JP2001299392A - Method of detecting catch22 syndromes - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a diagnostic technique for detecting CATCH22 syndrome by measuring the degree of gene expression simply, rapidly and inexpensively in comparison with the conventional techniques, for example, the FISH method and the CGH method. SOLUTION: The polynucleotide specimen including the base sequence of the exon 12 area in the UFD1L gene sequence given by the sequence No.1 (see the specification document of this invention) on the chromosome 22q11.2 is measured by the quantitative real time PCR detection technique whereby the defects in the base sequence are detected and the CATCH22 syndrome is detected and the oligonucleotide to be used as a probe or a primer for detecting the syndrome is provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for detecting CATCH22 syndrome, wherein the deletion or excess of a gene on a specific chromosome is detected by a quantitative real-time PCR detection method, and an oligonucleotide used for the detection.

[0002]

2. Description of the Related Art It has recently been found that various diseases such as cancer show changes such as deletion of tumor suppressor genes and overexpression of cancer-related genes. Conventionally, detection of these gene deletions and the like has been mainly performed by chromosome segregation, but this method cannot detect minute deletions of genes that cannot be detected by an optical microscope. As a method for detecting such a microdeletion of a gene, there is known fluorescence in situ hybridiz
ation: FISH) method is known, for example, using this method, DiGeorge syndrome or C
A method for detecting the deletion and mutation of a gene containing ATCH22 syndrome (CATCH-22 syndrome) has been proposed (Japanese Patent Application Laid-Open No. 7-501691).

That is, it is known that a CATCH22 syndrome patient has a deletion of about 3 Mb on chromosome 22q11.2, and the proposed method is based on the chromosome 22q11.2.
The above-mentioned CATCH syndrome is diagnosed by performing the FISH method using the N25DGCR probe having the partial nucleotide sequence present above.

However, this method (FISH method)
However, there are problems that detection takes a long time, a large amount of a required sample, and difficulty in handling and performing a sample, and there is a need in the art for development of an improved technique to replace this.

[0005] On the other hand, separately from the above, carrier diagnosis of gene deficiency in a family with a medical history is based on loss of heterozygosity analysis using polymorphic markers of the gene (loss of heterozygosity).
ygosity analysis: LOH). However,
Although this LOH method is convenient for carrier diagnosis of genes in a family with a history of disease, it requires a marker with a large amount of information near the target gene, and the determination of individuals unrelated to the family is difficult. There are drawbacks that are difficult.

[0006] Another method for determining gene excess is competitive genetic hybridization (Comparative Genetic Hybridization).
Genomic Hybridization: CGH; Kallioniemi, OP, et
al., Science, 258 , 818-821 (1992)), according to which a gene deletion or overexpression can be determined. However, this method requires a gene size of about 10 Mbp to determine the increase or decrease of one copy, and cannot determine trisomy 21 (trisomic), and furthermore, as in the FISH method, However, there are disadvantages that it takes time and the measurement procedure is troublesome.

[0007] In recent years, the PCR method developed for obtaining large amounts of gene fragments has been applied to the quantification of the expression level of RNA in tissues and the amount of viral DNA. (TaqMan)
A high-precision quantitative PCR method has been developed that can detect twice the gene amount difference by kinetic analysis that monitors the PCR reaction in real time using a probe.
(Homogeneous test system: Patent registration No. 2825976, ABI PR
ISM 7700 Sequence Detection System User's Manual:
5.10.5.13 (1996)).

[0008] The quantitative PCR method is called "quantitative real-time PCR detection method" and is applied to the measurement of various virus genes (measurement of EB virus gene: Japanese Patent Application Laid-Open No.
No. 137300, Measurement of HCV virus gene: JP-A-11-103
No. 899, measurement of HBV virus gene: JP-A-11-103897
issue). Furthermore, it has been reported that the method can be applied to the determination of X chromosome differences between men and women and deletion of tumor suppressor genes (Boulay, JL, et al., Biotechniques, 27 , 228-230).
(1999)), according to the results of identification of carriers in cancer families using DNA extracted from peripheral blood as a sample, the LOH
It is also reported that the method is simpler than the FISH method.
(Laurendaeau, I., et al., Clin. Chem., 45 (7) 982-
986 (1999)).

