CA2388563A1 - Diagnosis kit, method and microarray for determining human detoxification capacity - Google Patents
Diagnosis kit, method and microarray for determining human detoxification capacity Download PDFInfo
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Abstract
The invention relates to a diagnosis kit, a microarray and a method for determining human detoxification capacity. Said diagnosis kit contains at least one pair of oligonucleotides (reverse primer, forward primer), the two oligonucleotides of a pair being suitable for use as primers for amplifying one of the two complementary strands of a DNA segment that is sought by means of polymerase chain reaction, respectively and the DNA segment that is sought coding for at least one part of an enzyme of the animal detoxification metabolism.
Description
DIAGNOSTIC KIT, METHOD, AND MIRCOARRAY FOR DETERMINING THE
DETOXIFICATION ABILITY OF THE HUMAN BODY
The present invention relates to a diagnostic kit, a method, a microarray, and use thereof for determining the detoxification ability of the human body. Such diagnostic kits, methods, and microarrays are used in the field of environmental and occupational medicine and diseases encountered in these fields, such as neurodermatitis, asthma, MCS
(Multiple Chemical Sensitivity), or CFS (Chronic Fatigue Syndrome). They are also used in the f eld of pharmacology and for determining the metabolization of drugs or new products in the human body.
The environment of man is polluted by environmental chemicals (xenobiotics =
substances foreign to the living organism) of natural and anthropogeric origin which, when measured by today's standards, are not compatible with life of higher organisms, i.e., they are nonphysiological. Chemicals of this type cause malfunctions in ditlicult-to-regulate physiological systems, which are, if reparable at all, hard to repair. These foreign substances enter the human or animal organism via air, skin, or food. In most cases, water-soluble substances are rapidly eliminated while water-insoluble substances are not. On the contrary, these accumulate in the body, often in the fatty tissue (bioaccumulation), unless they are metabolized and broken down within a short time.
As a result, humans and higher organisms have a complex enzyme system which is responsible for ensuring that water-insoluble substances are eliminated. This complex enzyme system mediates the following reactions in two phases:
In the so-called phase I reaction, enzymes from the cytochrome system first convert the environmental toxins into reactive intermediate products which can, however, be acutely more toxic than the harmful substances as such. These intermediate products of the phase I
reaction can bind, e.g., to cellular components and thus cause injuries. As a rule, however, in most cases, chronic injuries tend to be attributable to the original water-insoluble substances.
The phase II reaction which follows the phase I reaction converts the reactive intermediate products into final products that can be eliminated.
Thus, phase I describes the conversion and activation, for example, of various known drugs and environmental chemicals (e.g., debrisoquine, opioids, benzo(a)pyrene, polycyclic and aromatic hydrocarbons) as well as a number of not yet known xenobiotics into electrophilic radical substances.
In the phase II reactions, there are several reaction pathways, such as glucoronidation, the formation of sulfate esters, and annino acid conjugation, in the course of which the enzymes of the most important phase II reaction pathway (glutathione S-transferases) deactivate the intermediate products from phase I (e.g., epoxides, nitrosamines) by conjugation with glutathione to form final products that can be eliminated.
DETOXIFICATION ABILITY OF THE HUMAN BODY
The present invention relates to a diagnostic kit, a method, a microarray, and use thereof for determining the detoxification ability of the human body. Such diagnostic kits, methods, and microarrays are used in the field of environmental and occupational medicine and diseases encountered in these fields, such as neurodermatitis, asthma, MCS
(Multiple Chemical Sensitivity), or CFS (Chronic Fatigue Syndrome). They are also used in the f eld of pharmacology and for determining the metabolization of drugs or new products in the human body.
The environment of man is polluted by environmental chemicals (xenobiotics =
substances foreign to the living organism) of natural and anthropogeric origin which, when measured by today's standards, are not compatible with life of higher organisms, i.e., they are nonphysiological. Chemicals of this type cause malfunctions in ditlicult-to-regulate physiological systems, which are, if reparable at all, hard to repair. These foreign substances enter the human or animal organism via air, skin, or food. In most cases, water-soluble substances are rapidly eliminated while water-insoluble substances are not. On the contrary, these accumulate in the body, often in the fatty tissue (bioaccumulation), unless they are metabolized and broken down within a short time.
As a result, humans and higher organisms have a complex enzyme system which is responsible for ensuring that water-insoluble substances are eliminated. This complex enzyme system mediates the following reactions in two phases:
In the so-called phase I reaction, enzymes from the cytochrome system first convert the environmental toxins into reactive intermediate products which can, however, be acutely more toxic than the harmful substances as such. These intermediate products of the phase I
reaction can bind, e.g., to cellular components and thus cause injuries. As a rule, however, in most cases, chronic injuries tend to be attributable to the original water-insoluble substances.
The phase II reaction which follows the phase I reaction converts the reactive intermediate products into final products that can be eliminated.
Thus, phase I describes the conversion and activation, for example, of various known drugs and environmental chemicals (e.g., debrisoquine, opioids, benzo(a)pyrene, polycyclic and aromatic hydrocarbons) as well as a number of not yet known xenobiotics into electrophilic radical substances.
In the phase II reactions, there are several reaction pathways, such as glucoronidation, the formation of sulfate esters, and annino acid conjugation, in the course of which the enzymes of the most important phase II reaction pathway (glutathione S-transferases) deactivate the intermediate products from phase I (e.g., epoxides, nitrosamines) by conjugation with glutathione to form final products that can be eliminated.
There is a correlation between the severity of the toxic symptoms and the reduction of the enzyme activities of the enzymes involved in the detoxification system.
For some of these en _rymes, genetic variations have been described which have a direct effect on the activity of these enzymes and therefore markedly influence the efrciency of these enzymes.
Thus, the individual genetically mediated variation in the activity of the individual enzymes makes it possible to assign the individual person to different stages of the detoxification potential. As a result, in addition to the toxin intake and the toxin metabolism, the toxin elimination is very important when it comes to determining which dose causes a toxic effect.
This also applies to the glutathione S-transferase which deactivates intermediate products from the phase I reaction to form final products, which can be eliminated, by conjugation with glutathione, the reduced form of which contains a nucleophilic SH group which, in combination with electrophilic C atoms, reacts readily to form thioether. A very high activity of the glutathione S-transferase ensures an efficient conjugation and a rapid detoxification while a reduced activity or the lack of a~~ activity of the glutathione S-transferase slows down the detoxification process.
The system of glutathione S-transferases contains several different enzymes, for example, glutathione S-transferase GSTM1 and glutathione S-tr~nsferase GSTTI.
Glutathione S-transferase GSTM1 Glutathione S-transferase GSTMI has a polymorphism in which the individual genotypes occur with equal chances side by side. Genotype 00 indicates the absence of the gene for GSTM1. If a functional gene is present, two different variants A and B can be distinguished. Both variants form active homodimeric enzymes (AA/BB, or, in the absence of a gene for an allele, AO/BO) or heterodimeric enzymes (AB), with the mixed form AB having the highest enzyme activity; this mixed form is, among other things, thought to be responsible for the "drug resistance" phenomenon in chemotherapy.
