JPH0568600A - Oligonucleotide and detection of ornithine transcarbamylase-mutated gene with the same - Google Patents

Abstract

PURPOSE:To provide an oligonucleotide which is useful as a probe fox multiplying a gene used for the detection of an ornithine transcarbamylase-mutated gene and which facilitates and ensures the diagnosis of the DNA of an ornithine transcarbamylase-deficient disease. CONSTITUTION:An oligonucleotide comprising a sequence of formula I or II, or an oligonucleotide comprising 15-40 bases partially having at least continuous >=15 bases in the sequence of formula I or II and containing a fragment of the sequence of formula I or II. The oligonucleotide is obtained by a known DNA synthesis method used for a DNA synthesis machine, etc. For the detection of an ornithine transcarbamylase-mutated gene, a specific nucleotide is multiplied, while the oligonucleotides of formulas I and II are used as a primer and as another primer, respectively, and the fragment of the multiplied nucleotide is sequenced.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an oligonucleotide used for detecting an ornithine transcarbamylase (hereinafter abbreviated as OTC) mutant gene and a method for detecting an OTC mutant gene using the oligonucleotide.
More specifically, the OTC gene is amplified by PCR using a specific oligonucleotide as a primer,
Then, it relates to a method for detecting an OTC mutant gene by detecting the amplified gene.

[0002]

OTC is one of the urea cycle enzymes that detoxify ammonia and synthesize urea. Deficiency of this enzyme causes convulsions, consciousness disorder, hyperammonemia, liver dysfunction, etc. He will die shortly after birth. In addition, even if one lives, most of them will be severely disabled children. It is also known that some of them develop at the age of 40 to 50 for the first time, and the onset age and clinical symptoms are not uniform. The incidence of this disease in Japan is more than 1 in 80,000, and it is one of the most frequent congenital amino acid metabolism disorders. Since OTC is found in the liver (slight in the small intestine) and its enzyme activity is only observed, conventionally, for confirmatory diagnosis, a part of the liver was collected by biopsy to measure the OTC activity. ..

On the other hand, since the expression of the OTC gene is limited to the liver and intestinal tract as described above, the mRNA of the patient
It was difficult to analyze by the method and prenatal diagnosis using amniotic fluid cells. However, in recent years D directly diagnoses genes in many diseases.
NA diagnosis is becoming possible. Since OTC deficiency is also one of genetic diseases, there has been a strong demand for development of a DNA diagnostic method for OTC deficiency, and various attempts have been made. Usually, when performing DNA analysis of a certain disease, mRNA of the expressed enzyme protein
The method of examining the base sequence of cDNA complementary to or the method of directly cloning genomic DNA for examination.

Similarly, for the diagnosis of OTC deficiency DNA, analysis of OTC cDNA is required. Or O
Since the TC gene is specifically expressed in the liver as described above, the analysis result of the genomic DNA is necessary to make a diagnosis using a material other than hepatocytes. These have already been reported by the inventors, but OTC is encoded by a gene existing on the X chromosome Xp21,
It has been revealed that a large gene of about 73 kb is composed of 10 exons (J. Biochem.
103, 302-308, 1988).

However, the method for analyzing OTC cDNA is not very desirable in view of the need for liver biopsy and the burden on the patient. Further, conventionally, when cells other than hepatocytes were used, it was necessary to first make a DNA library for each patient material and then analyze it. Therefore, it takes a long period of time to obtain the detection result, and a complicated processing step is required.
The value as a clinical diagnostic method was not necessarily high.

[0006] In addition, a primer was synthesized based on the nucleotide sequence reported by the inventors so far, and PCR was performed.
The method was not able to perform all the DNA amplifications of the 10 exons and the binding part of each exon with the intron, and thus was not useful for the diagnosis of all OTC deficiencies. Therefore, OTC deficiency DNA
Materials other than hepatocytes in the diagnosis (for example, peripheral leukocytes,
Even in the case of using cultured fibroblasts, amniotic fluid cells, and placental villi, there is a strong demand in the art for development of a method capable of diagnosing all the exons of the OTC gene and the mutations over the exons and intron junctions in a short period of time. However, the fact is that no useful method has been found yet.

[0007]

SUMMARY OF THE INVENTION The object of the present invention is to provide an OT.
An object of the present invention is to provide an oligonucleotide useful as a primer for gene amplification for diagnosing mutations in all exons and exons and intron junctions of OTC gene in DNA diagnosis of C deficiency. Another object of the present invention is to provide a method for detecting an ornithine transcarbamylase mutant gene.

