example, from the ATGC Culture Collection (ATCC 41 010), a pair of samples for "sandwich" hybridization was prepared. The restriction map of the gene has been described by Watt et al., PNAS 80 (1983), 6307-6311.

_f 25 Further treatment of the test nucleic acid, the c-myc gene was cleaved by the Hind III Rescriktionsenzyme, XbaI and PstI and the obtained fragments were isolated, purified and the production of "detector" from the agarose gel - and "capturing" samples subcloned into suitable vectors.

Test "detector" sample

The single-stranded ends of the 3, 1 kb Hind III-Xba I fragment from the c-myc gene were again made double stranded by treatment with DNA polymerase. To the resulting blunt-ended DNA fragment were with help

-ιοί. the T4 DNA ligase Hind III linker linked. After extraction with phenol, the DNA fragment was cut with Hind III and finally ligated into the Hind III site of pBR 322. The resulting plasmid was radioactively labeled in a nick translation by ^ J-dCTP.

Test "capturing" sample

The 1.1 kb Xbal-PstI fragment of the c-myc gene was ligated into the XbaI and PstI restriction sites of the phage vectors M13mp10 and mp11. The resulting DNA molecules were bound to a nitrocellulose filter (15 cng DNA / 1 cm).

b) Determination of the standard curve

The sample used to determine the standard curve consisted of an alkaline-denatured pBR 322 clone carrying the c-myc gene as an insert. The "sandwich" hybridization solution to which the test samples were added consisted of 4- χ SSC, 1 χ Denhardt's solution, 200 μg / ml herring sperm DNA, and 0.2 % SDS. The hybridization reaction was carried out at 65 ° C for a period of 17 to 19 hours. Subsequently, the filters were washed at 50 ^° C. in 0.1 × SSC, 0.2 × SDS. The marker (radioactivity) bound to the "sandwich" hybrids was determined in a gamma counter. The results are summarized in Table I.

Table I c pm sample c-myc filter Molecules / test 40 0 75 10 ⁶ 190 5 χ 10 ⁶ 340 10 ⁷ 2200 10 ⁸

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c) Determination of the number of genes

The samples contain cells from (1) a human placenta and (2) CoIo 320 cells, which can be obtained from the ATCC Culture Collection (ATCC-CCC220). The DNA was isolated from both samples and equal amounts thereof were denatured by boiling under alkaline conditions and then added to the two assay batches. Alkaline denaturation hydrolyzes the RNA present in the sample

 10

The test was performed by adding to each sample the two o-myc and c-Ki-ras I filters and the two (radioactively) labeled nucleic acids. This allowed standard and test DNAs to be measured for each sample. Based on the cKi-rasI determinations, it became clear that each test batch contains the same amount of DNA. Thus one can conclude that the c-myc gene is present in CoIo 320 cells in 16-20 fold higher copy numbers than in the normal tissue. The results are summarized in Table II.

Table II Sample c -Ki-rasI Filter c-myc Filter

0 R

cpm * cpm * number

human

Placenta cells 486 340 10 ⁷

colo 320 cells 432 3205 1.6 χ ΙΟ ⁸

Note: * "cmp values of the blank nitrocellulose filter (" background ¹ ^ background) were subtracted.

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Example 2

Quantitative determination of an amplified gene a) Nucleic acid samples and their preparation Standard samples

Cell standard nucleic acid

As control DNA, those sequences of the mouse metallothionein gene (MT gene) were used which span the promoter and the immediately adjacent "upstream" region. The structure of the MT gene was determined by Pavlakis and Hamer,. ¹ NAS 80 (1983), 397-15. The reference nucleic acid fragment is cloned in the pBPV-MMTneo (342-12) vector (ATCC 37,224) and can be obtained from the ATCC culture collection.

Further treatment of cell standard Nu.cleihsäure

The MT gene described above was cleaved for subcloning in the plasmid pAT 153 with the restriction enzymes KpnI, BglII and Eco RI. The KpnI ends were converted by a linker into Hind III ends.

Standard "detector" sample

The 1.2 kb Eco RI-Kpn I (Hind III) fragment derived from the upstream region of the metallothionein gene promoter was ligated into Eco RI and Hind III restriction sites of plasmid pAT 153. The resulting plasmid was radioactively labeled in a nick translation by ^ P-dNTP's.

Default "capturing" ^ robe

The 0.8 kb Kpnl-Bgl II fragment comprising the promoter and the immediately adjacent "upstream" region of the metallothionein gene was inserted into the restriction sites

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Kpnl and Bam HI of the phage vectors M15mp 18 and M 15 mp cloned. The resulting DNA molecules were bound to a nitrocellulose filter.

