JP2002101900A - Method for assaying enzyme involved in acetyl conjugation in human, and probe and kit each therefor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a new assaying technique for fractionating and quantifying an enzyme involved in an acetyl conjugation in humans, and to provide a probe and a kit for the technique. SOLUTION: The 1st objective probe, which is to be used in assaying an enzyme involved in an acetyl conjugation in humans, consists of an oligonucleotide hybridizable with a specific domain of a gene. The 2nd objective kit for assaying such an enzyme is selected from a combination of the probe with a primer pair of a forward primer and a reverse primer each consisting of an oligonucleotide hybridizable with a specific domain of a gene. The 3rd objective method for assaying such an enzyme comprises using the kit.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring an enzyme involved in acetyl conjugation in humans, and a probe and a kit therefor.

[0002]

2. Description of the Related Art Acetyl conjugation, one of the phase II reactions of drug metabolism, is carried out by various acetyltransferases.
It is well known to be catalyzed by e).

[0003] In developing a new drug, it is important to understand the movement (increase / decrease in expression level) of an enzyme involved in the phase II reaction when the new drug is administered. Because, II
Unless the fluctuation of the enzymes involved in the phase reaction is known, it is not possible to predict the increase or decrease in the efficacy or side effects of the concomitant drug used in combination with the new drug or the increase or decrease in the efficacy or side effects of the new drug due to the concomitant drug or other ingestion. This is because it is not possible to confirm the safety of the vehicle.

[0004] In the field of the development of new drugs and the like, there is a demand for the development of information relating to enzymes involved in the phase II reaction in humans, in particular, the development of measurement techniques capable of separately measuring each molecular species. If such a technology can be developed, for example, it is possible to easily understand the interaction between the new drug and other drugs or the effects during a special disease state, obtain useful information on the side effects of the new drug, and secure the safety of the new drug. be able to. On the other hand, polymerase chain reaction (P
CR) is widely known as a nucleic acid amplification method. For example, RT-PCR (Reverse Transcription-PCR), competitive RT-PCR and the like detect minute amounts of mRNA,
Demonstrates its power in quantification.

In recent years, a real-time quantitative detection method using PCR has been established (Taq Man PCR (Genome Res., 6 (1)
0), 986 (1996)) , ABI PRISM TM Sequence Detection Sy
stem (Applied Biosystems)). This is a method of measuring a nucleic acid using a specific fluorescently labeled probe (TaqMan prove). More specifically, for example, when a normal PCR reaction is performed in a state where a probe labeled with a reporter dye at the 5 ′ end and a quencher dye at the 3 ′ end are annealed to the target DNA, the extension reaction proceeds. Accordingly, the probe is hydrolyzed from the 5 'end by the 5'-3' exonuclease activity of the DNA polymerase. As a result, the reporter dye at the 5 'end separates from the quencher dye at the 3' end, which results in the FR being initially maintained at a constant spatial distance.
The effect of ET (Fluoresence Resonance Energy Transfer, a phenomenon of a decrease in the fluorescence intensity based on a decrease in the energy order of the reporter dye due to resonance of both fluorescent dyes) disappears, and the fluorescence intensity of the reporter dye controlled by the quencher dye increases. Therefore, by measuring the increase in the fluorescence intensity, the target nucleic acid can be selectively quantitatively detected in real time. According to this method,
There is no need for complicated steps such as analyzing the migration pattern by amplifying the amplified product after a PCR reaction, for example, by agarose gel electrophoresis, and there is an advantage that a variety of samples can be quantified in a short time and simultaneously.

In general, when a clinical test is performed at a clinical test center or the like, it is necessary to test a very large number of samples within a limited time. Therefore, there is a demand for the development of a method capable of performing the test efficiently. ing. The real-time detection method is expected to meet such a demand.

[0007] The present inventors have focused on the real-time detection method based on the above-mentioned PCR and conducted intensive research on the idea that this method can be used for detection of an enzyme involved in acetyl conjugation in humans.