[0008]

[Means for Solving the Problems] As a result of intensive studies to solve the above problems, the present inventors have carried out gene amplification using specific oligonucleotides as primers, and then sequenced the sequences of the amplified genes. By doing so, it was found that mutations can be diagnosed over all exons and exons and intron junctions in the OTC gene, and the present invention has been completed.

That is, the gist of the present invention is (1) an oligonucleotide consisting of a combination of the following sequences, A sequence (SEQ ID NO: 1) or a sequence (SEQ ID NO:
2) B sequence (SEQ ID NO: 3) or b sequence (SEQ ID NO :)
4) C sequence (SEQ ID NO: 5) or c sequence (SEQ ID NO: 5)
6) D sequence (SEQ ID NO: 7) or d sequence (SEQ ID NO:
8) E sequence (SEQ ID NO: 9) or e sequence (SEQ ID NO: 1)
0) F sequence (SEQ ID NO: 11) or f sequence (SEQ ID NO :)
12) G sequence (SEQ ID NO: 13) or g sequence (SEQ ID NO :)
14) H sequence (SEQ ID NO: 15) or h sequence (SEQ ID NO :)
16) I sequence (SEQ ID NO: 17) or i sequence (SEQ ID NO :)
18) In addition, among these respective oligonucleotides,
An oligonucleotide, which is a sequence fragment consisting of 15 to 40 bases each having at least 15 consecutive bases or more in part, (2) the oligonucleotide of (1) or the oligonucleotide of the fragment as a primer,
An ornithine transcarbamylase mutation characterized by amplifying a specific nucleotide fragment by repeating chain length reaction and separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. A method for detecting a gene, comprising: (3) cleaving a nucleotide fragment amplified by using the oligonucleotide described in (1) above as a primer with a restriction enzyme, and comparing the obtained cleaved fragment with a cleaved fragment of a normal gene. A method for detecting an ornithine transcarbamylase mutant gene, which is characterized in that (4) ornithine is characterized by using as a probe a nucleotide complementary to the sequence of the mutant portion of the mutant gene detected by the method described in (2) above. The present invention relates to a method for detecting a transcarbamylase mutant gene.

The oligonucleotide of the present invention is used for amplifying the genes in exons 1 to 10 shown in FIGS. 1 to 9, and specifically has the following sequences. (1) Primer for amplification of nucleotide fragment containing exon 1: A sequence: 5'-GAGTTTTCAAGGGCATAGAATCGTC-3 'a sequence: 5'-AGTTTTATGCATCACCATGATTCCT-3' (2) Primer for amplification of nucleotide fragment containing exon 2: B sequence: 5'-CACCATAGTACATGGGTCTTTTCTGA-3 'b sequence: 5'-AAAAAGGGGACTGGTAGTAATGGAAC-3' (3) Primer for amplification of nucleotide fragment containing exon 3: C sequence: 5'-CACTTATTTGGGGGCTAGTTATTACTTA-3 'c sequence: 5' -GTCTTCACCTTCAATCCCTCTCTTC-3 '(4) Primer for amplification of a nucleotide fragment containing exon 4: D sequence: 5'-GTTGAGATGATGGCCAATTCTTTGT-3' d sequence: 5'-CCATCAGATTCTGAAATCAGCTTTG-3 '(5) nucleotide fragment containing exon 5 Amplification primer: E sequence: 5'-GCATTATTAAGCATAATTATCTTAG-3 'e sequence: 5'-TTCACTTAAAGCAAGTCAGGAATTA-3' (6) Primer for amplification of nucleotide fragment containing exon 6: F sequence : 5'-TGGATTTCATCTCCTTCATCCCGTG-3 'f sequence: 5'-GTGATTTAAGAGAGGGGTTAGTTTC-3' (7) Primer for amplification of a nucleotide fragment containing exons 7 and 8: G sequence: 5'-CCTAAATAAGATTTAAATTCCTTCCT-3 'g sequence: 5' -GGAATTAATGAACCTGAGAGAGCAT-3 '(8) Primer for amplification of nucleotide fragment containing exon 9: H sequence: 5'-GCCACATATAATAGTCAAAAAGTGG-3' h sequence: 5'-ATCTCACTTGCTTATTATTTCCCCA-3 '(9) A nucleotide fragment containing exon 10 Amplification primers: I sequence: 5'-TCTAGGTCCCTAAGCAGACTGTCGC-3 'i sequence: 5'-CAACTCATTCTGTTACTGAAGAACA-3'

Further, the primer is not limited to the above-mentioned sequences as long as it can be used as a primer in the present invention, and usually 15 to 40 nucleotides each of which has at least 15 contiguous nucleotides or more in each of the above-mentioned sequences. Sequence fragments consisting of the bases of are also included in the scope of the present invention. Here, the primer chain length of 15 to 40 bases is a normal length used as a primer in the PCR method. As a primer, it is desirable to select a sequence of about 15 to 40 bases complementary to an intron site 10 to 20 bases apart from both ends of each exon.