[0009] As described above, conventionally, a CATCH22 syndrome patient has taken advantage of the fact that there is a deletion of about 3 Mb on chromosome 22q11.2 to obtain an N25DGCR probe having a partial base sequence existing on chromosome 22q11.2. Diagnosis has been made by the FISH method used (Tokuhei Hei 5-501691).
However, this method has the disadvantages that it takes a long time to detect, requires a large amount of sample, and has difficulties in handling and handling the sample. Instead, the CATC method is easier, simpler and shorter.
There is a need in the art to develop a technology that can detect, diagnose, etc., the H22 syndrome.

[0010]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a simpler and less time-consuming and improved technique for quantifying and detecting genetic diseases, which is required in the art.

In particular, the present invention relates to CATCH22 syndrome,
Quantification of genetic diseases such as trisomy 1 (Down syndrome)
It is an object of the present invention to provide the above-described improved technique capable of performing detection and diagnosis with higher sensitivity.

In view of the above problems, the present inventors have attempted to establish a simpler method of clinical diagnosis of CATCH22 syndrome instead of the FISH method. First, a gene on chromosome 22q11.2. About, to create an oligonucleotide having the partial base sequence, these were used as a probe or primer, and the idea of performing a quantitative real-time PCR detection method was conceived.
Even in diseases other than CATCH 22 syndrome and trisomy 21, there was a case where the same gene was microdeleted, and it was confirmed that a satisfactory result for clinical diagnosis could not always be obtained depending on the setting of primers and probes used.

However, as a result of intensive research,
According to a quantitative real-time PCR method using an oligonucleotide primer and a probe having the following specific partial base sequence, it has been found that the target CATCH syndrome is clearly distinguished from other diseases and detected. That is, the CATCH22 syndrome was suspected from clinical symptoms, but no gene deletion was observed according to the conventional FISH method. When the PCR method was performed, results consistent with the FISH method were obtained, and thus the CATCH2
It has been found that the two syndromes can be accurately diagnosed. The present invention has been completed based on such findings.

[0014]

According to the present invention, a UFD represented by SEQ ID NO: 1 on chromosome 22q11.2 is provided.
There is provided a method for detecting CATCH22 syndrome, comprising measuring a polynucleotide sample containing the nucleotide sequence of the exon 12 region of the 1L gene sequence by a quantitative real-time PCR detection method to detect a deletion of the nucleotide sequence.

In particular, according to the present invention, the above-described method for detecting CATCH22 syndrome, wherein a nucleotide sequence having a length of at least 15 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 1 is used as a probe or primer; Has the nucleotide sequence of 15 to 30 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 1, and the method for detecting CATCH22 syndrome; and the probe or primer has SEQ ID NO: 2, The method for detecting CATCH22 syndrome selected from those having the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 is provided.

Further, according to the present invention, the CATCH22 syndrome is detected by a quantitative real-time PCR detection method, which comprises a nucleotide sequence having a length of 15 to 30 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 1. Oligonucleotides used as probes or primers to perform

In particular, according to the present invention, there is provided the above-mentioned oligonucleotide having the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.

The method of the present invention can be used for CATCH22 syndrome or 2
It is an improved detection method that can accurately and easily detect clinical diagnosis of trisomy 1 with high sensitivity and that meets the needs of the art.

The method of the present invention can process a large number of specimens within a certain period of time as compared with the conventional method, and can improve the efficiency of the test and reduce the test time. It is also very useful in that an order inspection can be performed at the same time.

Further, according to the present invention, similarly, 2
A method for detecting trisomy 1 is provided.

The method comprises measuring a polynucleotide sample containing the S100β gene represented by SEQ ID NO: 5 on chromosome 21 by a quantitative real-time PCR detection method to detect gene excess in the sample. It is characterized by.

