CA1287558C - Quantitative determination of nucleic acid molecules and the reagent kitused - Google Patents
Quantitative determination of nucleic acid molecules and the reagent kitusedInfo
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- CA1287558C CA1287558C CA000530691A CA530691A CA1287558C CA 1287558 C CA1287558 C CA 1287558C CA 000530691 A CA000530691 A CA 000530691A CA 530691 A CA530691 A CA 530691A CA 1287558 C CA1287558 C CA 1287558C
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
ABSTRACT
The invention is related 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 determination is carried out by comparing the number of the test nucleic acid molecules potentially present in several copies in given unit to the number of chosen standard nucleic acid molecules advantageously present in a constant number per same unit.
The invention is related 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 determination is carried out by comparing the number of the test nucleic acid molecules potentially present in several copies in given unit to the number of chosen standard nucleic acid molecules advantageously present in a constant number per same unit.
Description
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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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~Z~375~3 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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1~875S8 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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- . . . . .. ' ' ~ - , .:. , ' ' ~LX8'7558 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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- ~l287SS8 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 ` ' :, ~2~71~s8 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.
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.
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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 1o6 75 5 x 106 190 107 3~0 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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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.
Quantification of amplified gene a) Nucleic acid reagents and_their preparation :- ~
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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375S~3 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 lo6 ~5 -3 x 106 79 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 ~ ` , , ` ` ', ` ' `: ` ' `
~87~58 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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~28755~3 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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~LZ87~;8 ~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 5 x 106 ~5 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.
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The results are shown in Table 6. ;
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1~7558 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.
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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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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~87~5~3 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 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 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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' 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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~Z~375~3 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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37~
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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1~875S8 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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- . . . . .. ' ' ~ - , .:. , ' ' ~LX8'7558 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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- ~l287SS8 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 ` ' :, ~2~71~s8 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.
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.
. :
~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.
;
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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lX8755t~
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 1o6 75 5 x 106 190 107 3~0 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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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.
Quantification of amplified gene a) Nucleic acid reagents and_their preparation :- ~
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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375S~3 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 lo6 ~5 -3 x 106 79 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 ~ ` , , ` ` ', ` ' `: ` ' `
~87~58 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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~28755~3 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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~LZ87~;8 ~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 5 x 106 ~5 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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1~7558 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.
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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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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~87~5~3 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 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 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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Claims (13)
1. A quantitative method for determination of nucleic acid molecules by a sandwich or solution hybridization method, c h a r a c t e r i z e d i n t h a t the number of given nucleic acid molecules per given unit is determined by comparing the number of the test nucleic acid molecules potentially present in several copies in the unit to the number of chosen standard nucleic acid molecules advantageously present in a constant number per same unit.
2. The method according to claim 1 c h a r a c t e r i z e d i n t h a t the nucleic acids present in the sample a) are rendered, if necessary, into a form whereby they can participate in the hybridization reaction, b) any nucleic acids potentially disturbing the hybridization reaction are rendered, if necessary, into a form whereby they cannot interfere with the hybridization test, c) are brought into contact, either undivided or, when necessary divided, with at least one test probe pair sufficiently homologous to the nucleic acid potentially present in several copies and with at least one chosen and suitable standard probe pair sufficiently homologous to the nucleic acid molecule advantageously present in a constant number; the detector probes of the said test probe pair and said standard probe pair are labelled with a suitable, detectable label and the capturing probes have been affixed to a suitable carrier or a substance has been affixed to the said capturing probes which enables isolation of the resulting hybrids;
d) after the hybridization reaction or reactions have taken place the test hybrid and standard hybrid are separated when necessary and the said attached label measured; the number of nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.
d) after the hybridization reaction or reactions have taken place the test hybrid and standard hybrid are separated when necessary and the said attached label measured; the number of nucleic acid molecules per given unit is obtained by comparing the test and standard nucleic acid numbers.
3. The method according to Claim 1 or 2, c h a r a c -t e r i z e d i n t h a t the test and the standard nucleic acids are deoxyribonucleic acids.
4. The method according to claim 1 or 2, c h a r a c -t e r i z e d i n t h a t the test nucleic acid is ribonucleic acid and the standard nucleic acid is deoxyribonucleic acid.