The oligonucleotide of the present invention is a known D
It can be easily prepared by the NA synthesis method. For example, it can be synthesized using a DNA synthesizer manufactured by Applied Biosystems. The synthesized oligonucleotide is purified by a known method using HPLC using a reverse phase column, a DNA adsorbing column or polyacrylamide gel electrophoresis, and used as a primer.

[0013] The present invention provides a method for rapidly detecting any mutation in the OTC gene of an OTC deficient patient as described above. To this end, it is necessary to have a method that can detect abnormalities at the boundary site between exons and introns, which could not be detected until now. There are 10 exons in the OTC gene, and the gene at the boundary between each exon and the intron is isolated according to the following method.

(1) Extraction of chromosomal DNA Fresh, frozen or formalin-fixed specimens obtained from liver, whole blood, isolated leukocytes, amniotic fluid cells, placental ciliated tissues, hair roots, cultured cells, etc., are enzymes such as proteinases. After digesting the protein by treatment, DNA is extracted by a conventional method such as phenol extraction and ethanol precipitation treatment.

(2) Polymerase chain reaction (PCR) Using the synthesized oligonucleotide of the present invention as a primer on the chromosomal DNA obtained in (1), Taq DNA
Polymerase is added and the polymerase chain reaction reaction is performed according to a conventional method to obtain all exons and amplified DNA fragments at the boundary between exons and introns. Specifically, PCR is generally performed in the following steps. Explaining the detection of a DNA fragment containing exon 1 as an example, the target DNA in a sample is hybridized with an oligonucleotide consisting of an A sequence (SEQ ID NO: 1) or an oligonucleotide which is an A sequence fragment as one primer. (Annealing step) to carry out chain length reaction (extension step). The double-stranded nucleotides thus obtained are separated into single strands (daturation step), and their complementary strands function as a template for the other primer. As the other primer, an oligonucleotide consisting of a sequence (SEQ ID NO: 2) or a
A specific nucleotide fragment is amplified by repeating the separation operation from the template of the chain length reaction product using these two primers using the oligonucleotide which is a sequence fragment. For the chain length reaction with a primer, a known thermostable DNA polymerase as described above is usually used. The operation of separating the chain length reaction product from the template is carried out by various known methods, but it is preferably carried out by heat denaturation.
The amount of target DNA in the sample used for PCR is usually 0.1 to 1
About μg is the standard, and the amount of primer is usually 50 pmol each.
Use degree. The PCR conditions are usually 94 ° C. for 1 to 2 minutes in the denaturation step and 35 in the annealing step.
The conditions are as follows: ~ 65 ° C for 1 to 3 minutes, preferably 37 to 55 ° C for 1 to 2 minutes, and the extension step is usually 70 to 74 ° C for 2 to 3 minutes. Usually 25
~ 40 cycles, preferably 30 cycles.

(3) Cloning The amplified DNA fragment obtained in (2) is subjected to electrophoresis after phosphorylating the 5'end and blunting both ends of the DNA fragment with Klenow fragment. This is the DNA obtained
Since there may be non-specific DNA fragment contamination in
This is to remove it. A band having a chain length estimated from the used primer is cut out from the gel subjected to electrophoresis to obtain a DNA fragment having the target chain length.
Then, the obtained DNA fragment is inserted into a commercially available plasmid, and the plasmid is introduced into an appropriate host such as Escherichia coli. The chain length of the insert DNA of the plasmid contained in the transformant is examined, and the clone having the insert DNA of interest is determined to be positive.

(4) Sequencing The nucleotide sequence of the insert DNA of the positive clone is sequenced by a known method. For example, it is determined by the dideoxy chain termination method. The nucleotide sequence of the intron containing each exon of the OTC gene thus determined is shown in FIG.
It shows in FIG. In this way, the base sequence of the intron containing each exon of the OTC gene in the chromosomal DNA obtained from each sample can be known.

The above-mentioned oligonucleotide used as a primer in the present invention contains an exon and also covers an intron at the boundary between the exon and the intron.
Since it is designed to amplify the NA fragment, it is possible to easily detect not only the exon of the OTC gene but also the mutant gene at the boundary between the exon and the intron by sequencing the nucleotide sequence of the insert DNA of the positive clone. You can In addition, the 7th exon and the 8th exon are prepared by using a primer based on the DNA sequence of the intron on the 5'side of the 7th exon and the 3'side of the 8th exon, It is also possible to detect the exon and intron boundary sites at the same time.