In the detection of trisomy 21, a nucleotide sequence having a length of at least 15 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 5 is used as a probe or primer. The nucleotide sequence of the probe or primer is more preferably 15 to 30 nucleotides in length from the nucleotide sequence shown in SEQ ID NO: 5, and specific examples thereof are SEQ ID NO: 6, SEQ ID NO: : 7 and SEQ ID NO: 8.

Furthermore, according to the present invention, SEQ ID NO:
3 from consecutive 15 bases in the base sequence represented by 5
Oligonucleotides consisting of a base sequence of 0 bases and used as probes or primers for detecting trisomy 21 by quantitative real-time PCR detection method, in particular, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8 The probe or primer having the nucleotide sequence represented by

[0024]

BEST MODE FOR CARRYING OUT THE INVENTION The quantitative real-time PCR detection method employed in the detection method of the present invention can basically follow a known method. The operation etc., specifically,
For example, it is described in Japanese Patent No. 2825976. Also, PE
The description in the ABI PRISM 7700 Sequencing System User Manual from Biosystems can also be followed.

[0025] The method of the present invention comprises the quantitative real-time PC
In the R detection method, as a probe and a primer,
It is important to use one having a specific base sequence. Specific examples of the base sequence include the following. (1) Contiguous one of the nucleotide sequences represented by SEQ ID NO: 1 (the nucleotide sequence of the exon 12 region of the UFD1L gene)
An oligonucleotide (forward primer) having a base sequence of 5 to 30 bases in length, (2) a base sequence of 15 to 30 bases in sequence of the base sequence shown in SEQ ID NO: 1 (3) an oligonucleotide probe having a nucleotide sequence having a length of 15 to 47 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 1, and (4) an oligonucleotide probe having the nucleotide sequence shown in SEQ ID NO: 1. ), A reporter fluorescent dye and a quencher fluorescent dye are bound to the oligonucleotide probe, and when the reporter fluorescent dye is bound to the probe to which the quencher fluorescent dye is bound, the reporter fluorescent dye The resonance energy transfer suppresses the fluorescence intensity, and the quencher fluorescent dye is bound to the bound probe. The have a state, in which the fluorescence intensity is not suppressed, the probe.

The present invention also provides the forward primer (1), the reverse primer (2)
UF to be measured in the test sample using the probe of (4)
UFD1L in a test sample consists of performing a PCR reaction using the gene in the exon 12 region of the D1L gene sequence as a template, and measuring the fluorescence from the reaction solution in real time.
A method for measuring an exon 12 region gene of a gene sequence is also provided. The present invention also provides a method for diagnosing CATCH22 syndrome using a measurement value obtained by the measurement method.

More specifically, the 5'-side primer (forward primer) used in the method of the present invention has a length of at least 15 bases, which is a contiguous sequence of the base sequence represented by SEQ ID NO: 1, as described above. Has a base sequence of 15 to 30 bases in length. Among them, particularly preferable ones include those having a base sequence of 18 bases represented by SEQ ID NO: 2.

The base sequence represented by SEQ ID NO: 1
Reported all cases had been deleted in CATCH22 syndrome
(Pizzuti, A., et al., Hum.Mol. Genet.,6
(2), 259-265 (1997): Yamagishi, H., et al., Scienc.
e,283(5405), 1158-1161 (1999)), from 1156 bases long
UFD1L gene (human ubiquitin fusion-degradation 1-like
Protein (Homo sapiens ubiquitin fusion-degradation 1 l
ike protein): Accession number: Exon of U64444)
12 (its base sequence is shown in SEQ ID NO: 1)
It is an existing base sequence.

The entire nucleotide sequence of the UFD1L gene is known (for example, Novelli, G., et al., Biochem. Bioph.
ys Acta, 1396 (2) 158-162 (1998), Gene Bank
(GenBank) accession number: U64444).