5. The method according to Claim 1 or 2, c h a r a c -t e r i z e d i n t h a t the test and standard nucleic acids are ribonucleic acids.
6. The method according to Claim 1 or 2, c h a r a c -t e r i z e d i n t h a t the test nucleic acid is deoxyribonucleic acid and the standard nucleic acid is ribonucleic acid.
7. The method according to Claim 1 or 2, c h a r a c t e r i z e d i n t h a t the detector probe of the standard probe pair - a recombinant plasmid comprising a 1.1 kb BglII-BglII fragment of the HindIII
fragment of the human c-Ki-rasI gene, said HindIII
fragment being cloned into the pBR322 plasmid and said BglII-BglII fragment being subcloned into the restriction site of the BamHI restriction enzyme of the pBR322 plasmid - and the capturing probes - recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the HindIII
fragment of the human c-Ki-rasI gene, said HindIII
fragment being cloned into the pBR322 plasmid and said BglII-HindIII fragment being subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes - are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
fragment of the human c-Ki-rasI gene, said HindIII
fragment being cloned into the pBR322 plasmid and said BglII-BglII fragment being subcloned into the restriction site of the BamHI restriction enzyme of the pBR322 plasmid - and the capturing probes - recombinant phages comprising a 0.5 kb BglII-HindIII fragment of the HindIII
fragment of the human c-Ki-rasI gene, said HindIII
fragment being cloned into the pBR322 plasmid and said BglII-HindIII fragment being subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes - are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
8. The method according to Claim 1 or 2, c h a r a c t e r i z e d i n t h a t the detector probe of the standard probe pair - a recombinant plasmid comprising a 1.2 kb EcoRI-KpnI(HindIII) fragment from upstream of the promoter area of the mouse metallothionine gene, which fragment has been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes - and the capturing probes - recombinant phages comprising a 0.8 kb Kpnl-BglII fragment from the promoter area of the metallothionine gene and the area upstream of it, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes - are brought, either individually or together with the test probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
9. The method according to Claim 1 or 2, c h a r a c t e r i z e d in t h a t in order to determine the degree of amplification of the c-myc oncogene and/or the number of messenger RNA molecules corresponding to this gene, the detector probe of the test probe pair - a recombinant plasmid comprising a 3.7 kb HindIII-XbaI fragment of the c-myc gene, said fragment being subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme - and the capturing probes - recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, which fragment has been subcloned into the M13 mp10 and M13 mp11 vectors between the restriction sites of the XbaI
and PstI restriction enzymes - are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
and PstI restriction enzymes - are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
10. The method according to Claim 1 or 2, c h a r a c t e r i z e d i n t h a t in order to determine the degree of amplification of the dihydrofolate reductase or DHFR gene and/or the number of messenger RNA molecules corresponding to this gene, the detector probe of the test probe pair - a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment coding for the DHFR gene of the pMTVdhfr plasmid, which fragment has been subcloned into the pAT153 plasmid vector between the restriction sites of the HindIII and BamHI restriction enzymes - and the capturing probes -recombinant phages comprising a 1.4 kb HindIII fragment of the MMTV gene area of the pMTVdhfr plasmid, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors at the restriction site of the HindIII
restriction enzyme - are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
restriction enzyme - are brought, either individually or together with the standard probe pair, into contact with an undivided or when necessary divided nucleic acid sample.
11. A reagent kit for the quantitative determination of nucleic acid molecules c h a r a c t e r i z e d i n t h a t the kit contains at least one tect probe pair and at least one standard probe pair, the detector probes of both the test probe pair and the standard probe pair being labelled with a suitable label and the capturing probes having been affixed to a suitable carrier or a substance having been affixed to the said capturing probes, enabling isolation of sandwich hybrids.