In the present invention, each primer is used to
Although a DNA fragment containing each exon of the OTC gene is detected, all of these exons may be detected, or some of the exons may be combined in any combination. It is preferable to perform all the detections in order to make various diagnoses.

In addition, in order to detect the OTC mutant gene in the present invention, in addition to the above-mentioned method, the oligonucleotide of the present invention is used as a primer to amplify a nucleotide fragment, which is then cleaved with a restriction enzyme to obtain The mutant gene can also be easily detected by comparing the digested fragment with the digested fragment of the normal OTC gene by the restriction enzyme. That is, if there is a mutation in the recognition site of the restriction enzyme, the mutation can be detected by the different bands when the cleaved fragment is subjected to electrophoresis. For example, as described in the examples below, the sixth
A single nucleotide substitution with an amino acid change was found in the exon, which eliminates the restriction enzyme FokI recognition site.

Further, the OTC mutant gene can be easily detected by cleaving the sequence of the mutant portion of the mutant gene detected by the above method and preparing a nucleotide complementary to the sequence to prepare a probe. can do.

By examining whether the mutation thus detected changes the property of the amino acid encoded by this site or whether the mutation in the family is associated with the onset of the disease, it is possible to obtain high reliability. Can diagnose. Furthermore, when a liver biopsy is performed, it can be confirmed that the mutation causes the pathology depending on whether the activity and the characteristics of the OTC protein in the liver are changed.

[0023]

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. (1) Extraction of DNA 10 Place in a blood sampler containing an anticoagulant such as heparin or EDTA-Na.
About 30 ml of blood was collected from the patient. After white blood cells are separated, they are washed with PBS (-) etc. and frozen, and then DN
A was collected. 5 to 9 times the amount of cell lysis cooled to 4 ℃
buffer (0.32M sucrose 1% (v / v) triton X-100, 5mM MgC
The sample was placed in l _2, 10 mM Tris-HCl (pH 7.5)) and homogenized. The nuclear fraction was precipitated by centrifugation at 2500 g for 15 minutes at 4 ° C. Next, 5 ml of Salin-EDTA solution (25 mM EDTA (pH 8.
0), 75 mM NaCl) was added to dissolve the precipitate. 10% sodium
After adding 0.5 ml of dodecyl sulfate and stirring, 10 mg / ml
55 μl of proteinase K was added and incubated at 37 ° C. for 2 to 4 hours. The mixture was mixed with an equal amount of phenol for 10 minutes and then centrifuged for 10 minutes to collect the aqueous layer. The same operation was repeated to extract the protein with an equal amount of chloroform: phenol (1: 1) and an equal amount of chloroform. DNA was precipitated by adding 100% ethanol stored at -20 ° C, which is twice the amount of the aqueous layer. Remove ethanol to avoid losing the precipitate,
It was washed with 70% ethanol and then vacuum dried. 1 ml of 50 mM
It was dissolved in Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM EDTA. Add RNase A to 50 μg / ml for 45 minutes
Incubated at 37 ° C. 10% sodium dodecyl sulfa
Add 100 μl of te and add 10 μg of 10 mg / ml proteinase K
1 and the mixture was incubated at 37 ° C for 60 minutes. It was extracted with chloroform-isoamino alcohol (24: 1). One-tenth amount of 3M sodium Acetate was added, and the DNA was precipitated with 2.5 times amount of 100% ethanol. Ethanol was removed so as not to lose the precipitate, the DNA was washed with 70% ethanol, and then vacuum dried. The resulting precipitate was added to TE buffer (10 mM Tris-HCl (pH 7.5),
It was dissolved in 1 mM EDTA).

(2) Polymerase chain reaction (PCR) PCR was performed for each exon of the 10 OTC genes (however, the 7th exon and the 8th exon were reacted simultaneously with one set of primers). The following solution was added to 1 μl (μg / ml) of the chromosomal DNA obtained in the above step (1). 10 × reaction buffer 10 μl primer (10 pmol / μl) 5 μl primer (10 pmol / μl) 5 μl dNTP (each 1.25 mM) 16 μl Taq DNA polymerase (5 units / μ1) (Takara Shuzo) 0.5 μl sterile distillation Water 62.5 μl

Here, the composition of 10 × reaction buffer is
100 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgC
L ₂ , 0.1% (w / v) gelatin was used. The DNA sequences of the primers used are shown in FIG. P
The CR reaction conditions are 94 ° C for the denaturation step 1
Min, 55 ° C for 1 minute in the annealing process (37 ° C for the 5th, 7th and 8th exons), 72 for the extension process
℃ 2 minutes as one cycle, after 30 cycles,
An additional reaction of 5 minutes at 72 ° C was added. In the reaction, mineral oil was layered on the reaction solution to prevent evaporation.