Glutathione S-transferase M1 plays an important role in the liver, the lungs, and the skin, with the null genotype occurring in approximately 40% of the Caucasian population.
This null genotype is thought to be associated with the development of diverse tumor diseases (lung, skin, and bladder cancer) and chronic diseases with environmental factors (asbestosis, chronic bronchitis).
Glutathione S-transferase GSTT1 Glutathione S-tr.~nsl:erase T1 is present, among other things, in the liver and in erythrocytes and mediates the glutathione conjugation with halogenated hydrocarbons and epoxides. 1n approxin~ltely 25% of the German population tested so far, no enzyme activity of the glutathione S-trlnsferase was found in erythrocytes. Genotype 00 indicates the absence of this gene for this glutathione S-transferase, therefore the null genotype is considered to be a risk factor since the GSTT1-mediated activity against DNA-damaging peroxides and other metabolites is reduced. Known toxic substrates of GSTT1 are, among other things, methyl bromide, ethylene oxide, and methylene chloride.
The simultaneous absence ofboth enzymes, i.e., the genetic null type for GSTM1 and GSTT1, was found in 10% of the Caucasian population. Thus, this population group has a higher risk due to the reduced detoxification capacity.
In the presently applicable maximum permissible occupational exposure levels, the percentage of people who are potentially genetically susceptible have not been taken into consideration.
In order to determine the load of exposure to environmental chemicals for individuals and to prescribe the appropriate therapy for ill patients, in prior art, an exposure test is carried out, on the basis of which it is possible to estimate the detoxification potential and thus the enzyme activity of the individual. In prior art, this test involves the administration of a test substance which is decomposed by the enzymes to be tested and the subsequent determination of the clearance of this substance in the individual.
The disadvantage of such a test is that the enzyme activities can be determined only by subjecting the person to an additional exposure to a chemical. A method of determination of this type, however, is clearly contraindicated especially for children or individuals with an impaired general state of health, or individuals of whom it is known that they are already highly contaminated with noxious substances. As a result, this method cannot be used in all cases, and especially not in those cases in which, due to the existing contamination with noxious substances, a determination of the enzyme activity would be especially desirable so as to be able to prescribe the most effective treatment.
German Patent DE 197 38 908, attributable to the same inventor as the invention discussed above, proposes to determine genetic polymorphisms of genes in individuals which code for an en-_ryme from the detoxification metabolism In this case, the polymorphism can occur both in the gene and in various allelic variabilities of one or several polymorphic genes.
The method proposed in DE 197 38 908, however, is extremely complicated and can be performed in only a very few highly specialized laboratories. One of the reasons is that this method requires that a multiplication of the DNA of an individual be carried out, the prerequisite of which is a thorough familiarity with molecular biology and the highest level of experimental experience since the development of reaction batches, for example, for a polymerase chain reaction, is extremely complicated and requires experience and knowledge far beyond the average ability of those skilled in the art if the outcome is to be accurate and successful. Thus, the disadvantage of the method proposed in DE 197 38 908 is that it cannot be carried out by just any laboratory physician or by just any medical laboratory.
Thus, the problem to be solved by the present invention is to make available a diagnostic kit, a microarray, a method and applications thereof, by means of which it will be possible to carry out standardized determinations of the human detoxification potential in a simple manner and in any laboratory.
This problem is solved by the diagnostic kit as claimed in Claim 1, the microarray as claimed in Claim 21, the method as claimed in Claim 28, and the use as claimed in Claim 45.
Useful further developments follow from the dependent claims.
The diagnostic kit according to the present invention contains the substances as offered by a number of different manufacturers which are required to carry out a polymerase chain reaction. In addition, the diagnostic kit also contains a minimum of one pair of oligonucleotides which are required as reverse primers and forward primers in the amplification by means of a polymerase chain reaction. These oligonucleotides are also used in the methods according to the present invention. According to the present invention, the oligonucleotides of the pairs are selected to ensure that a DNA segment, which codes at least for part of the GSTT1 enzyme of the animal detoxification metabolism, is amplified. Thus, once an amplification of a DNA segment has been carried out, it can be easily determined whether a gene for the GSTTI enzyme is present. This makes it possible to deterntine the null type.
To be able to also detect different alleles of the same gene, for each of the alleles, different oligonucleotides are used in the methods according to the present invention and included in the diagnostic kit, thus making it possible to detect an a111p11()Cat1011 Of an allele.
This can also be done, for example, by providing that the oligonucleotides for the individual alleles differ in such a way that those regions of the DNA of the alleles are amplified which differ, for example, with respect to a cut site for a restriction enzyme.
The identification of a gene and/or its alleles can also be carried out by means of the microarray according to the present invention, e.g., a DNA chip, where the individual cells of the chip have oligonucleotides which hybridize specifically with certain segments of the genes that are to be determined. In this context, the European Patent EP 0 373 203, in which the construction and use of DNA chips is discussed, is hereby mentioned by way of a general example. The determination can be carried out either directly without amplification or a(ler amplification of the sought-a(1er DNA segment. Similarly, the determination can be carried out without or after the restriction digestion of the DNA or an amplified DNA
segment thereof into shorter DNA segments.
An advantage of the diagnostic kit according to the present invention, the method according to the present invention, and the microarray according to the present invention is that every laboratory physician, every medical laboratory, and every scientific laboratory engaged in such tests will receive the substances, all of which complement one another, possibly even within one single diagnostic kit, and that the laboratories will not need to spend time on preparatory activities and will be able to determine the polymorphism in an easy and simple manner. In particular, since the primer oligonucleotides and the oligonucleotides of the microarray have already been tested, the major work involved in the development of appropriate oligonucleotides is rendered unnecessary.
Thus, it is possible for the first time to determine the polymorphisms and the individual detoxification potential of individual patients on a large scale and at the patient's location in a simple manner and at a reasonable cost. The reason is that the laboratory physician and/or the medical laboratory now needs) only a microarray according to the present invention, a conventional thermoscycler [sic; thermocycler] (PCR
machine) and/or a device for the separation of the individual amplified DNA fragments. Such a device may be, for example, a gel electrophoresis unit or a capillary electrophoresis device.
A conventional capillary electrophoresis device of this type is manufactured, for example, under the name of "PE ABI Prism Genetic Analyzer 310"TM by the firm of Perkin Elmer BiosystemsTM.
Thermocyclers for the amplif cation of the DNA are manufactured, for example, under the name of "GenAmp 2400"TM and "GenAmp 9600"TM by the firm of Perkin Elmer Biosysten~sTM.
To facilitate the detection of the amplified DNA segments, at least one of the oligonucleotides of one pair can be labeled with a iluorophor, thus making possible an automatic evaluation on the basis of the specific fluorescence emission of the fluorophors used.