12. The reagent kit according to Claim 11, c h a r a c -t e r i z e d i n t h a t the detector probe of the test probe pair used for the determination of the degree of amplification of the c-myc oncogene and/or the number of messenger RNA molecules corresponding to this gene is a recombinant plasmid comprising a 3.7 kb HindIII-XbaI
restriction fragment of the c-myc gene, which fragment has been subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme, and the capturing probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, which fragment has been subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the XbaI and PstI restriction enzymes, and the detector probe of the standard probe pair is a 1.1 kb BglII BglII
fragment of the HindIII fragment of the human c-Xi-rasI
gene, said HindIII fragment having been cloned into the pBR322 plasmid and said BglII-BglII fragment having been subcloned into the pBR322 plasmid at the restriction site of the BamHI restriction enzyme, and the capturing probes are recombinant phages comprising a 0.5 kb BglII-HindIII
fragment of the HindIII fragment of the c-Ri-rasI gene, said HindIII fragment having been subcloned into the pBR322 plasmid of the c-Ki-rasI gene, and said BglII-HindIII fragment having been subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes.
restriction fragment of the c-myc gene, which fragment has been subcloned into the pBR322 plasmid at the restriction site of the HindIII restriction enzyme, and the capturing probes are recombinant phages comprising a 1.1 kb XbaI-PstI fragment of the c-myc gene, which fragment has been subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the XbaI and PstI restriction enzymes, and the detector probe of the standard probe pair is a 1.1 kb BglII BglII
fragment of the HindIII fragment of the human c-Xi-rasI
gene, said HindIII fragment having been cloned into the pBR322 plasmid and said BglII-BglII fragment having been subcloned into the pBR322 plasmid at the restriction site of the BamHI restriction enzyme, and the capturing probes are recombinant phages comprising a 0.5 kb BglII-HindIII
fragment of the HindIII fragment of the c-Ri-rasI gene, said HindIII fragment having been subcloned into the pBR322 plasmid of the c-Ki-rasI gene, and said BglII-HindIII fragment having been subcloned into the M13 mp10 and M13 mp11 phage vectors between the restriction sites of the BamHI and HindIII restriction enzymes.
13. The reagent kit according to Claim 11, c h a r a c -t e r i z e d i n t h a t the detector probe of the test probe pair used for determination of the degree of amplification of the dihydrofolate reductase or DHFR
gene and/or the number of messenger RNA molecules corresponding to this gene is a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment coding for the DHFR gene of the pMTVdhfr plasmid, which fragment has been subcloned into the pAT153 plasmid vector between the restriction sites of the HindIII and BamHI
restriction enzymes, and the capturing probes are recombinant phages comprising a 1.4 kb HindIII fragment of the MMTV gene area of the pMTVdhfr plasmid, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors at the restriction site of the HindIII restriction enzyme, and the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-KpnI fragment from upstream of the promoter area of the mouse metallothionine gene, which fragment has been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb KpnI-BglII
fragment of the metallothionine gene formed by the promoter area and the area upstream of it, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes.
gene and/or the number of messenger RNA molecules corresponding to this gene is a recombinant plasmid comprising a 0.75 kb HindIII-BglII fragment coding for the DHFR gene of the pMTVdhfr plasmid, which fragment has been subcloned into the pAT153 plasmid vector between the restriction sites of the HindIII and BamHI