[0008] However, currently known enzymes involved in acetyl conjugation, which metabolize chemical substances, have known mRNA sequences, but do not have overlapping sequence portions with each other as target genes. And the same P
It was considered that it was difficult to search for an oligonucleotide as a primer pair capable of sufficiently amplifying under the CR conditions and hybridizing to a specific position of the target gene. In addition, the construction of a probe that can hybridize to a specific region sandwiched by such a pair of primers earlier under the same PCR conditions than those primers and that is specific to an enzyme involved in acetyl conjugation is also performed in the same manner. Was considered difficult.

In particular, the ease of hybridization between two nucleic acids whose nucleotide sequences are known can be estimated to some extent by calculating the melting point (Tm), but the combination of the primer and probe selected based on this estimation is Since it is known that a good result is not necessarily obtained in DNA measurement, each primer pair for real-time detection by PCR can be separately measured for enzymes involved in acetyl conjugation in humans. It was expected that the selection of a combination of a probe and a probe would require extensive experiments and the like by trial and error of a skilled person.

[0010]

SUMMARY OF THE INVENTION An object of the present invention is to establish a method for measuring the enzyme involved in acetyl conjugation in humans by PCR, in particular, to establish a real-time detection method, and to provide a probe and primer pair therefor.

The present inventors have conducted extensive experiments and studies on enzymes involved in acetyl conjugation in humans for the above purpose, and as a result, provided the desired primer pairs and probe combinations for each of the four enzymes. Thus, the present invention having the following gist has been completed.

[0012]

According to the present invention, there is provided a probe used for measuring an enzyme involved in acetyl conjugation in humans, which hybridizes to each of the following regions (1) to (4): A probe selected from oligonucleotides; particularly, a probe having a reporter dye and a quencher dye bound thereto; a probe having a base sequence length of 20 to 40; The probe comprising the sequence shown; and the probe having the sequence shown in SEQ ID NO: 1 to SEQ ID NO: 4. (1) 203-229 region of AANAT gene (2) 446-473 region of NAT1 gene (3) 659-684 region of NAT2 gene (4) 130-155 region of ARD1 gene

According to the present invention, the following (1) to (4)
A primer pair of a forward primer and a reverse primer each comprising an oligonucleotide that hybridizes to each of the following regions of a gene encoding an enzyme involved in acetyl conjugation in humans, and an oligo that hybridizes to the following region sandwiched between both primers A kit for measuring an enzyme involved in acetyl conjugation in humans, which is selected from a combination of probes consisting of nucleotides; in particular, the above-mentioned measurement kit in which the probe is further bound to a reporter dye and a quencher dye is provided. (1) Primer pair; 145-164 region of AANAT gene and
295-277 region, probe; 203-229 region (2) Primer pair; 426-444 region and 56 of NAT1 gene
8-543 region, probe; 446-473 region (3) Primer pair; 631-657 region and 78 of NAT2 gene
1-756 region, probe; 659-684 region (4) primer pair; 92-114 region and 194 of ARD1 gene
-173 region, probe; 130-155 region

The combination (set) of the primer pair and the probe constituting the above-mentioned kit is preferably a combination containing a sequence represented by each sequence number selected from the following (1) to (4), more preferably Those having the sequence shown by SEQ ID NO: can be mentioned. (1) primer pair; SEQ ID NOs: 5 and 6, probe; SEQ ID NO: 1 (2) primer pair; SEQ ID NOs: 7 and 8, probe; SEQ ID NO: 2 (3) primer pair; SEQ ID NOs: 9 and 10, probe; SEQ ID NO: 3 (4) primer pair; SEQ ID NOS: 11 and 12, probe;
SEQ ID NO: 4

Each of the above sets can be used for measuring the following enzymes involved in acetyl conjugation. (1) set; for AANAT measurement (2) set; for NAT1 measurement (3) set; for NAT2 measurement (4) set; for ARD1 measurement For a sample containing a gene of an enzyme involved in conjugation, polymerase chain reaction (PC
R, RT-PCR), and measuring the presence or absence of hydrolysis of the probe used. The method for measuring enzymes involved in acetyl conjugation in humans, The above-mentioned measurement method is provided which is performed depending on the presence or absence of fluorescent coloration.