(3) Cloning 1) Kination reaction The PCR reaction product of the above step (2) was treated with phenol. Add 1/10 volume of 3M sodium acetate
The DNA was precipitated with 100% ethanol, ethanol was removed so as not to lose the precipitate, the mixture was washed with 70% ethanol, and then vacuum dried. The obtained precipitate was dissolved in 7 to 50 μl of sterile distilled water. 7 μl of this solution was mixed with the following solution. 10 × kination buffer 1 μl T4 polynucleothiode kinase (10 units / μ1) (Takara Shuzo) 1 μl 50 mM ATP 1 μl

Here, the composition of 10 × kination buffer is
0.5M Tris-HCl (pH 7.6), 0.1M MgCl ₂ , 50 mM dithiot
hreitol, 1 mM spermidine HCl, 1 mM EDTA (pH 8.
0) was used. The mixed solution was reacted at 37 ° C for 30 minutes to phosphorylate the 5'end. After treating the reaction solution with phenol, add 1/10 volume of 3M sodium acetate and precipitate the DNA with 2.5 volumes of 100% ethanol. Remove the ethanol to prevent precipitation and wash with 70% ethanol, then vacuum dry. did. The precipitate obtained is sterilized in distilled water 20.5.
It was dissolved in μl.

2) Blunting of the ends of the PCR reaction product The above solution was mixed with the following solution in order to completely blunt the ends of the PCR reaction product. 10 × Klenow buffer 2.5 μl Large Fragment E.coli DNA Polymerase I (Takara Shuzo) 1 μl dNTP (2 mM each) 1 μl Here, the composition of 10 × Klenow buffer is 0.5 M Tris-HC.
1 (pH 7.6) and 0.1M MgCl ₂ was used. After reacting the above mixed solution at room temperature for 30 minutes, 70 ° C 5
The enzyme activity was deactivated in minutes.

3) Electrophoresis To remove non-specific products, enzymes, dNTPs, etc., 3 μl of a solution of bromophenol blue / xylene cyanol / ficol was added to the above solution, and a low melting point agarose gel containing ethidium bromide was used. After electrophoresis, the desired double-stranded DNA is cut out under long-wavelength ultraviolet light, DNA is extracted from the gel, and TE buffer (10 mM Tris
-Dissolved in HCl (pH 7.5), 1 mM EDTA).

4) Ligation into vector (ligati
on) Reaction A DNA solution was prepared with TE buffer to a concentration of 0.1 to 0.5 pmol / μl, and 1 μl thereof was mixed with the following solution. 10 × ligation buffer 1 μl CIAP-treated Hinc II digested pUC18 (0.1 μg / μl) 1 μl sterilized distilled water 6 μl T4 DNA ligase (350 units / μ1) 1 μl Here, the composition of 10 × ligation buffer is 0.66M Tris-
HCl (pH 7.6), 66mM MgCl ₂ , 100mM dithiothreitol, 1
The one consisting of mM ATP was used. This mixed solution was reacted at room temperature for 3 hours.

5) Transformation Ca ⁺⁺ treated E. coli and the above ligation reaction solution (5 to 5)
10 μl), mix and leave on ice for 30 minutes, then 50 μg /
LB agar medium containing ml ampicillin (Bacto-try
ptone 10g, Bacto-yeast extract 5g, NaCl 10g, Agar
15 g was dissolved in distilled water, adjusted to pH 7.5 with NaOH and adjusted to 1 l, and then autoclaved). The transformed cells were recovered. The transformed colonies were transferred to LB liquid medium (LB
Agar medium composition excluding Agar) 8 in 5 ml
Cultured for more than an hour.

1.5 to 5 ml of this was centrifuged. The resulting pellet was mixed with 50 μl of 50 mM Glucose, 25 mM Tris-HCl.
(pH 8.0), 10 mM EDTA dissolved uniformly in ice. 10 mg
/ Ml Lysozyme, 50 mM Glucose, 25 mM Tris-HCl (pH
8.0), 50 μl of 10 mM EDTA was added, and the mixture was left at room temperature for 5 minutes.
200 μl of 0.2N NaOH and 1% SDS solution was added, mixed gently, and then left on ice for 5 minutes. A 5M potassium acetate solution (pH 4.8) was added, mixed gently and allowed to stand in ice for 5 minutes or more.