Thus, overall, the deternunation of the polymorphism of detoxification enzymes by means of the diagnostic kit, the microarray, and the method according to the present invention can now be carried out by anyone in a simple, safe, and standardized manner.
According to the present invention, the diagnostic kit can be further developed in that not only oligonucleotides for the determination of a specific enzyme but also other oligonucleotide pairs for the determination of other genes of detoxification enzymes can be included in the diagnostic kit. Similarly, the microarray according to the present invention can also contain f elds which have oligonucleotides with which other genes of detoxification enzymes can be determined.
In this case, it is possible a~~d simple for the user not only to carry out a null type determination or an allele determination of a specific enzyme but at the same time to determine other enzymes as well. Thus, for example, the user can obtain information about GSTM1 as well as GSTTI, which makes it possible, for example, to predict the "drug resistance" phenomenon in the chemotherapeutic treatment of a patient.
Below, a few examples of diagnostic kits according to the present invention and methods according to the present invention will be described.
A first diagnostic kit enables a multiplex null type determination by means of a PCR
(polymerase chain reaction) of the genes for GSTM1 and GSTTI with labeled oligonucieotides as primers for a detection by means of the Perkin Elmer ABI
Prism Genetic Analyzer 310 device.
G
The diagnostic kit contains as the substances required for the PCR the following components which are packaged in separate containers:
pL of PCR bul~'er (10 ~ buffer, e.g., n»nufactured by the firm of Promega) 8 ItL of MgCl2 (25 mM) 2 yL of dNTPs (10 mM) (deo~inucleotide triphosphates, e.g., guanine (G), adenine (A), cytosine (C), thymine (T)) 2 pL of primer GSTM1 fw (10 pmol/pL) 2 pL of primer GSTM 1 rv ( 10 pmoUpL) 2 pL of primer albumin fw (10 pmol/pL) 2 pL of primer albumin rv ( 10 pmoUpL) 2 pL of primer GSTT1 fw (10 pmoUpL) 2 pL of primer GSTT1 rv (10 pmol/yL) G pL of template DNA (83 ng/pL) G1.5 pL oftwice distilled H20 0.5 pL of Taq polymerise (5 U/yL) (e.g., manufactured by the firm of Promega) The oligonucleotides GSTM1 fiv which serve as forward primers are labeled with the dye FAM (carboayfluorescein), the forward primer for albumin is labeled with NED
(fluorescent dye manufactured by the Finn of PE Biosystems), and the forward primer for GSTT1 is labeled with HEX (4,7,2',4',5',7'-heaachloro-G-carboayfluorescein).
The individual primers have the following nucleotide sequences:
GSTM 1 fw: GAA CTC CCT GAA AAG CTA AAG C;
GSTMI rv: GTT GGG CTC AAA TAT ACG GTG G;
GSTT1 fw: TTC CTT ACT GGT CCT CAC ATC TC;
GSTT1 rv: TCA CCG GAT CAT GGC CAG CA;
Albumin fw: GCC CTC TGC TAA CAA GTC CTA C, and Albumin rv: GCC CTA AAA AGA AAA TCG CCA ATC.
In addition, the diagnostic kit may also contain a fluorescent standard (ROX
standard, ROX = carboy-X-rhodamine) as a length standard for the fragment size assignment.
In addition, the diagnostic kit contains the description of the method for the amplification of DNA with the following parameters:
2 min :L min 1 min 1 min 5 min o0 Preheat 30 Hold Hold 2 x 1 The evaluation can now be carried out by means ofgel electrophoresis. For this purpose, a 2.5'%~ gel (Se~ICem LETM, BiozymTM) is cast, a nulrker (100 by ladder) is applied, and for the separation, the gel is run for 45 min at a voltage of 100 mV. The internal control for the gene for albumin has a length of 350 by and has to be positive in every batch. GSTM 1 has a length of 219 bp, and GSTT1 has a length of 480 bp. The last two genes can be seen as DNA bands in the gel only if a functional gene is present; otherwise, the respective null type is present.
Figure 1 shows a gel in which the bands for GSTT1 (T1) can be clearly seen in tracks 3 and 4 at 480 bp, for the gene for albumin (A) at 350 bp, and for the gene GSTM1 (M1) at 219 bp. Track 2 contains the marker substances as the standard. Thus, in this case, both the gene for GSTM1 and the GSTT1 gene are present.
Figure 2 shows a corresponding evaluation by means of capillary electrophoresis with the PE ABI Prism Genetic Analyzer 310TM device. Again, the bands can be clearly seen at the fragment lengths (in base pairs). The height of the peaks of the individual bands are dependent on the fluorescent markers used and therefore as such do not provide any quantitative information. The other bands represent the ROX standard which makes it possible to assign the fragment sizes.
Figure 3 shows the results of additional studies by means of capillary electrophoresis.
Here, in addition to the bands of the ROX standard, only the bands of the albumin standard (A) and of G5TT1 (T1) are present. As to GSTM1, a null type is obviously present.
In another example, a diagnostic kit for the determination of the variants of with labeled oligonucleotides as primers was used.
Glutathione S-transferase M 1 is characterized by a genetic polymorphism in two ways. First, one or both of the GSTM1 genes (located on the paired chromosomes) which code for glutathione S-transferase M1 can be absent, thus making it possible for a homozygotic t)~pe to develop if two GSTM1 genes are present, for a heterozygotic ype to develop if only one GSTM1 gene is present, or for a homozygotic null type to develop if both genes are absent. Secondly, the exchange of guanine (G) for cytosine (C) in position 2619 leads from an enzyme type B to an enzyme type A, with the heterozygotic AB
type having the highest potential enzyme activity. By identifying the genomic allele variations of an individual, it is therefore possible to determine the potential detoxification ability and thus the potential exposure limits, and consequently the treatment that is suitable for this particular individual.
The diagnostic kit which serves to determine this allele variability contains the following substances required for the PCR, each of which is packaged in separate containers:
pL of PCR bufT'er (10 x buffer, e.g., manufactured by the firm of Promega) 8 pL of MgCl2 (25 mM) 2 pL of dNTPs (10 mM) (guanine (G), cytosine (C), adenine (A) thymine (T)) 4 pL of primer GSTM1 fiv (10 pmoUyL) 2 ~tL of primer GSTM1-A (10 pmoUpL) 2 pL ofprimer GSTMI-B (10 pmoUyL) G pL of template DNA (83 ng/yL) 65.5 pL of twice distilled H20 0.5 pL ofTaq polymerase (5 U/yL) (e.g., manufactured by the firm of Promega) The oligonucleotides GSTM1-A are labeled with the fluorophor HEX, and the oligonucleotides GSTM1-B are labeled with the fluorophor FAM. The oligonucleotides have the following base sequence:
GST'M1 fw: GCT TCA CGT GTT ATG GAG GTT C for both alleles of GSTM1;
GSTM1-A: TTG GGA AGG CGT CCA AGC GC;
GSTMl-B: TTG GGA AGG CGT CCA AGC AG.