More specifically, using a primer pair of a forward primer and a reverse primer, and a probe having a reporter dye and a quencher dye and hybridizing with a template nucleic acid in a region sandwiched between the two primers, D having 3 'exonuclease activity
Polymerase chain reaction (PC
Performing R) provides a real-time detection method for measuring the genes of enzymes involved in acetyl conjugation in humans.

The method for real-time detection of an enzyme involved in acetyl conjugation in humans according to the present invention comprises the same PCR
In addition, various enzymes can be separated and quantified simply and quickly in real time under RT-PCR reaction conditions, and the establishment thereof is very useful in the clinical field and pharmaceutical field. For example, the method of the present invention can easily quantify the expression of mRNA in a human sample of an enzyme involved in acetyl conjugation of drug metabolism, which is important in kinetic tests in drug development. Thus, changes in enzymes involved in acetyl conjugation when exposed to chemicals such as pharmaceuticals can be known.

In addition, when examining the mRNA expression of an enzyme involved in acetyl conjugation in a tissue removed by surgery or a tissue removed by biopsy, various types of samples can be processed quickly and extracted from a few pieces of tissue. Detectable with total RNA.

Further, with respect to enzymes involved in specific acetyl conjugation, changes in the expression level due to enzyme induction in the liver, kidney, etc., correlate with changes in the value in blood (cells having nuclei contained in blood). Although there is a possibility, the expression level in this blood is small and a large amount of blood is required for measurement.
However, if the primers and probes designed by the present inventors are used, the measurement can be performed with a device capable of measuring with a small number of samples, which is useful for measuring the expression level in blood. In addition, for enzymes involved in specific acetyl conjugation, changes in expression levels due to enzyme induction in the liver, kidney, etc. may correlate with changes in the value of cells having nuclei contained in secretions such as saliva. However, the expression level in cells having nuclei contained in secretions such as saliva is small, and a large amount of secretions such as saliva is required for measurement. However, if the primers and probes designed by the present inventor are used, the measurement can be performed with a device capable of measuring with a small number of samples, which is useful for measuring the expression level in saliva and the like.

Hereinafter, the probe and primer pairs used in the method of the present invention will be described in detail. As described above, each probe and primer is selected from oligonucleotides that hybridize to a specific region of the gene of an enzyme involved in acetyl conjugation. Is done.

Here, "hybridize" means to hybridize under PCR (RT-PCR) conditions described later.

The common requirements of the probe and the primer according to the present invention are that the amplification product is 50 to 400 base pairs in length, that the primer and the probe are as close as possible, and that the G It can be mentioned that the ratio of (guanine) and C (cytosine) is around 50% as much as possible, and that four or more G (guanine) are not continuous. Other requirements for the probe of the present invention include a Tm value of about 70 ° C. Similarly, the primer has a Tm value of about 60 ° C.
Before and after.

MRNA of each enzyme involved in acetyl conjugation
The sequences are known and each is GeneBank
Registered with the following registration number. (1) AANAT gene; NM — 001088 (2) NAT1 gene; NM — 000662 (3) NAT2 gene; NM — 000015 (4) ARD1 gene; AF085355

In the present specification, the designation of the specific region of each enzyme is in accordance with the sequence of the gene registered with each of the above-mentioned registration numbers.

[0025] The number of nucleotides of the primer and probe oligonucleotides is at least 15, usually 15 to 50, and preferably 20 to 40. If the number of nucleotides of the primers and probes becomes too large, the single-stranded DNA
If it is too small, the specificity of hybridization decreases.

Examples of preferred specific sequences of specific nucleotide sequences of primers and probes are as shown in SEQ ID NOs: 1 to 4 (for probes) and SEQ ID NOs: 5 to 12 (for primers), as described above. is there.

It is known that a primer or a probe consisting of, for example, 20 nucleotides hybridizes even if there is a small number of mismatches with the template strand, and can function as a PCR primer or a detection probe. I have. Therefore, the primers and probes of the present invention are not limited to those having the above specific nucleotide sequence, but include those as a part thereof, and substitution, deletion and / or substitution of, for example, 2 or less nucleotides in this sequence. Alternatively, a sequence modified by addition can be included.