The supernatant was recovered by centrifugation at 12000 rpm and 4 ° C. for 10 minutes. After mixing with an equal amount of phenol and centrifuging for 5 minutes, the aqueous layer was collected. The same operation was repeated with an equal amount of chloroform: phenol (1: 1) and an equal amount of chloroform.
DNA was precipitated by adding 2.5 volumes of 100% ethanol.
After centrifugation, remove ethanol to prevent precipitation and remove 70%
After washing with ethanol, it was vacuum dried. 50 μl TE buffe
It was dissolved in r (10 mM Tris-HCl (pH 7.5), 1 mM EDTA).
1 μl of 0.5 mg / ml Rnase A was added and incubated at 37 ° C. for 30 minutes. 30% 20% PEG6000, 2.5M NaCl solution
After adding μl and mixing well, the mixture was left in ice for 1 hour or more.

After centrifugation, the supernatant was removed so as not to lose the precipitate, the mixture was washed with 70% ethanol, and then vacuum dried. After drying, 30 to 50 μl of TE buffer (10 mM Tris-HCl (pH 7.5),
1 mM EDTA) was used as the plasmid DNA solution. 2 μg of DNA is taken from the plasmid DNA solution,
To this, 2 μl of 2N NaOH and sterile distilled water were added to make 20 μl. After standing at room temperature for 5 minutes, 8 μl of 5M ammonium acetate solution was added. After 100 μl of 100% ethanol was added and mixed, the mixture was left at −80 ° C. for 5 minutes. After centrifugation, ethanol was removed so that the precipitate was not lost, the precipitate was washed with 70% ethanol, and then vacuum dried.

(4) Sequencing 1) Determination of Mutation in Patient Nine kinds of clones containing the first to tenth exons obtained in the above step (3) and the boundary sites between each exon and intron were analyzed by the dideoxy chain termination method. Were determined. as a result,
A single base substitution involving an amino acid change was found in the 6th exon. This mutation is in GGATG at the recognition site for the restriction enzyme FokI.
Substitution of A for T, resulting in the change of 196th aspartic acid to valine. Since there are no other base substitutions, the mutated 196th aspartic acid is conserved across species, and the properties of amino acids change significantly, this base substitution is considered to be the cause of mutant OTC. It was

2) Kindred analysis The sixth exon in which the mutation in the patient and the parent exist and the boundary site between the exon and the intron are amplified by the PCR method using the primers shown in FIG. 10, and the difference in the cleavage due to the restriction enzyme FokI digestion is compared. did. In addition, two kinds of synthetic oligonucleotides complementary to the normal and mutant sequences consisting of 19 bases were used as probes, and the ASO method (Allele specific oligonucleotide
hybridization) was performed.

The sixth exon of parents, children and normal women was amplified, cleaved with restriction enzyme FokI, and electrophoresed. Amplified DN
A is a 201 bp band and is normally cleaved to 107 bp and 94 bp by FokI, but the FokI recognition site has disappeared in the patient.
It remained at 201 bp. The father was normal with two 107 bp and 94 bp detected, while the mother had three bands of 201, 107 and 94 bp, which revealed that one of the alleles had this mutation. Furthermore, by the ASO method, a band was detected with the mutant probe in the patient, with the normal probe in the father and normal woman, and in the mother with both probes.
It was in agreement with the result of family analysis by cutting.

[0038]

EFFECT OF THE INVENTION Gene amplification is carried out using the oligonucleotide of the present invention as a primer, and the sequence of the amplified gene is then sequenced to detect all mutations in the exons and the exons and introns of the OTC gene. The existence can be confirmed. Therefore, according to the present invention, DNA diagnosis of OTC deficiency can be easily and surely performed.

[0039]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GAGTTTTCAA GGGCATAGAA TCGTC 25

SEQ ID NO: 2 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence AGTTTTATGC ATCACCATGA TTCCT 25

SEQ ID NO: 3 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence CACCATAGTA CATGGGTCTT TTCTGA 26

SEQ ID NO: 4 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence AAAAAGGGGA CTGGTAGTAA TGGAAC 26

SEQ ID NO: 5 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence CACTTATTTG GGGGCTAGTT ATTACTTA 28

SEQ ID NO: 6 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GTCTTCACCT TCAATCCCTC TCTTC 25

SEQ ID NO: 7 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GTTGAGATGA TGGCCAATTC TTTGT 25

SEQ ID NO: 8 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence CCATCAGATT CTGAAATCAG CTTTG 25

SEQ ID NO: 9 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GCATTATTAA GCATAATTAT CTTAG 25

SEQ ID NO: 10 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence TTCACTTAAA GCAAGTCAGG AATTA 25

SEQ ID NO: 11 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence TGGATTTCAT CTCCTTCATC CCGTG 25

SEQ ID NO: 12 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GTGATTTAAG AGAGGGGTTA GTTTC 25

SEQ ID NO: 13 Sequence length: 26 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence CCTAAATAAG ATTTAAATTC CTTCCT 26