Test samples

Test nucleic acid

To prepare the sample pair for "sandwich" hybridization, the commercially available plasmid pMTVdhfr (Bethesda Research Laboratories, Product No. 5569 SS) was used. The structure of this plasride has been described by Lee et al., Nature 294- (1981), 228-252.

Further treatment of the test nucleic acid The pMTVdhfr-Plaaraid, which has the cDNA of the dihydrofolate reductase gene (DHB ¹ R), was cleaved with the restriction enzymes Hind III and Bgl II.

Test "detector" sample

The 0.75 kb Hind III-Bgl II fragment comprising the region of the DHFR gene in the plasmid pMTVdhfr was ligated into the restriction sites Hind III and Bgl II of the vector pAT. The resulting plasmid was in a Nicktrans-

7.0 '' ion by ^ P-dNTP's radioactively labeled.

Test "capturing" sample

A 1.4 kb Hind III fragment from the MMTV gene region of the plasmid pMTVdhfr was cloned into the phage vectors M I5 mp and M 15 mp19.

b) Best of the standard curve

The sample used to determine the standard curve was purified DNA from the pMTVdhfr plasmid. The test was carried out as described in Example 1 b), except that a liquid scintillation counter was used to determine the radioactivity. The resulting standard curve is shown in Table III.

- 14 Table III

sample

c pm

Molecules / Test DHE ¹ R filter

0 17

10 ⁶ 45

3 χ 10 ⁶ 79

¹ ^ ²¹ °

c) Determination of the number of genes

Mouse fibroblast cells (NIH 3T3, ATCC No. CRL 1658) transfected with various amounts of DHFR cDNA were maintained in cell culture plates and used as samples. The cells were lysed by SDS and their DNA sheared by pushing through a fine needle of a subcutaneous injection syringe. A 250 μl sample of about 10 cells was taken from the homogenate and 50 μl sodium hydroxide solution was added.

The sample was boiled and neutralized with acetic acid and the hybridization mixture. The total volume was 0.5 ml. The total samples described above were used simultaneously in the hybridization reaction. As a background control, an "empty" nitrocellulose filter was added Hybridization, washing, and determination of radioactivity were performed as described in Example 1 (b), except that a liquid scintillation counter was used for the radioactivity measurement The results are summarized in Table IV.

- 15 Table IV

cell MT number the Ze cpm * DHFR number of cpra * in the Number of Mo copies sample molecules in the sample control cell (no DHFR 21 - cDNA) 182 1.05 χ 10 ⁶ 80 <10 ⁶ 3 Line I 13 * 0.9 χ 10 ⁶ 732 3 χ 10 ⁶ 40 Line II 21u 1.25 χ 10 ^b 5 χ 10 ⁷

Note: ^ ' cpm values of the blank nitrocellulose filter ("background") were subtracted

The metallothionein gene (MT gene) is an internal marker that measures the number of cells in a Λ-top. The results show that in this assay 1C> ⁶ cells give an MT-specific signal of 165 + 20 cpm. The DHFR samples measure the amount of DHFR cDNA. The number of cells was derived from the MT-specific signal. It was thus possible to determine the number of DHFR cDNA copies in the different cell lines as shown in Table IV.

Example 3 Quantitative determination of mRNA

a) Nucleic Acid Samples and their Preparation Using the test samples described in Example 2, it is also possible to measure the amount of mRNA that is being copied from the DHFR cDNA. The pMTVdhfr plasmid is designed to begin transcription of the DHFR gene on the MMTV promoter. The resulting mRNAs have a length of about 1.0 kb. Of these, about 0.25 kb comes from the MMTV promoter region and the rest

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from the DHFR cDNA; see. Lee et al., Nature 29t- (1981), 228-232.

Standard samples

As cell standard nucleic acid, standard "detector" and standard "capturing" sample, those described in Example 2 were used.

Test samples

As the test nucleic acid, test "detector" and test "capture" sample, those described in Example 2 were also used.

b) Determination of the statistical curve

To determine the standard curve, that sample was used which has the corresponding mRNA as a result of the in vitro transcription of the dihydrofoate reductase gene. The DNA used for the transcription was prepared by inserting the 1.4 kb Hind III fragment of the MMTV promoter from the plasmid pMTVdhfr together with the 0.75 kb Hind III-Bgl II fragment (DH5R cDNA) into the restriction sites Hind III and Bam HI of the plasmid pSP64 (Promega Biotec). The sample RNA was stored in aqueous 0.2 $ SDS solution. The "sandwich" hybridization test was carried out in the manner described in Examples 1 b) and 2 b), only the denaturation was left blank. The results are summarized in Table V.