The above-mentioned oligonucleotides as probes and primers of the present invention can be easily synthesized by an automatic synthesizer, for example, using a DNA synthesizer (Perkin Elmer) according to a conventional method. If necessary, purification can be performed using a commercially available purification cartridge or the like.

The probe for real-time detection of the present invention comprises:
A reporter dye is attached at one end, eg, the 5'-end, and a quencher dye is attached at the other end, eg, the 3'-end. The reporter dye is, for example, a substance that emits fluorescence upon irradiation with excitation light, and the quencher acts on the reporter dye when present in close proximity to the reporter dye, and has a function of eliminating the generation of the fluorescence. Can be something. Examples of the reporter dye include, for example, 6-carboxy-fluorescein (FAM), tetrachloro-6-carboxyfluorescein (TET), 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein. Inn (JOE),
Hexochloro-6-carboxyfluorescein (HE
X) and the like. Examples of quencher dyes include:
For example, 6-carboxy-tetramethyl-rhodamine (T
AMRA) and the like.

The probe of the present invention can be prepared by binding a reporter dye and a quencher dye to the probe oligonucleotide having the specific sequence. For example, on the 5 'side of the probe, usually several methylene chains are used as a linker, and a FAM molecule can be bound to a terminal phosphate group in the form of a phosphate ester. Further, a TAMRA molecule can be bound to the 3 'side by an amide bond via a structural unit shown below.

[0031]

Embedded image

Hereinafter, a real-time detection method by PCR using the primer pair and the probe of the present invention will be described in detail.

The method of the present invention essentially requires the use of the above-described primer pair and probe of the present invention, and the others are known PCR methods (see, for example, Science, 230, 1350 (1985)), and RT-PCR (Genome Res. ., 6 (10), 986 (1996)
Especially real-time detection methods (eg TaqMan PC
R, ABI PRISMTM 7700 SEQUENCE DETECTION SYSTEM, App
lied Biosystems, Ver1, June 1996).

This method is particularly useful as a method for measuring the expression level of an enzyme involved in acetyl conjugation targeted by the present invention.
This is useful when measuring NA. In this case, a biological tissue expressing the specific enzyme is collected, and all RNs are collected according to a conventional method.
After extracting A and then synthesizing cDNA complementary thereto in accordance with a conventional method, PCR may be performed using the primer pair of the present invention and the probe. Alternatively, RT-PCR can be directly performed using the primers and probes of the present invention after extracting total RNA according to a conventional method.

The PCR reaction and the RT-PCR reaction can be basically performed according to a known method. The reaction conditions can also be appropriately determined according to a known method, and are not particularly different. Specifically, the conditions described in the following examples can be preferably adopted.

The detection can also be performed basically in accordance with a conventional method, for example, by irradiating the PCR reaction solution with an argon laser beam and detecting the emitted fluorescence using a CCD camera.

Thus, by carrying out the method of the present invention, the enzymes involved in the desired acetyl conjugation can be measured and detected easily and quickly, and separately for each enzyme.

The present invention further provides a kit for performing the above method. This kit comprises the above primer pair and probe. The kit of the present invention may further include a known nucleic acid that can be used as a control, and a primer pair and a probe for measuring the nucleic acid.

According to the method of the present invention, the genes of the enzymes involved in acetyl conjugation can be classified and measured and detected for each enzyme, whereby the enzymes involved in acetyl conjugation can be quantified quickly and accurately. be able to. In this manner, basic data relating to the interaction of the new drug with other drugs, contraindications, and the like can be efficiently obtained.

[0040]

The present invention will be described below in more detail with reference to test examples.

[0041]

[Test example] Preparation of calibration curve by real-time detection method (1) Test method Real-time one-step RT-PC adopted in this test
In the R method, up to 96 different reactions can be performed simultaneously using 96 wells, and up to 96 samples can be measured at a time. In addition, the RT reaction and the PCR reaction can be performed in one tube.