SEQ ID NO: 14 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GGAATTAATG AACCTGAGAG AGCAT 25

SEQ ID NO: 15 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence GCCACATATA ATAGTCAAAA AGTGG 25

SEQ ID NO: 16 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence ATCTCACTTG CTTATTATTT CCCCA 25

SEQ ID NO: 17 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence TCTAGGTCCC TAAGCAGACT GTCGC 25

SEQ ID NO: 18 Sequence length: 25 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence CAACTCATTC TGTTACTGAA GAACA 25

[Brief description of drawings]

FIG. 1 shows the nucleotide sequence of the OTC gene containing exon 1. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 2 shows the nucleotide sequence of the OTC gene containing exon 2. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 3 shows the nucleotide sequence of the OTC gene containing exon 3. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 4 shows the nucleotide sequence of the OTC gene containing exon 4. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 5 shows the nucleotide sequence of the OTC gene containing exon 5. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 6 shows the nucleotide sequence of the OTC gene containing exon 6. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 7 shows the nucleotide sequence of the OTC gene containing exons 7 and 8. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 8 shows the nucleotide sequence of the OTC gene containing exon 9. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 9 shows the nucleotide sequence of the OTC gene containing exon 10. The underlined portion indicates the primer and the doubled line indicates the exon.

FIG. 10 shows a list of A sequences and a sequences to I sequences and i sequences used as primers.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 15/10 15/54 // C12N 9/10 7823-4B (72) Inventor Kazunori Shimada Osaka Prefecture 4-6-1-609 Yamada Nishi, Suita City (72) Minoru Matsuura Exhibition 5-20-23 Black Hair, Kumamoto City, Kumamoto Prefecture

Claims (22)