In addition, the diagnostic kit contains instructions with the following PCR
program:
3min 45sec 1 min 1, 5min30sec 30 45sec* o sec Pre- 4 x 30 Hold heat x 1 * plus 3 sec per cycle The DNA segments that are amplified with the PCR program mentioned have a length of 132 bp. The detection again takes place by means of capillary electrophoresis, the results of which are shown in Figures 4 and 5. MI-B denotes the band which forms as the result of the 132 by long DNA segment of variant B of GSTM1 and which is labeled with the fluorophor FAM, while MI-A denotes the band which forms as a result of the 132 by long DNA segment of variant A of GSTM 1.
As an alternative, the evaluation can also be carried out by means of gel electrophoresis. For this purpose, a 2.5% gel (SeaKem LE BiozymTM) is cast, and subsequently a marker (100 by ladder) is applied in addition to the PCR
product so as to be able to check the PCR success. The gel runs for 45 min at a voltage of 100 mV.
To be able to distinguish between variant A and B by means of gel electrophoresis, the PCR
product is subjected to a restriction digestion. Variant A contains a cut site for Haetl and is cut into two fragments with a length of 1 I6 by and 1G bp, while varia~~t B does not contain a cut site for the restriction enzyme HaeII. Subsequently, a 4% gel is cast for the separation of smaller fragments (BioRad), and again a marker (100 by ladder) is applied. The 4% gel runs for one hour at a voltage of 1(10 mV. With this gel, it is now possible to see the bands at I IG by and IG by for va~-iv~t A a«d at 132 by for varia~~t B of the GSTM1 gene.
Figure 6 shows such a gel for various samples in different gel tracks, for which both the I IG by band ("GSTMI-A" for variant A) and the 132 by band ("GSTM1-B" for variant B) can be seen. The second track from the left contains marker substances as the standard.
Tracks 4, G, and 9 correspond to samples which contain the gene for the GSTM1 variant B
while tracks 3, 5, 7, 8 and 10 through 14 contain samples with the gene for the GSTM 1 variant A.
SEQUENCE LISTING
<110> Adnagen GmbH
<120> DIAGNOSTIC RIT, METHOD AND MICROARRAY FOR DETERMINING THE
DETOXIFICATION ABILITY OF THE HUMAN BODY
<130> 5031-122 <140>
<141>
<150> PCT/EP00/10478 <151> 2000-10-25 <150> DE 199 55 024.7 <151> 1999-11-16 <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 1 gaactccctg aaaagctaaa gc 22 <210> 2 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 2 gttgggctca aatatacggt gg 22 <210> 3 <211> 23 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 3 ttccttactg gtcctcacat ctc 23 <210> 4 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Deecription of the artificial sequence: PCR- Primer <400> 4 tcaccggatc atggccagca 20 <210> 5 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 5 gccctctgct aacaagtcct ac 22 <210> 6 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 6 gccctaaaaa gaaaatcgcc aatc 24 <210> 7 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 7 gcttcacgtg ttatggaggt tc 22 <210> 8 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial eequence: PCR- Primer <400> 8 ttgggaaggc gtccaagcgc 20 <210> 9 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequences PCR- Primer <400> 9 ttgggaaggc gtccaagcag 20
For some of these en _rymes, genetic variations have been described which have a direct effect on the activity of these enzymes and therefore markedly influence the efrciency of these enzymes.
Thus, the individual genetically mediated variation in the activity of the individual enzymes makes it possible to assign the individual person to different stages of the detoxification potential. As a result, in addition to the toxin intake and the toxin metabolism, the toxin elimination is very important when it comes to determining which dose causes a toxic effect.
This also applies to the glutathione S-transferase which deactivates intermediate products from the phase I reaction to form final products, which can be eliminated, by conjugation with glutathione, the reduced form of which contains a nucleophilic SH group which, in combination with electrophilic C atoms, reacts readily to form thioether. A very high activity of the glutathione S-transferase ensures an efficient conjugation and a rapid detoxification while a reduced activity or the lack of a~~ activity of the glutathione S-transferase slows down the detoxification process.
The system of glutathione S-transferases contains several different enzymes, for example, glutathione S-transferase GSTM1 and glutathione S-tr~nsferase GSTTI.
Glutathione S-transferase GSTM1 Glutathione S-transferase GSTMI has a polymorphism in which the individual genotypes occur with equal chances side by side. Genotype 00 indicates the absence of the gene for GSTM1. If a functional gene is present, two different variants A and B can be distinguished. Both variants form active homodimeric enzymes (AA/BB, or, in the absence of a gene for an allele, AO/BO) or heterodimeric enzymes (AB), with the mixed form AB having the highest enzyme activity; this mixed form is, among other things, thought to be responsible for the "drug resistance" phenomenon in chemotherapy.
Glutathione S-transferase M1 plays an important role in the liver, the lungs, and the skin, with the null genotype occurring in approximately 40% of the Caucasian population.
This null genotype is thought to be associated with the development of diverse tumor diseases (lung, skin, and bladder cancer) and chronic diseases with environmental factors (asbestosis, chronic bronchitis).
Glutathione S-transferase GSTT1 Glutathione S-tr.~nsl:erase T1 is present, among other things, in the liver and in erythrocytes and mediates the glutathione conjugation with halogenated hydrocarbons and epoxides. 1n approxin~ltely 25% of the German population tested so far, no enzyme activity of the glutathione S-trlnsferase was found in erythrocytes. Genotype 00 indicates the absence of this gene for this glutathione S-transferase, therefore the null genotype is considered to be a risk factor since the GSTT1-mediated activity against DNA-damaging peroxides and other metabolites is reduced. Known toxic substrates of GSTT1 are, among other things, methyl bromide, ethylene oxide, and methylene chloride.
The simultaneous absence ofboth enzymes, i.e., the genetic null type for GSTM1 and GSTT1, was found in 10% of the Caucasian population. Thus, this population group has a higher risk due to the reduced detoxification capacity.
In the presently applicable maximum permissible occupational exposure levels, the percentage of people who are potentially genetically susceptible have not been taken into consideration.
In order to determine the load of exposure to environmental chemicals for individuals and to prescribe the appropriate therapy for ill patients, in prior art, an exposure test is carried out, on the basis of which it is possible to estimate the detoxification potential and thus the enzyme activity of the individual. In prior art, this test involves the administration of a test substance which is decomposed by the enzymes to be tested and the subsequent determination of the clearance of this substance in the individual.
The disadvantage of such a test is that the enzyme activities can be determined only by subjecting the person to an additional exposure to a chemical. A method of determination of this type, however, is clearly contraindicated especially for children or individuals with an impaired general state of health, or individuals of whom it is known that they are already highly contaminated with noxious substances. As a result, this method cannot be used in all cases, and especially not in those cases in which, due to the existing contamination with noxious substances, a determination of the enzyme activity would be especially desirable so as to be able to prescribe the most effective treatment.