restriction enzymes, and the capturing probes are recombinant phages comprising a 1.4 kb HindIII fragment of the MMTV gene area of the pMTVdhfr plasmid, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors at the restriction site of the HindIII restriction enzyme, and the detector probe of the standard probe pair is a recombinant plasmid comprising a 1.2 kb EcoRI-KpnI fragment from upstream of the promoter area of the mouse metallothionine gene, which fragment has been subcloned into the pAT153 plasmid between the restriction sites of the EcoRI and HindIII restriction enzymes, and the capturing probes are recombinant phages comprising a 0.8 kb KpnI-BglII
fragment of the metallothionine gene formed by the promoter area and the area upstream of it, which fragment has been subcloned into the M13 mp18 and M13 mp19 phage vectors between the restriction sites of the KpnI and BamHI restriction enzymes.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FIFI-860836 | 1986-02-27 | ||
| FI860836A FI76119C (en) | 1986-02-27 | 1986-02-27 | Quantitative determination of nucleic acid molecules and reagent packaging used in the process |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1287558C true CA1287558C (en) | 1991-08-13 |
Family
ID=8522222
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000530691A Expired - Lifetime CA1287558C (en) | 1986-02-27 | 1987-02-26 | Quantitative determination of nucleic acid molecules and the reagent kitused |
Country Status (26)
| Country | Link |
|---|---|
| JP (1) | JPS62205800A (en) |
| KR (1) | KR870008033A (en) |
| AT (1) | AT393511B (en) |
| AU (1) | AU603562B2 (en) |
| BE (1) | BE1001168A4 (en) |
| CA (1) | CA1287558C (en) |
| CH (1) | CH675593A5 (en) |
| DD (1) | DD270383A5 (en) |
| DE (1) | DE3706285A1 (en) |
| DK (1) | DK174784B1 (en) |
| ES (1) | ES2061411A6 (en) |
| FI (1) | FI76119C (en) |
| FR (1) | FR2594849B1 (en) |
| GB (1) | GB2187283B (en) |
| HU (1) | HU201808B (en) |
| IE (1) | IE66904B1 (en) |
| IL (1) | IL81695A (en) |
| IS (1) | IS3197A7 (en) |
| IT (1) | IT1202581B (en) |
| LU (1) | LU86792A1 (en) |
| NL (1) | NL195097C (en) |
| NO (1) | NO175380C (en) |
| NZ (1) | NZ219421A (en) |
| PT (1) | PT84368B (en) |
| SE (1) | SE468816B (en) |
| ZA (1) | ZA871401B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9347944B2 (en) | 2010-03-31 | 2016-05-24 | Yamaguchi Technology Licensing Organization, Ltd. | Method for detecting pneumonia causative bacteria using nucleic acid chromatography |
Families Citing this family (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5714380A (en) | 1986-10-23 | 1998-02-03 | Amoco Corporation | Closed vessel for isolating target molecules and for performing amplification |
| IL86724A (en) | 1987-06-19 | 1995-01-24 | Siska Diagnostics Inc | Method and kits for the amplification and detection of nucleic acid sequences |
| EP0304845A3 (en) * | 1987-08-28 | 1991-03-06 | Profile Diagnostic Sciences Inc. | Method and kit for assaying gene expressions |
| DE68921918T2 (en) * | 1988-05-09 | 1995-09-07 | Univ Temple | Procedure for predicting the effectiveness of antineoplastic treatment in individual patients. |
| AU629845B2 (en) * | 1988-08-30 | 1992-10-15 | Abbott Laboratories | Detection and amplification of target nucleic acid sequences |
| AU5731790A (en) * | 1989-05-18 | 1990-12-18 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Rna probe for detecting c-fes mrna |
| US5232829A (en) * | 1989-09-29 | 1993-08-03 | Hoffmann-La Roche Inc. | Detection of chlamydia trachomatis by polymerase chain reaction using biotin labelled lina primers and capture probes |
| DK138090D0 (en) * | 1990-06-06 | 1990-06-06 | Novo Nordisk As | DIAGNOSTIC METHOD OF ANALYSIS |
| DE69214243T2 (en) * | 1991-09-23 | 1997-02-06 | Pfizer | Methods for the detection of specific mRNA and DNA in cells |
| US6300058B1 (en) | 1992-01-29 | 2001-10-09 | Hitachi Chemical Research Center, Inc. | Method for measuring messenger RNA |
| GB9210916D0 (en) * | 1992-05-21 | 1992-07-08 | Isis Innovation | Nucleic acid quantification |
| CA2140763A1 (en) * | 1992-07-28 | 1994-02-03 | Masato Mitsuhashi | Gene detection system |
| US5580971A (en) * | 1992-07-28 | 1996-12-03 | Hitachi Chemical Company, Ltd. | Fungal detection system based on rRNA probes |