- 17 Table V

Sample molecules / test

c pm

DHFR filter

0 5 χ 10 ⁽

10 '5 χ 10'

10 *

130

390

653

c) Determination of Number of mNRA Molecules The number of mENA molecules produced by transcription of the DHFR gene was determined in the cell line described in Example 2.

The cells were lysed by SDS and their DNA slightly sheared by pushing through a fine needle of a subcutaneous injection syringe. A 250 μΐ sample of about 5 x 10 cells was taken from the homogenate. The homogenate was then used without denaturation in the "sandwich" hybridization assay. The "sandwich" hybridization was performed as described in Example 2c) and 1b), except that only the DEFR-Frogs were added to the hybridization solution. In a parallel sample of 250 μM homogenate, the cell number was determined using the metallothionein sample described in Example 2c). The results are summarized in Table VI.

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I II MT 3 4 table VI cpra * Number of molecules sample DHFR Number per cell cell c pm * Cell number in the 146b 4800 3.45 χ 2 χ the Mo- in the 100 500 38Ü 430 sample 10 ⁸ 10 ⁹ Line line .5 χ 10 ⁶ .2 χ 10 ⁶

Note .: 'crap values of the empty nitrocellulose filter ( "background") were subtracted

The results show that only about 100 mRNA molecules are deleted from the DHFR genes per cell of line I, but that about 500 of these mRNA molecules are formed per cell of line II.

Example 4 · Determination of the number of amplified genes by "solution"

Hybridization 25

a) Nucleic acid frogs and their preparation

Standard samples

As cell standard nucleic acid, standard "Detect.or" and standard "capturing" sample, those described in Example 2 were used. The 1.2 kb Eco RI-KpnI (Hind III) fragment in plasmid pAT 153 was transformed into a nick-translated

12S tion radioactively labeled by ^ JdCTP. The 0.8 kb Kpnl-Bgl II fragments in the phage vectors M 13 mp 18 and M 13 mp 19 were modified with biotin using the Photoprobe reagent (Vector Laboratories, CA, USA, Product No. SP-1000) ,

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Test samples

As the test nucleic acid, test "detector" and test "capture" sample, those described in Example 2 were used. The 0.75 kb Hind III-Bgl II fragment in plas-125 mid pAT 153 was radioactively labeled in a nick translation by ↑ JdCTP. The 1.4 kb Hind III fragments in the phage vectors M 13 mp 18 and M 13 mp 19 were

TM biotinylated with photoprobe.

b) Determination of standard curves

A standard cell curve was prepared with a known amount of cells. The hybridization signal was. measured with the standard samples that recognize the MT gene. A test nucleic acid standard curve was prepared with the Plasmi ^'1 pMTVdhfr and the test samples which recognize this plasmid. Hybridizations were performed for 90 minutes at 70 ⁰ C in a 200 μΐ solution carried out (0.6 M NaCl, 20 mM phosphate buffer, pH 7.5, 1 mM EDTA, polyethylene glycol # 4 6000). After the reaction, 50 μl streptavidin-agarose (Bethesda Research Laboratories, Maryland, USA, Product No. 5942SA) was added 1 M NaCl, 10 mM sodium phosphate, pH 7.5, 1 mM EDTA to a final volume of 500 μl. The hybrids were collected for 15 minutes at 37 ° C on the streptavidin-agarose. The agarose was then washed once for 5 minutes with the buffered 1 M NaCl solution at 37 ° C and twice for 2 minutes with 15 mM NaCl, 1.5 mM sodium citrate at 55 ° C. The amount of bound hybrids was determined by measuring the agarose in a gamma counter; see. Syvänen et al., Nucleic Acids Res., 14 (1986), 5037-5048. The results are summarized in Tables VII and VIII .:

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- 20 Table VII

sample cpm Number of genes Cells / test MT samples 0.8 χ 10 ⁶ 1.6 χ 10 ⁶ 3 χ 10 ⁶ 162 216 298 Table VIII sample cpm Molecules / test DHFR samples 106 5 χ 10 ⁶ 5 χ 10 ⁷ 148 394 2240 Determination of

Samples of the cell line described in Example 2 were treated in a similar manner, the sample volume, corresponding to about 2 x 10 cells, was 125 μl. The determination of the number of cells and the number of test nucleic acid molecules was carried out in separate vessels. The Zeil sample, the corresponding "detector" and "capturing" samples and the components of the hybridization mixture were added to a final volume of 200 μΐ. Control tests without Zeil standard or test DNA were also performed. Hybridization, collection of hybrids, washing and measurement were carried out according to Example 4- b). The results are read from standard curves which were prepared in parallel according to Example 4- b). The results are summarized in Table IX.