The 3 'primer (reverse primer) used in the method of the present invention has a length of at least 15 consecutive nucleotides, preferably 15 to 30 consecutive nucleotides in the nucleotide sequence shown in SEQ ID NO: 1. Is selected from those having the base sequence of Of these, particularly preferred ones include those having a base sequence of 19 bases represented by SEQ ID NO: 3. This corresponds to exon 12 1109-11 of the UFD1L gene.
This corresponds to the 27th sequence.

The probe used in the method of the present invention is a probe in which a reporter fluorescent dye and a quencher fluorescent dye are bound to an oligonucleotide. The oligonucleotide portion of the probe is selected from those having a continuous base sequence of at least 15 bases, preferably 15 bases to 30 bases in the base sequence shown in SEQ ID NO: 1. Among them, particularly preferable ones include those having a base sequence of 28 bases shown in SEQ ID NO: 4. The base sequence represented by SEQ ID NO: 4 corresponds to exon 12 of the UFD1L gene.
This corresponds to the nucleotide sequence at positions 1066 to 1093 in (base sequence represented by SEQ ID NO: 1).

The region within exon 12 of the UFD1L gene to which the oligonucleotide portion of the above probe hybridizes does not partially overlap with the region to which the 5′-side primer hybridizes, but it is necessary to carry out the actual PCR reaction. It is necessary to select a combination of the primer and the probe so that the regions where the 5′-side primer and the probe to be used hybridize do not overlap each other.

When the reporter fluorescent dye is bound to the same probe as the quencher fluorescent dye, the fluorescence intensity of the reporter fluorescent dye is suppressed by fluorescence resonance energy transfer, and the same as the quencher fluorescent dye. When the probe is not bound to the probe, the fluorescence intensity is not suppressed. As the reporter fluorescent dye, any known various dyes can be used, and for example, a fluorescein-based fluorescent dye such as FAM (6-carboxy-fluorescein) is preferable.
As the quencher fluorescent dye, various known dyes can be used, and for example, a rhodamine-based fluorescent dye such as TAMRA (6-carboxy-tetramethyl-rhodamine) is preferable. As these fluorescent dyes, those contained in commercially available kits for real-time detection PCR can also be used as they are.

[0034] The binding positions of the reporter fluorescent dye and the quencher fluorescent dye are not particularly limited. Usually, the reporter fluorescent dye is provided at one end (preferably the 5 'end) of the oligonucleotide portion of the probe, and the quencher fluorescent dye is provided at the other end. Should be combined. The binding between the oligonucleotide and the fluorescent dye can be performed according to a known method (for example, Noble et al., (1984), Nuc. A).
cids Res. 12 : 3387-3403; Iyer et al., (1990), J. A.
m. See the method described in Chem. Soc. 112 : 1253-1254).

In the method of the present invention, the 5′-side primer, the 3′-side primer, and the probe are used,
Exon 1 of UFD1L gene to be measured in test sample
Using the inside of 2 as a template, reverse transcription PCR (RT-PCR) is performed, and the fluorescence from the reaction solution is measured in real time. The real-time detection PCR method itself is known, and apparatuses and kits for the real-time detection PCR method are also commercially available. The present invention can be carried out using such commercially available devices and kits.

In the PCR reaction, for example, DNA or RNA extracted and prepared from a subject is used as a sample, and the above 5 ′ primer, 3 ′ primer and probe, and a heat-resistant DNA polymerase (which also has reverse transcriptase activity) ), DATP, dGTP, dCTP and dTTP, and can be carried out according to a conventional method.
If the sample is RNA, use dUTP instead of dTTP.
And contaminating DNA from the PCR product can be degraded by adding uracil-N-glycosylase (UNG).

The conditions for the PCR reaction can be the same as those known to be employed in this kind of real-time PCR detection method. The specific conditions are described in detail in Examples below.

The sample is a CATC to be detected.
It may be derived from a patient suspected of having H syndrome, ie, a patient suspected of having a desired gene deficiency, and may be, for example, a body fluid such as blood of the patient.

In the PCR reaction, for example, a genomic DN is extracted from a peripheral blood sample using a commercially available DNA extraction kit or the like.
A can be prepared and used as it is as DNA for amplification.