German Patent DE 197 38 908, attributable to the same inventor as the invention discussed above, proposes to determine genetic polymorphisms of genes in individuals which code for an en-_ryme from the detoxification metabolism In this case, the polymorphism can occur both in the gene and in various allelic variabilities of one or several polymorphic genes.
The method proposed in DE 197 38 908, however, is extremely complicated and can be performed in only a very few highly specialized laboratories. One of the reasons is that this method requires that a multiplication of the DNA of an individual be carried out, the prerequisite of which is a thorough familiarity with molecular biology and the highest level of experimental experience since the development of reaction batches, for example, for a polymerase chain reaction, is extremely complicated and requires experience and knowledge far beyond the average ability of those skilled in the art if the outcome is to be accurate and successful. Thus, the disadvantage of the method proposed in DE 197 38 908 is that it cannot be carried out by just any laboratory physician or by just any medical laboratory.
Thus, the problem to be solved by the present invention is to make available a diagnostic kit, a microarray, a method and applications thereof, by means of which it will be possible to carry out standardized determinations of the human detoxification potential in a simple manner and in any laboratory.
This problem is solved by the diagnostic kit as claimed in Claim 1, the microarray as claimed in Claim 21, the method as claimed in Claim 28, and the use as claimed in Claim 45.
Useful further developments follow from the dependent claims.
The diagnostic kit according to the present invention contains the substances as offered by a number of different manufacturers which are required to carry out a polymerase chain reaction. In addition, the diagnostic kit also contains a minimum of one pair of oligonucleotides which are required as reverse primers and forward primers in the amplification by means of a polymerase chain reaction. These oligonucleotides are also used in the methods according to the present invention. According to the present invention, the oligonucleotides of the pairs are selected to ensure that a DNA segment, which codes at least for part of the GSTT1 enzyme of the animal detoxification metabolism, is amplified. Thus, once an amplification of a DNA segment has been carried out, it can be easily determined whether a gene for the GSTTI enzyme is present. This makes it possible to deterntine the null type.
To be able to also detect different alleles of the same gene, for each of the alleles, different oligonucleotides are used in the methods according to the present invention and included in the diagnostic kit, thus making it possible to detect an a111p11()Cat1011 Of an allele.
This can also be done, for example, by providing that the oligonucleotides for the individual alleles differ in such a way that those regions of the DNA of the alleles are amplified which differ, for example, with respect to a cut site for a restriction enzyme.
The identification of a gene and/or its alleles can also be carried out by means of the microarray according to the present invention, e.g., a DNA chip, where the individual cells of the chip have oligonucleotides which hybridize specifically with certain segments of the genes that are to be determined. In this context, the European Patent EP 0 373 203, in which the construction and use of DNA chips is discussed, is hereby mentioned by way of a general example. The determination can be carried out either directly without amplification or a(ler amplification of the sought-a(1er DNA segment. Similarly, the determination can be carried out without or after the restriction digestion of the DNA or an amplified DNA
segment thereof into shorter DNA segments.
An advantage of the diagnostic kit according to the present invention, the method according to the present invention, and the microarray according to the present invention is that every laboratory physician, every medical laboratory, and every scientific laboratory engaged in such tests will receive the substances, all of which complement one another, possibly even within one single diagnostic kit, and that the laboratories will not need to spend time on preparatory activities and will be able to determine the polymorphism in an easy and simple manner. In particular, since the primer oligonucleotides and the oligonucleotides of the microarray have already been tested, the major work involved in the development of appropriate oligonucleotides is rendered unnecessary.
Thus, it is possible for the first time to determine the polymorphisms and the individual detoxification potential of individual patients on a large scale and at the patient's location in a simple manner and at a reasonable cost. The reason is that the laboratory physician and/or the medical laboratory now needs) only a microarray according to the present invention, a conventional thermoscycler [sic; thermocycler] (PCR
machine) and/or a device for the separation of the individual amplified DNA fragments. Such a device may be, for example, a gel electrophoresis unit or a capillary electrophoresis device.
A conventional capillary electrophoresis device of this type is manufactured, for example, under the name of "PE ABI Prism Genetic Analyzer 310"TM by the firm of Perkin Elmer BiosystemsTM.
Thermocyclers for the amplif cation of the DNA are manufactured, for example, under the name of "GenAmp 2400"TM and "GenAmp 9600"TM by the firm of Perkin Elmer Biosysten~sTM.
To facilitate the detection of the amplified DNA segments, at least one of the oligonucleotides of one pair can be labeled with a iluorophor, thus making possible an automatic evaluation on the basis of the specific fluorescence emission of the fluorophors used.
Thus, overall, the deternunation of the polymorphism of detoxification enzymes by means of the diagnostic kit, the microarray, and the method according to the present invention can now be carried out by anyone in a simple, safe, and standardized manner.
According to the present invention, the diagnostic kit can be further developed in that not only oligonucleotides for the determination of a specific enzyme but also other oligonucleotide pairs for the determination of other genes of detoxification enzymes can be included in the diagnostic kit. Similarly, the microarray according to the present invention can also contain f elds which have oligonucleotides with which other genes of detoxification enzymes can be determined.
In this case, it is possible a~~d simple for the user not only to carry out a null type determination or an allele determination of a specific enzyme but at the same time to determine other enzymes as well. Thus, for example, the user can obtain information about GSTM1 as well as GSTTI, which makes it possible, for example, to predict the "drug resistance" phenomenon in the chemotherapeutic treatment of a patient.
Below, a few examples of diagnostic kits according to the present invention and methods according to the present invention will be described.
A first diagnostic kit enables a multiplex null type determination by means of a PCR
(polymerase chain reaction) of the genes for GSTM1 and GSTTI with labeled oligonucieotides as primers for a detection by means of the Perkin Elmer ABI
Prism Genetic Analyzer 310 device.
G
The diagnostic kit contains as the substances required for the PCR the following components which are packaged in separate containers:
pL of PCR bul~'er (10 ~ buffer, e.g., n»nufactured by the firm of Promega) 8 ItL of MgCl2 (25 mM) 2 yL of dNTPs (10 mM) (deo~inucleotide triphosphates, e.g., guanine (G), adenine (A), cytosine (C), thymine (T)) 2 pL of primer GSTM1 fw (10 pmol/pL) 2 pL of primer GSTM 1 rv ( 10 pmoUpL) 2 pL of primer albumin fw (10 pmol/pL) 2 pL of primer albumin rv ( 10 pmoUpL) 2 pL of primer GSTT1 fw (10 pmoUpL) 2 pL of primer GSTT1 rv (10 pmol/yL) G pL of template DNA (83 ng/pL) G1.5 pL oftwice distilled H20 0.5 pL of Taq polymerise (5 U/yL) (e.g., manufactured by the firm of Promega) The oligonucleotides GSTM1 fiv which serve as forward primers are labeled with the dye FAM (carboayfluorescein), the forward primer for albumin is labeled with NED
(fluorescent dye manufactured by the Finn of PE Biosystems), and the forward primer for GSTT1 is labeled with HEX (4,7,2',4',5',7'-heaachloro-G-carboayfluorescein).