The principle of this quantification is to use two types of adjacent fluorescent dyes and to determine the wavelength range between the fluorescence wavelength of one dye (reporter dye) and the excitation light wavelength of the other dye (quencher dye). Are used when FRET occurs. A probe (TaqMan probe) in which two types of fluorescent dyes that generate FRET are bound to both ends is hybridized to cDNA derived from a specific P450 molecular species amplified by PCR, and in this state, PCR elongation reaction starts, and Taq DNA The TaqMan probe is hydrolyzed by the 5′-3 ′ endonuclease activity possessed by the polymerase, the reporter dye is eliminated, and a physical distance is generated between the TaqMan probe and the quencher dye, and the fluorescence intensity of the reporter dye suppressed by FRET is reduced. Since the increase in the fluorescence intensity is proportional to the amount of increase in the PCR amplification product, a desired quantification can be performed by measuring this for each PCR reaction.

In this test, FAM was used as a reporter dye at the 5 'end of the probe, and TAMRA was used as a quencher dye at the 3' end. The binding of each dye and the production of a TaqMan probe using the dye were performed according to the method described in the literature (Genome Res., 6 (10), 986 (1996)).

Oligonucleotides as primers and probes were used as ABs for automatic synthesis.
Using a DNA / RNA synthesizer manufactured by Company I, the substrate (dN
TP) and the specified reagents.

As the sample RNA, total RNA purified from adult brain, adult kidney, adult lung, adult small intestine, fetal liver, and adult liver pool was used. In addition, all R
All NAs are Clontech Laborator
ies, Inc.).

Total RNA purified from adult brain, adult kidney, adult lung, adult small intestine, fetal liver, and adult liver pool was diluted with RNase-free water to 20 μg /
mL. Then, these six types are mixed in equal amounts,
It was used for preparing a calibration curve. Thereafter, the resultant was diluted at a 5-fold common ratio using 50 μg / mL yeast tRNA (Yeast tRNA, manufactured by GIBCO). 5 μL was used for the measurement.

The RT-PCR reaction was performed using a 300 nM forward primer, a 900 nM reverse primer, and 2
TaqMan One-Step RT-PC containing 00nM TaqMan probe
R Master Mix Reagents Kit (PE Applied Biosystems)
ABI PRI in 50 μL / tube system
SM ^TM 7700 Sequence Detection System (PE Appl
ied Biosystems).

The temperature conditions are as follows: after keeping the temperature at 48 ° C. for 30 minutes and at 95 ° C. for 10 minutes, at 95 ° C. for 15 seconds and at 60 ° C.
A 50 minute cycle was performed 50 times, and the fluorescence intensity was measured for each cycle.

(2) Test Results Table 1 shows the results (calibration curves) of the above-mentioned tests performed on each of the following enzymes using a primer pair and a probe having the sequence shown in each SEQ ID NO: (1) arylalkylamine N-acetyltransferase (a
rylalkylamine N-acetyltransferase, AANAT) (2) N-acetyltransferase 1,
NAT1) (3) N-acetyltransferase 2
(2, NAT2) (4) N-terminal acetyltransferase complex
Subunit (N-terminal acetyltransferase complex
ard1 subunit, ARD1)

[0050]

[Table 1]

From Table 1, 100,000 pg total RNA / 5
When a calibration curve diluted at a 5-fold common ratio was prepared from the 0 μL reaction solution volume, 800 pg of total RN
It was quantitative up to A / 50 μL reaction volume, and the correlation coefficient (r) of the calibration curve of other enzymes was ≥0.99 with 6.4 pg to 4000 pg total RNA as the lower limit of quantification.

[0052]