| ES2055661B1 (en) * | 1993-01-20 | 1995-03-01 | Univ Malaga | DETERMINATION OF THE GENE EXPRESSION BY SPECIFIC CAPTURE OF RNA AND ITS DIRECT QUANTIFICATION BY CAPILLARY ELECTROPHORESIS IN A FREE ZONE. |
| GB9309966D0 (en) * | 1993-05-14 | 1993-06-30 | Nordion Int Inc | Detection of prostrate-specific antigen in breast tumors |
| GB9415489D0 (en) * | 1994-08-01 | 1994-09-21 | Nordion Int Inc | Detection of prostate-specific antigen in amniotic fluid |
| AT401062B (en) * | 1994-09-26 | 1996-06-25 | Immuno Ag | Method for quantifying nucleic acids |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4442203A (en) * | 1981-06-30 | 1984-04-10 | Massachusetts Institute Of Technology | Gene amplification assay for detecting tumor promoters |
| FI63596C (en) * | 1981-10-16 | 1983-07-11 | Orion Yhtymae Oy | MICROBIA DIAGNOSIS FOERFARANDE SOM GRUNDAR SIG PAO SKIKTSHYBRIDISERING AV NUCLEINSYROR OCH VID FOERFARANDET ANVAENDA KOMBINATIONER AV REAGENSER |
| AU555146B2 (en) * | 1982-03-15 | 1986-09-11 | Trustees Of Columbia University In The City Of New York, The | Method for intorducing cloned, amplifiable genes into eucaryotic cells and for producing proteinaceous products materials |
| CA1252046A (en) * | 1982-11-04 | 1989-04-04 | Martin J. Cline | Methods for oncogenic detection |
| FI71768C (en) * | 1984-02-17 | 1987-02-09 | Orion Yhtymae Oy | Enhanced nucleic acid reagents and process for their preparation. |
| US4675283A (en) * | 1984-07-19 | 1987-06-23 | Massachusetts Institute Of Technology | Detection and isolation of homologous, repeated and amplified nucleic acid sequences |
| FI72146C (en) * | 1985-01-02 | 1987-04-13 | Orion Yhtymae Oy | Procedure for Identifying Nucleic Acids. |
| FR2583771B1 (en) * | 1985-06-21 | 1988-12-09 | Centre Nat Rech Scient | DNA SEQUENCES OF ARCHAEBACTERIA HOMOLOGATED TO ONCOGEN V-MYC, PROTEINS ENCODED BY THESE SEQUENCES, PROCESSES FOR OBTAINING SAME AND IMMUNOLOGICAL APPLICATIONS |
-
1986
- 1986-02-27 FI FI860836A patent/FI76119C/en not_active IP Right Cessation
-
1987
- 1987-02-25 JP JP62042442A patent/JPS62205800A/en active Granted
- 1987-02-26 IE IE49687A patent/IE66904B1/en not_active IP Right Cessation
- 1987-02-26 NZ NZ219421A patent/NZ219421A/en unknown
- 1987-02-26 FR FR878702541A patent/FR2594849B1/en not_active Expired - Lifetime
- 1987-02-26 CA CA000530691A patent/CA1287558C/en not_active Expired - Lifetime
- 1987-02-26 AT AT431/87A patent/AT393511B/en not_active IP Right Cessation
- 1987-02-26 IT IT19501/87A patent/IT1202581B/en active
- 1987-02-26 KR KR870001680A patent/KR870008033A/en not_active Application Discontinuation
- 1987-02-26 IS IS3197A patent/IS3197A7/en unknown
- 1987-02-26 BE BE8700177A patent/BE1001168A4/en not_active IP Right Cessation
- 1987-02-26 SE SE8700821A patent/SE468816B/en not_active IP Right Cessation
- 1987-02-26 LU LU86792A patent/LU86792A1/en unknown
- 1987-02-26 DK DK198700993A patent/DK174784B1/en not_active IP Right Cessation
- 1987-02-26 ES ES08700776A patent/ES2061411A6/en not_active Expired - Fee Related
- 1987-02-26 DE DE19873706285 patent/DE3706285A1/en active Granted
- 1987-02-26 NO NO870794A patent/NO175380C/en unknown
- 1987-02-26 GB GB8704517A patent/GB2187283B/en not_active Expired - Lifetime
- 1987-02-26 ZA ZA871401A patent/ZA871401B/en unknown
- 1987-02-26 CH CH756/87A patent/CH675593A5/de not_active IP Right Cessation
- 1987-02-26 HU HU87750A patent/HU201808B/en unknown
- 1987-02-26 AU AU69275/87A patent/AU603562B2/en not_active Expired
- 1987-02-26 NL NL8700483A patent/NL195097C/en not_active IP Right Cessation
- 1987-02-26 PT PT84368A patent/PT84368B/en unknown
- 1987-02-27 DD DD87300286A patent/DD270383A5/en unknown
- 1987-02-27 IL IL81695A patent/IL81695A/en not_active IP Right Cessation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9347944B2 (en) | 2010-03-31 | 2016-05-24 | Yamaguchi Technology Licensing Organization, Ltd. | Method for detecting pneumonia causative bacteria using nucleic acid chromatography |
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