The individual primers have the following nucleotide sequences:
GSTM 1 fw: GAA CTC CCT GAA AAG CTA AAG C;
GSTMI rv: GTT GGG CTC AAA TAT ACG GTG G;
GSTT1 fw: TTC CTT ACT GGT CCT CAC ATC TC;
GSTT1 rv: TCA CCG GAT CAT GGC CAG CA;
Albumin fw: GCC CTC TGC TAA CAA GTC CTA C, and Albumin rv: GCC CTA AAA AGA AAA TCG CCA ATC.
In addition, the diagnostic kit may also contain a fluorescent standard (ROX
standard, ROX = carboy-X-rhodamine) as a length standard for the fragment size assignment.
In addition, the diagnostic kit contains the description of the method for the amplification of DNA with the following parameters:
2 min :L min 1 min 1 min 5 min o0 Preheat 30 Hold Hold 2 x 1 The evaluation can now be carried out by means ofgel electrophoresis. For this purpose, a 2.5'%~ gel (Se~ICem LETM, BiozymTM) is cast, a nulrker (100 by ladder) is applied, and for the separation, the gel is run for 45 min at a voltage of 100 mV. The internal control for the gene for albumin has a length of 350 by and has to be positive in every batch. GSTM 1 has a length of 219 bp, and GSTT1 has a length of 480 bp. The last two genes can be seen as DNA bands in the gel only if a functional gene is present; otherwise, the respective null type is present.
Figure 1 shows a gel in which the bands for GSTT1 (T1) can be clearly seen in tracks 3 and 4 at 480 bp, for the gene for albumin (A) at 350 bp, and for the gene GSTM1 (M1) at 219 bp. Track 2 contains the marker substances as the standard. Thus, in this case, both the gene for GSTM1 and the GSTT1 gene are present.
Figure 2 shows a corresponding evaluation by means of capillary electrophoresis with the PE ABI Prism Genetic Analyzer 310TM device. Again, the bands can be clearly seen at the fragment lengths (in base pairs). The height of the peaks of the individual bands are dependent on the fluorescent markers used and therefore as such do not provide any quantitative information. The other bands represent the ROX standard which makes it possible to assign the fragment sizes.
Figure 3 shows the results of additional studies by means of capillary electrophoresis.
Here, in addition to the bands of the ROX standard, only the bands of the albumin standard (A) and of G5TT1 (T1) are present. As to GSTM1, a null type is obviously present.
In another example, a diagnostic kit for the determination of the variants of with labeled oligonucleotides as primers was used.
Glutathione S-transferase M 1 is characterized by a genetic polymorphism in two ways. First, one or both of the GSTM1 genes (located on the paired chromosomes) which code for glutathione S-transferase M1 can be absent, thus making it possible for a homozygotic t)~pe to develop if two GSTM1 genes are present, for a heterozygotic ype to develop if only one GSTM1 gene is present, or for a homozygotic null type to develop if both genes are absent. Secondly, the exchange of guanine (G) for cytosine (C) in position 2619 leads from an enzyme type B to an enzyme type A, with the heterozygotic AB
type having the highest potential enzyme activity. By identifying the genomic allele variations of an individual, it is therefore possible to determine the potential detoxification ability and thus the potential exposure limits, and consequently the treatment that is suitable for this particular individual.
The diagnostic kit which serves to determine this allele variability contains the following substances required for the PCR, each of which is packaged in separate containers:
pL of PCR bufT'er (10 x buffer, e.g., manufactured by the firm of Promega) 8 pL of MgCl2 (25 mM) 2 pL of dNTPs (10 mM) (guanine (G), cytosine (C), adenine (A) thymine (T)) 4 pL of primer GSTM1 fiv (10 pmoUyL) 2 ~tL of primer GSTM1-A (10 pmoUpL) 2 pL ofprimer GSTMI-B (10 pmoUyL) G pL of template DNA (83 ng/yL) 65.5 pL of twice distilled H20 0.5 pL ofTaq polymerase (5 U/yL) (e.g., manufactured by the firm of Promega) The oligonucleotides GSTM1-A are labeled with the fluorophor HEX, and the oligonucleotides GSTM1-B are labeled with the fluorophor FAM. The oligonucleotides have the following base sequence:
GST'M1 fw: GCT TCA CGT GTT ATG GAG GTT C for both alleles of GSTM1;
GSTM1-A: TTG GGA AGG CGT CCA AGC GC;
GSTMl-B: TTG GGA AGG CGT CCA AGC AG.
In addition, the diagnostic kit contains instructions with the following PCR
program:
3min 45sec 1 min 1, 5min30sec 30 45sec* o sec Pre- 4 x 30 Hold heat x 1 * plus 3 sec per cycle The DNA segments that are amplified with the PCR program mentioned have a length of 132 bp. The detection again takes place by means of capillary electrophoresis, the results of which are shown in Figures 4 and 5. MI-B denotes the band which forms as the result of the 132 by long DNA segment of variant B of GSTM1 and which is labeled with the fluorophor FAM, while MI-A denotes the band which forms as a result of the 132 by long DNA segment of variant A of GSTM 1.
As an alternative, the evaluation can also be carried out by means of gel electrophoresis. For this purpose, a 2.5% gel (SeaKem LE BiozymTM) is cast, and subsequently a marker (100 by ladder) is applied in addition to the PCR
product so as to be able to check the PCR success. The gel runs for 45 min at a voltage of 100 mV.
To be able to distinguish between variant A and B by means of gel electrophoresis, the PCR
product is subjected to a restriction digestion. Variant A contains a cut site for Haetl and is cut into two fragments with a length of 1 I6 by and 1G bp, while varia~~t B does not contain a cut site for the restriction enzyme HaeII. Subsequently, a 4% gel is cast for the separation of smaller fragments (BioRad), and again a marker (100 by ladder) is applied. The 4% gel runs for one hour at a voltage of 1(10 mV. With this gel, it is now possible to see the bands at I IG by and IG by for va~-iv~t A a«d at 132 by for varia~~t B of the GSTM1 gene.
Figure 6 shows such a gel for various samples in different gel tracks, for which both the I IG by band ("GSTMI-A" for variant A) and the 132 by band ("GSTM1-B" for variant B) can be seen. The second track from the left contains marker substances as the standard.
Tracks 4, G, and 9 correspond to samples which contain the gene for the GSTM1 variant B
while tracks 3, 5, 7, 8 and 10 through 14 contain samples with the gene for the GSTM 1 variant A.