[Sequence List] SEQUENCE LISTING <110> Ostuka Pharmaceutical Factory Inc. <120> Method for measuring enzymes involved in acetyl conjugation in humans, probes and kits therefor <130> 00P1073 <160> 12 <210> 1 <211> 27 <212> DNA <213> human AANAT gene <400> 1 atgagatccg gcacttcctg accctat 27 <210> 2 <211> 28 <212> DNA <213> human NAT1 gene <400> 2 tcttccgttt gacggaagag aatggatt 28 <210> 3 <211 > 26 <212> DNA <213> human NAT2 gene <400> 3 catcattttg ttccttgcag acccca 26 <210> 4 <211> 26 <212> DNA <213> human ARD1 gene <400> 4 attgttgcag aggcacctgg tggaga 26 <210> 5 <211> 20 <212> DNA <213> human AANAT gene <400> 5 tttgagatcg agcgtgaagc 20 <210> 6 <211> 19 <212> DNA <213> human AANAT gene gagcgagcc gatgatgaa 19 <210> 7 <211> 19 <212> DNA <213> human NAT1 gene 400> 7 tcagcctcag gtgccttgt 19 <210> 8 <211> 26 <212> DNA <213> human NAT1 gene <400> 8 agatttttcg gtatttgctg tcttct 26 <210> 9 <211> 27 <212> DNA <213> human NAT2 gene <400> 9 acgtctccaa catcttcatt tataacc 27 <210> 10 <211> 26 <212> DNA <213> human NAt2 gene <400> 10 cctcagtgag agttttaaac tcgacc 26 <210> 11 <211> 23 <212> DNA <213> human ARD1 gene <400> 11 tctacctaca atacctcgcc cac 23 <210> 12 <211> 22 <212> DNA <213> human ARD1 gene <400> 12 actgagcctt ctgctttacc ca 22

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Claims (12)

[Claims] 1. A probe used for measuring an enzyme involved in acetyl conjugation in a human, comprising the following (1) to (4):
A probe selected from oligonucleotides that hybridize to each of the regions. (1) 203-229 region of AANAT gene (2) 446-473 region of NAT1 gene (3) 659-684 region of NAT2 gene (4) 130-155 region of ARD1 gene 2. The probe according to claim 1, wherein the reporter dye and the quencher dye are bound. 3. The probe according to claim 1, wherein the base sequence has a length of 20 to 40. 4. The probe according to claim 1, which comprises any one of the sequences shown in SEQ ID NOs: 1 to 4. 5. The probe according to claim 1, which is any one of the sequences shown in SEQ ID NOs: 1 to 4. 6. A primer pair of a forward primer and a reverse primer each comprising an oligonucleotide that hybridizes to each of the following regions of a gene encoding an enzyme involved in acetyl conjugation in human, as described in the following (1) to (4): And a kit for measuring an enzyme involved in acetyl conjugation in human, which is selected from a combination of probes consisting of oligonucleotides that hybridize to the following regions sandwiched between the above primers. (1) Primer pair; 145-164 region of AANAT gene and
295-277 region, probe; 203-229 region (2) Primer pair; 426-444 region and 56 of NAT1 gene
8-543 region, probe; 446-473 region (3) Primer pair; 631-657 region and 78 of NAT2 gene
1-756 region, probe; 659-684 region (4) primer pair; 92-114 region and 194 of ARD1 gene
-173 region, probe; 130-155 region 7. The measurement kit according to claim 6, wherein the probe further comprises a reporter dye and a quencher dye. 8. The measurement according to claim 7, wherein the measurement is selected from a combination of a primer pair consisting of an oligonucleotide containing a sequence represented by each SEQ ID No. and a probe of a set represented by the following (1) to (4): kit. (1) primer pair; SEQ ID NOs: 5 and 6, probe; SEQ ID NO: 1 (2) primer pair; SEQ ID NOs: 7 and 8, probe; SEQ ID NO: 2 (3) primer pair; SEQ ID NOs: 9 and 10, probe; SEQ ID NO: 3 (4) primer pair; SEQ ID NOS: 11 and 12, probe;
SEQ ID NO: 4 9. The measurement kit according to claim 8, wherein
The set (1) is for AANAT measurement, and (2)
Set is for measurement of NAT1 and set (3) is for NAT
A measurement kit for measuring 2, and the set (4) is for measuring ARD1. 10. A set of (1) to (4) according to claim 8, which is selected from a combination of a primer pair consisting of an oligonucleotide having a sequence represented by each SEQ ID NO: and a probe. 10. The measurement kit according to 9. 11. A polymerase chain reaction (PC) using a measurement kit according to claim 6 for a sample containing an enzyme involved in acetyl conjugation in humans.
R), and measuring the presence or absence of hydrolysis of the probe used, the method for measuring enzymes involved in acetyl conjugation in humans. 12. The method according to claim 11, wherein the presence / absence of hydrolysis is determined by the presence / absence of fluorescence emission by irradiation with excitation light.