- 21 Table IX

cell

MT DHFR

cpm Number of cells in the sample Number of Mo number of molecules in the copies Sample

control cells

Line I

15 line II

210

237 2.3 χ 10

1.5 x 10 ¹

73

233

<ΙΟ "

3.8 χ

2.1 χ ΙΟ ⁶ 3059 8.8 χ ΙΟ ⁷

Note: ^ cpm values from non-Zeil standard or test nucleic acid control tests were subtracted

Claims (2)

2 7 O 3 S 3 21- Invention claims 1. A method for the quantitative determination of nucleic acid molecules according to the "sandwich" or "solution" hybridization principle, characterized in that the number of given nucleic acid molecules per given unit is determined by the number of test nucleic acid molecules that can be present in multiple copies in the unit with the number of standard nucleic acid molecules selected preferably have a constant copy number in the same unit compared to. 2. The method according to claim 1, characterized in that the nucleic acids present in the sample a) if necessary, be converted into a form in which they can be used in the hybridization reaction, b) nucleic acids which possibly interfere with the hybridization reaction, if necessary be converted into a form in which they can not interfere with the hybridization assay, Ϊ70383 · - 23- c) are contacted, either undivided or divided if necessary, with at least one test sample pair sufficiently homologous to the nucleic acid possibly present in larger copies, and at least one one selected and suitable standard pair of probes sufficiently homologous to the nucleic acid and preferably in constant copy number, the "detector" samples of the test sample pair and the standard sample pair being labeled with a detectable one Marker and wherein the capturing samples are coupled to a carrier or a substance that allows isolation of the resulting hybrids, d) and after the hybridization reaction the test and standard hybrids are separated if necessary and the bound label is measured and the number of nucleic acid molecules per given unit is determined by comparing the number of test and standard nucleic acids. 3. The method according to claim 1 and 2, characterized in that the test and standard nucleic acids are deoxyribonucleic acids. 4. The method according to claim 1 and 2, characterized in that the test nucleic acid is a ribonucleic acid and the standard nucleic acid is a deoxyribonucleic acid. 5. The method according to claim 1 and 2, characterized in that the test and Standara nucleic acids are ribonucleic acids. 6. The method according to claim 1 and 2, characterized in that the test nucleic acid is a deoxyribomic and the standard nucleic acid is a ribonucleic acid. 7 · Method according to claims 1 to 4, characterized in that the "detector" sample of the standard pair of samples - a rekoiubinantes plasmid with a 1.1 kb Bgl II - Bgl II fragment from the Hind III fragment of the human c ki-ra? I-gene, wherein the Hind III fragment was cloned in the plasmid pBR 322 and the Bgl II Bgl II fragment was subcloned in the Bam HI restriction site of the plasmid pBR 322 - and the capturing samples - recombinant phage a 0.5 kb Bgl II-Hind III fragment from the Hind III fragment of the human c-Ki-ras I gene, where the Hind III fragment in the PIt, smid pBR 322 was cloned and the Bgl II fragment Hind III fragment was subcloned into the restriction sites Bam HI and Hind III of the phage vectors M 13 mp 10 and M 13 mp 11, either individually or in combination with the test sample pair, with an undivided or if necessary divided nucleic acid sample , 8. The method according to claim 1 to 4, characterized in that the "detector" sample of the standard sample pair - a recombinant plasmid with a 1.2 kb Eco RI -Kpnl (Hind III) fragment of the "upstream" region the promoter region from the mouse metallothionein gene, wherein the fragment was subcloned into the restriction sites Eco RI and Hind III of the plasmid pAT 153 - and the capturing samples - recombinant phages with a size of 0.8 kb Kpnl-Bgl II fragment from the promoter and the adjacent "upstream" region of the metallothionein gene, wherein the fragment was subcloned into the restriction sites Kpn I and Bam HI of the phage vectors M 13 mp 18 and M 13 mp 19 entv / eder individually or together with the test sample pair with an undivided or, if necessary, with a divided nucleic acid sample in contact. 