SEQUENCE LISTING
<110> Adnagen GmbH
<120> DIAGNOSTIC RIT, METHOD AND MICROARRAY FOR DETERMINING THE
DETOXIFICATION ABILITY OF THE HUMAN BODY
<130> 5031-122 <140>
<141>
<150> PCT/EP00/10478 <151> 2000-10-25 <150> DE 199 55 024.7 <151> 1999-11-16 <160> 9 <170> PatentIn Ver. 2.1 <210> 1 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 1 gaactccctg aaaagctaaa gc 22 <210> 2 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 2 gttgggctca aatatacggt gg 22 <210> 3 <211> 23 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 3 ttccttactg gtcctcacat ctc 23 <210> 4 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Deecription of the artificial sequence: PCR- Primer <400> 4 tcaccggatc atggccagca 20 <210> 5 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 5 gccctctgct aacaagtcct ac 22 <210> 6 <211> 24 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 6 gccctaaaaa gaaaatcgcc aatc 24 <210> 7 <211> 22 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequence: PCR- Primer <400> 7 gcttcacgtg ttatggaggt tc 22 <210> 8 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial eequence: PCR- Primer <400> 8 ttgggaaggc gtccaagcgc 20 <210> 9 <211> 20 <212> DNA
<213> Artificial sequence <220>
<223> Description of the artificial sequences PCR- Primer <400> 9 ttgggaaggc gtccaagcag 20
Claims (37)
1. A diagnostic kit for determining the detoxification ability of the human body with at least two pairs of oligonucleotides (reverse primers, forward primers), with the two oligonucleotides of one pair being suitable as primers for the amplification by means of a polymerase chain reaction of one of the two complementary strands of a sought-after DNA
segment and with the sought-after DNA segment of one pair coding at least for part of the GSTT1 enzyme and with the sought-after DNA segment of the other pair coding at least for part of the GSTM1 enzyme which contains position 2619.
segment and with the sought-after DNA segment of one pair coding at least for part of the GSTT1 enzyme and with the sought-after DNA segment of the other pair coding at least for part of the GSTM1 enzyme which contains position 2619.
2. The diagnostic kit as claimed in the preceding claim, characterized by the fact that it contains the substances necessary for carrying out a polymerase chain reaction.
3. The diagnostic kit as claimed in one of the preceding claims, characterized by the fact that it contains a buffer solution, magnesium chloride, deoxynucleotide triphosphates, and a heat-stable polymerase as the substances necessary for carrying out a polymerase chain reaction.
4. The diagnostic kit as claimed in the preceding claim, characterized by the fact that it contains a polymerase of Thermus aquaticus (Taq polymerase) as the heat-stable polymerase.
5. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that at least one of the two oligonucleotides of at least one pair of oligonucleotides is labeled with a fluorophor.
6. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it contains a DNA sample with the sought-after DNA segment as the positive control.
7. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it also contains a segment of a DNA which codes for an albumin and two oligonucleotides which are suitable as primers for the amplification of at least a segment of one of the two complementary strands of the DNA that code for the albumin so as to allow a reference determination.
8. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it contains two or several pairs of oligonucleotides for DNA
segments of two or more different genes or gene variants.
segments of two or more different genes or gene variants.
9. The diagnostic kit as claimed in the preceding claim, characterized by the fact that the several genes or gene variants code for different enzymes or for different variants of the same enzyme.
10. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that to determine the presence of the GSTM1 enzyme, it has one pair of oligonucleotides with the following sequences:
GAA CTC CCT GAA AAG CTA AAG C and GTT GGG CTC AAA TAT ACG GTG G.
GAA CTC CCT GAA AAG CTA AAG C and GTT GGG CTC AAA TAT ACG GTG G.
11. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that to determine the presence of the GSTT1 enzyme, it has one pair of oligonucleotides with the following sequences.
TTC CTT ACT GGT CCT CAC ATC TC and TCA CCG GAT CAT GGC CAG CA.
TTC CTT ACT GGT CCT CAC ATC TC and TCA CCG GAT CAT GGC CAG CA.
12. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that to determine the presence and possibly the present variants of the GSTM1 enzyme, it has two pairs of oligonucleotides with the following sequences:
GCT TCA CGT GTT ATG GAG GTT C, which is identical for both pairs, and TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG.
GCT TCA CGT GTT ATG GAG GTT C, which is identical for both pairs, and TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG.
13. The diagnostic kit as claimed in the preceding claim, characterized by the fact that each of the two oligonucleotides TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG
is labeled with a different fluorophor.
is labeled with a different fluorophor.
14. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it also contains instructions for carrying out the polymerase chain reaction and instructions for carrying out a fragment analysis.
15. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it contains instructions for carrying out a restriction digestion with a subsequently following gel electrophoresis.
16. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it contains a chart for evaluating the measured results obtained.
17. The diagnostic kit as claimed in any one of the preceding claims, characterized by the fact that it contains a microarray (DNA chip), with this microarray having a number of cells (fields) and with an oligonucleotide, which hybridizes with one of the sought-after DNA
segments, being located in at least one cell.
segments, being located in at least one cell.
18. The diagnostic kit as claimed in the preceding claim, characterized by the fact that a second oligonucleotide is located in a minimum of one other cell and that the sequence of the oligonucleotide which is contained in the minimum of one cell differs from the sequence of the other oligonucleotide.
19. The diagnostic kit as claimed in one of the two preceding claims, characterized by the fact that one oligonucleotide each is located in at least two cells, with the oligonucleotides located in the different cells hybridizing with different sought-after DNA
segments.
segments.
20. A microarray, for example, a DNA chip, with an arrangement of several cells (fields) that are separated from one another, characterized by the fact that oligonucleotides are located in at least two cells, with the oligonucleotides of the one cell hybridizing with a DNA segment which codes for part of the GSTT1 enzyme and with the oligonucleotides of the other cell hybridizing with a DNA segment which codes for part of the GSTT1 enzyme that contains position 2619.
21. The microarray as claimed in the preceding claim, characterized by the fact that at least one of the sought-after DNA segments of GSTM1 has a cytosine or a guanine in position 2619.
22. The microarray as claimed in the preceding claim, characterized by the fact that a sought-after DNA segment has a guanine in position 2619 and another sought-after DNA
segment has a cytosine in position 2619.
segment has a cytosine in position 2619.
23. A method for determining the detoxification ability of the human body, characterized by the fact that the presence of the gene which codes for the GSTT1 enzyme and the presence and the allelic variability of position 2619 of the gene which codes for the GSTM1 enzyme is determined.
24. The method as claimed in the preceding claim, characterized by the fact that it is possible to detect whether a guanine or a cytosine is present in position 2619 of one or of both human genes of GSTM1.