[Claims] 1. An oligonucleotide consisting of the following A sequence (SEQ ID NO: 1) or a sequence (SEQ ID NO: 2):
Or 15 to 40, each of which has at least 15 contiguous bases or more in part of the A sequence or the a sequence
An oligonucleotide which is an A sequence fragment or a sequence fragment consisting of 1 base. A sequence: 5'-GAGTTTTCAAGGGCATAGAATCGTC-3 'a sequence: 5'-AGTTTTATGCATCACCATGATTCCT-3' 2. An oligonucleotide consisting of the following B sequence (SEQ ID NO: 3) or b sequence (SEQ ID NO: 4),
Or 15 to 40, each of which has at least 15 contiguous bases or more in part of the B sequence or the b sequence
An oligonucleotide which is a B sequence fragment or a b sequence fragment consisting of one base. B sequence: 5'-CACCATAGTACATGGGTCTTTTCTGA-3 'b sequence: 5'-AAAAAGGGGACTGGTAGTAATGGAAC-3' 3. An oligonucleotide consisting of the following C sequence (SEQ ID NO: 5) or c sequence (SEQ ID NO: 6),
Or 15 to 40, each of which has at least 15 contiguous bases or more in part of the C sequence or the c sequence
An oligonucleotide which is a C sequence fragment or a c sequence fragment consisting of one base. C sequence: 5'-CACTTATTTGGGGGCTAGTTATTACTTA-3 'c sequence: 5'-GTCTTCACCTTCAATCCCTCTCTTC-3' 4. An oligonucleotide consisting of the following D sequence (SEQ ID NO: 7) or d sequence (SEQ ID NO: 8):
Or 15 to 40, each of which has at least 15 contiguous bases in the D sequence or the d sequence
An oligonucleotide which is a D sequence fragment or a d sequence fragment consisting of one base. D sequence: 5'-GTTGAGATGATGGCCAATTCTTTGT-3 'd sequence: 5'-CCATCAGATTCTGAAATCAGCTTTG-3' 5. An oligonucleotide consisting of the following E sequence (SEQ ID NO: 9) or e sequence (SEQ ID NO: 10), or a part of at least 15 consecutive bases of the E sequence or the e sequence. Have in 15 ~
An oligonucleotide which is an E sequence fragment or an e sequence fragment consisting of 40 bases. E sequence: 5'-GCATTATTAAGCATAATTATCTTAG-3 'e sequence: 5'-TTCACTTAAAGCAAGTCAGGAATTA-3' 6. An oligonucleotide comprising the following F sequence (SEQ ID NO: 11) or f sequence (SEQ ID NO: 12), or a part of at least 15 consecutive bases of each of the F sequence or the f sequence. Have in 15 ~
An oligonucleotide which is an F sequence fragment or f sequence fragment consisting of 40 bases. F sequence: 5'-TGGATTTCATCTCCTTCATCCCGTG-3 'f sequence: 5'-GTGATTTAAGAGAGGGGTTAGTTTC-3' 7. An oligonucleotide consisting of the following G sequence (SEQ ID NO: 13) or g sequence (SEQ ID NO: 14), or a part of at least 15 consecutive bases of the G sequence or g sequence, respectively. Have in 15 ~
An oligonucleotide which is a G sequence fragment or a g sequence fragment consisting of 40 bases. G sequence: 5'-CCTAAATAAGATTTAAATTCCTTCCT-3 'g sequence: 5'-GGAATTAATGAACCTGAGAGAGCAT-3' 8. An oligonucleotide consisting of the following H sequence (SEQ ID NO: 15) or h sequence (SEQ ID NO: 16), or a part of at least 15 consecutive bases of the H sequence or the h sequence. Have in 15 ~
An oligonucleotide which is an H sequence fragment or an h sequence fragment consisting of 40 bases. H sequence: 5'-GCCACATATAATAGTCAAAAAGTGG-3 'h sequence: 5'-ATCTCACTTGCTTATTATTTCCCCA-3' 9. An oligonucleotide consisting of the following I sequence (SEQ ID NO: 17) or i sequence (SEQ ID NO: 18), or a part of at least 15 consecutive bases of the I sequence or i sequence. Have in 15 ~
An oligonucleotide which is an I sequence fragment or an i sequence fragment consisting of 40 bases. I sequence: 5'-TCTAGGTCCCTAAGCAGACTGTCGC-3 'i sequence: 5'-CAACTCATTCTGTTACTGAAGAACA-3' 10. An oligonucleotide consisting of the A sequence according to claim 1 or an oligonucleotide which is an A sequence fragment is used as one primer, and an oligonucleotide consisting of the a sequence according to claim 1 or an oligonucleotide which is an a sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 11. An oligonucleotide consisting of the B sequence according to claim 2 or an oligonucleotide which is a B sequence fragment is used as one primer, and an oligonucleotide consisting of the b sequence according to claim 2 or an oligonucleotide which is a b sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 12. An oligonucleotide comprising the C sequence according to claim 3 or an oligonucleotide which is a C sequence fragment as one primer, and an oligonucleotide comprising the c sequence according to claim 3 or an oligonucleotide which is a c sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 13. An oligonucleotide consisting of the D sequence according to claim 4 or an oligonucleotide which is a d sequence fragment is used as one primer, and an oligonucleotide consisting of the d sequence according to claim 4 or a d sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 14. An oligonucleotide consisting of the E sequence according to claim 5 or an oligonucleotide which is an E sequence fragment is used as one primer, and an oligonucleotide consisting of the e sequence according to claim 5 or an oligonucleotide which is an e sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 15. An oligonucleotide comprising the F sequence according to claim 6 or an oligonucleotide which is an F sequence fragment is used as one primer, and an oligonucleotide comprising the f sequence according to claim 6 or an oligonucleotide which is an f sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 16. An oligonucleotide consisting of the G sequence according to claim 7 or an oligonucleotide which is a G sequence fragment is used as one primer, and an oligonucleotide consisting of the g sequence according to claim 7 or an oligonucleotide which is a g sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 17. An oligonucleotide comprising the H sequence according to claim 8 or an oligonucleotide which is an H sequence fragment is used as one primer, and an oligonucleotide comprising the h sequence according to claim 8 or an oligonucleotide which is an h sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 18. An oligonucleotide consisting of the I sequence according to claim 9 or an oligonucleotide which is an I sequence fragment is used as one primer, and an oligonucleotide consisting of the i sequence according to claim 9 or an oligonucleotide which is an i sequence fragment. As the other primer
An ornithine transfection characterized by amplifying a specific nucleotide fragment by repeating a chain length reaction with one primer and a separation operation of a chain length reaction product from a template, and then sequencing the sequence of the amplified nucleotide fragment. Method for detecting carbamylase mutant gene. 19. A method for detecting an ornithine transcarbamylase mutant gene, which comprises using all or part of the detection methods according to claims 10 to 18 in combination. 20. A nucleotide fragment amplified by using the oligonucleotide according to any one of claims 1 to 9 as a primer is cleaved with a restriction enzyme, and the obtained cleavage fragment and a fragment of a normal ornithine transcarbamylase gene cleaved by the restriction enzyme. And a method for detecting an ornithine transcarbamylase mutant gene, comprising: 21. The detection method according to claim 20, wherein the primer is the nucleotide according to claim 6 and the restriction enzyme is FokI. 22. A method for detecting an ornithine transcarbamylase mutant gene, which comprises using a nucleotide complementary to the sequence of the mutant portion of the mutant gene detected by the detection method according to claim 10 as a probe.