17 03 8 3   25 9. The method according to claims 1, 2, 3, 6, 7 and 8, characterized in that for determining the amplification of the c-myo oncogene and / or the number of this gene corresponding mRNA molecules, the "detector" sample of the test sample pair - a recombinant plasmid with a 3.7 kb Hind III-XbaI fragment from the c-myc gene, the fragment being subcloned in the Hind III restriction site of plasmid pBR 322 - and the 'capturing' Samples - recombinant phage with a 1.1 kb Xbal-PstI fragment from the c-myc gene, the fragment being subcloned into the restriction sites Xbal and PstI of the phage vectors M mp 10 and M 13 mp 11, either alone or together with the standard pair of samples are contacted with an undivided or, if necessary, a divided nucleic acid sample. 10. The method according to claims 1, 2, 3 »6, 7 and 8, characterized in that for determining the amplification of the dihydrofolate reductase gene (DHFR gene) and / or the number of this gene corresponding mRNA molecules, the "Detector" sample of the test sample pair - a recombinant plasmid having a 0.75 kb Hind III-Bgl II fragment coding for the DHFR gene and derived from the plasmid pMTVdhfr, the fragment being inserted into the restriction sites Hind III and Bam HI of the plasmid pAT 158 was subcloned - and the "capturing" samples - recombinant phage with a 1.4 kb Hind III fragment of the MMTV gene region from the plasraid pMTVdhfr, the fragment in the restriction site Hind III of the phage vectors M 13 mp 18 and M 13 mp 19 were either individually or together with the standard sample pair with an undivided or, if necessary, contacted with a divided nucleic acid sample. 11. reagent for the quantitative determination of nucleic acid molecules, characterized in that it contains at least one test sample pair and at least one standard pair of samples, wherein the "detector" samples of the test-sample pair and the standard pair of samples coupled to a marker and the capturing samples are coupled to a carrier or substance, thereby enabling the isolation of "sandwich" hybrids. 12. Reagent kit according to claim 11, characterized in that the "detector" sample of the test sample pair, which was used for the determination of the amplification of the c-myc oncogene and / or the number of mRNA molecules corresponding to this gene, a recombinant Plasmid with a 3.7 kb Hind III-XbaI fragment from the c-myc gene, the fragment being subcloned into the restriction site Hind III of plasmid pBR 322, and the capturing samples containing recombinant phage 1 , 1 kb Xbal-PstI fragment from the c-myc gene, the fragment being subcloned into the restriction sites Xbal and PstI of the phage vectors M 13 mp 10 and M 13 mp 11, and the "detector" sample of the standard Pair of samples is a 1.1 kb BglII-BglII fragment from the Hind III fragment of the human c-Ki-ras I gene, the Hind III fragment cloned into the plasmid pBR 322 and the BglII-BglII fragment which is located in the restriction site Bam HI of the plasmid pB R 322 was subcloned and the capturing samples are recombinant phage with a 0.5 kb BglII-Hind III fragment from the Hind III fragment of the human cKi rasI gene, the Hind III fragment was cloned in the plasmid pBR 322 and the Bglll HindIII fragment in the restriction sites Bam HI and Hind III of the phage vectors M 13 mpio and M 13 mp 11 was subcloned.
35 -η- 13. Reagent kit according to claim 11, characterized in that the "detector" sample of the test sample pair used for the determination dör amplification of the dihydrofolate reductase gene (DHFR gene) and / or the number of mRNA that corresponds to this gene Molecules used is a recombinant plasmid with a 0.75 kb long, coding for the DHFR gene uni the plasmid pMTVdhfr derived Hind ΙΙΙ-κκΙ II fragment, wherein the fragment in the restriction sites Hind III and Bam HI of the plasmid pAT 153 was subcloned, and the capturing probes are recombinant phage with a 1.4 kb Hind III fragment of the MMTV gene region from the plasmid pMTVdhfr, the fragment being in the restriction site Hind III of the phage vectors M 13 mp 18 and M 13 mp 19 subcloned ! 5, and the "standard" sample pair "detector" probe is a recombinant plasmid with a 1.2 kb Eco RI-Kpn I (Hind III) fragment of the upstreara region of the murine metaalothionein gene promoter region in which the fragment ii was subcloned to the restriction sites Eco RI and Hind III of the plasmid pAT 153, and the capturing probes recombinant phages with a 0.8 kb Kpn I-Bgl II fragment from the promoter and the adjacent " upstream region of the metallothionein gene, wherein the fragment in the restriction sites Kpn.I and Bam HI of ² ^ Fnagenvektoren M 13 mp 18 and M 13 mp 19 was subcloned.