25. The method as claimed in one of the two preceding claims, characterized by the fact that the detoxification ability is determined in the increasing sequence 00 < A0, B0, AA, BB < AB
where 00 stands for the absence of both genes of GSTM1, A0 and B0 stand for the presence of one gene of GSTM1, and AA stands for the presence of one or of two genes of GSTT1.
where 00 stands for the absence of both genes of GSTM1, A0 and B0 stand for the presence of one gene of GSTM1, and AA stands for the presence of one or of two genes of GSTT1.
26. The method as claimed in one of the three preceding claims, characterized by the fact that the detoxification ability is determined in the increasing sequence 00 < A0, AA, where 00 stands for the absence of both genes of GSTT1 and A0 and AA stand for the presence of one gene of GSTM1, AA, BB stand for the presence of two genes, with cytosine in position 2619 (a) and guanine in position 2619 (B), and AB stands for the presence of one gene of GSTM1 each, with cytosine (A) and guanine (B), respectively, in position 2619.
27. The method as claimed in one of Claims 23 through 26, characterized by the fact that DNA or c-DNA is isolated from a sample of an individual and is at least partially multiplied and that subsequently the allelic variability and/or the presence or absence of a gene which codes for an enzyme of the detoxification metabolism is determined.
28. The method as claimed in the preceding claim, characterized by the fact that the DNA is multiplied by means of a polymerase chain reaction.
29. The method as claimed in the preceding claim, characterized by the fact that the polymerase chain reaction is carried out with one pair of oligonucleotides which have the following sequences:
TTC CTT ACT GGT CCT CAC ATC TC and TCA CCG GAT CAT GGC CAG CA.
TTC CTT ACT GGT CCT CAC ATC TC and TCA CCG GAT CAT GGC CAG CA.
30. The method as claimed in the preceding claim, characterized by the fact that one of the two oligonucleotides is labeled with a fluorescent dye.
31. The method as claimed in one of Claims 28 through 30, characterized by the fact that the polymerase chain reaction is carried out with one pair of oligonucleotides which have the following sequences:
GAA CTC CCT GAA AAG CTA AAG C and GTT GGG CTC AAA TAT ACG GTG G.
GAA CTC CCT GAA AAG CTA AAG C and GTT GGG CTC AAA TAT ACG GTG G.
32. The method as claimed in the preceding claim, characterized by the fact that one of the two oligonucleotides is labeled with a fluorescent dye.
33. The method as claimed in one of Claims 28 through 32, characterized by the fact that the polymerase chain reaction is carried out with two pairs of oligonucleotides which have the following sequences:
GCT TCA CGT GTT ATG GAG GTT C, which is identical for both pairs, and TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG.
GCT TCA CGT GTT ATG GAG GTT C, which is identical for both pairs, and TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG.
34. The method as claimed in the preceding claim, characterized by the fact that each of the two oligonucleotides TTG GGA AGG CGT CCAAGC GC and TTG GGA AGG CGT CCA AGC AG
is labeled with a different fluorescent dye.
is labeled with a different fluorescent dye.
35. The method as claimed in any one of Claims 23 through 34, characterized by the fact that the multiplied DNA is digested by means of suitable restriction enzymes and that based on the obtained DNA fragments, the allelic variability and the presence or absence of a gene which codes for an enzyme of the detoxication metabolism is determined.
36. The method as claimed in one of Claims 30, 32, or 34, characterized by the fact that to determine the multiplied DNA, the fluorescent radiation emitted by the multiplied DNA is detected.
37. The method as claimed in any one of Claims 23 through 36, characterized by the fact that the determination of the allelic variability and the presence or absence of a gene is carried out by means of a microarray as claimed in one of Claims 20 through 22.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19955024A DE19955024C2 (en) | 1999-11-16 | 1999-11-16 | Diagnostic Kit |
| DE19955024.7 | 1999-11-16 | ||
| PCT/EP2000/010478 WO2001036670A2 (en) | 1999-11-16 | 2000-10-25 | Diagnosis kit, method and microarray for determining human detoxification capacity |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2388563A1 true CA2388563A1 (en) | 2001-05-25 |
Family
ID=7929173
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002388563A Abandoned CA2388563A1 (en) | 1999-11-16 | 2000-10-25 | Diagnosis kit, method and microarray for determining human detoxification capacity |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1230386A2 (en) |
| JP (1) | JP2003517147A (en) |
| AU (1) | AU7922300A (en) |
| CA (1) | CA2388563A1 (en) |
| DE (1) | DE19955024C2 (en) |
| WO (1) | WO2001036670A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE10219373A1 (en) * | 2002-04-30 | 2004-02-19 | Adnagen Ag | Diagnostic kit, useful for assessing a subject's tolerance of drugs, comprises reagents for determining alleles of genes encoding detoxification enzymes |
| DE10246339A1 (en) * | 2002-10-04 | 2004-04-15 | Adnagen Ag | Predicting risk, and diagnosis, of chemical-induced disease, e.g. chronic fatigue syndrome, comprises determining allelic status of genes involved in the detoxification process |
| EP3385393A1 (en) * | 2017-04-05 | 2018-10-10 | Eckart Schnakenberg | In vitro method for diagnosing of a person's risk for developing an aerotoxic syndrome and kit for carrying out the method |
| CN107475397A (en) * | 2017-09-06 | 2017-12-15 | 江苏省疾病预防控制中心 | A kind of method and its kit for detecting susceptible occupational noise deaf gene |
Family Cites Families (3)
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|---|---|---|---|---|
| DE19738908B4 (en) * | 1997-09-05 | 2004-01-29 | Adnagen Gmbh | Procedure for determining individual genetic influences for pollutant effects and / or planning a therapy concept for people at risk or suffering from neurodermatitis and / or asthma |
| US5919627A (en) * | 1997-10-29 | 1999-07-06 | Incyte Pharmaceuticals, Inc. | Microsomal glutathione-S-transferase |
| JP2003528564A (en) * | 1998-06-06 | 2003-09-30 | ジェノスティック ファーマ リミテッド | Probes used for genetic profiling |
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1999
- 1999-11-16 DE DE19955024A patent/DE19955024C2/en not_active Expired - Fee Related
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2000
- 2000-10-25 JP JP2001538548A patent/JP2003517147A/en active Pending
- 2000-10-25 EP EP00969543A patent/EP1230386A2/en not_active Withdrawn
- 2000-10-25 CA CA002388563A patent/CA2388563A1/en not_active Abandoned
- 2000-10-25 AU AU79223/00A patent/AU7922300A/en not_active Abandoned
- 2000-10-25 WO PCT/EP2000/010478 patent/WO2001036670A2/en not_active Application Discontinuation
Also Published As
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|---|---|
| WO2001036670A3 (en) | 2001-12-06 |
| DE19955024A1 (en) | 2001-06-13 |
| DE19955024C2 (en) | 2003-01-16 |
| WO2001036670A2 (en) | 2001-05-25 |
| EP1230386A2 (en) | 2002-08-14 |
| JP2003517147A (en) | 2003-05-20 |
| AU7922300A (en) | 2001-05-30 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued | ||
| FZDE | Discontinued |
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