Since the amplified DNA contains a region complementary to the probe used in the present invention, the probe hybridizes with the single-stranded amplified DNA. In the state where the probe is completely hybridized, when extension using the single-stranded DNA to which the probe is hybridized as a template occurs, the probe is hydrolyzed from the 5 'end side by the exonuclease activity of DNA polymerase. As a result of this degradation, the reporter fluorescent dye and the quencher fluorescent dye bound to the oligonucleotide portion of the probe are separated, and the reporter fluorescent dye, which has been suppressed by the fluorescence resonance energy transfer caused by the quencher fluorescent dye, The fluorescence intensity increases. On the other hand, when the DNA in the exon 12 region of the UFD1L gene is not present in the sample, the amplification of the DNA does not occur, so that the probe does not hybridize to the DNA and is not hydrolyzed by the DNA polymerase. For this reason,
Fluorescence from the reporter fluorescent dye remains suppressed by the quencher fluorescent dye and the fluorescence intensity does not increase. Utilizing such a principle, by measuring the fluorescence intensity,
It is possible to detect a predetermined gene in exon 12 of the UFD1L gene in the specimen sample.

In the method of the present invention, the fluorescence intensity is measured in real time. That is, while measuring the fluorescence intensity, the PC
Perform the R reaction. The measured fluorescence intensity exceeds the detection limit after a certain number of cycles and increases rapidly. And
D of exon 12 region of UFD1L gene in specimen sample
As the NA amount increases, the fluorescence intensity increases rapidly with a small number of cycles. Therefore, by examining the number of cycles after which the rapid increase in the fluorescence intensity starts, the quantitative measurement of the DNA of the predetermined gene in the specimen sample can be performed.

More specifically, for example, 10 times the standard deviation of the fluorescence intensity in each cycle (for example, 3 to 15 cycles) in a negative control containing no DNA in the exon 12 region of the UFD1L gene is set as a threshold value, By examining the number of cycles in which the intensity exceeds this threshold, exon 1 of the UFD1L gene in the specimen sample is determined.
It is possible to accurately and quantitatively measure DNA in two regions. DN of exon 12 region of UFD1L gene in specimen sample
When the common logarithm of the number A is plotted on the horizontal axis and the number of cycles when the threshold value is exceeded is plotted on the vertical axis, the measurement results are almost completely on a straight line. Therefore, if such a calibration curve is prepared, the number of cycles in which the threshold value is exceeded can be checked to determine the number of U in the sample sample.
The amount of DNA in the exon 12 region of the FD1L gene can be quantitatively measured. As described above, according to the method of the present invention, unlike the conventional semi-quantitative PCR method, there is no need to perform an operation of performing electrophoresis after PCR to check amplification, and the method of the present invention is very simple in this regard.

[0043]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Reference Examples and Examples, but the present invention is not limited to these Examples.

[0044]

[Reference Example 1] Detection of CATCH22 syndrome using the FISH method Facial abnormalities, hypocalcemia, tetralogy of fallot (Tetralogy of
Fallot, TOF), Pulmonary Atresin, P
A), CAT based on clinical symptoms such as congenital heart disease (TOF + PA)
About 24 patients suspected of CH22 syndrome, N25
(D22S75) Using a probe (manufactured by Oncor),
It was tested by the ISH method whether there was a deletion in the region of 22q11.2. The FISH method is described in Tokuhei Hei 5-501.
This was performed according to the method described in JP-A-691.

As a result, in all of the 24 cases, 22q1
It was confirmed that there was a deletion in the 1.2 region. That is, the fluorescent probe normally binds to two chromosomes q11.2 and two signals are detected, but only one signal is detected in the CATCH22 patient.

[0046]

Example 1 Method for Detecting CATCH22 Syndrome of the Present Invention As a sample for measurement, C-diagnosed in Reference Example 1 was used.
Peripheral blood samples obtained from 24 patients with ATCH22 syndrome, 22 patients with the same CATCH syndrome diagnosed by G-banding staining, and 40 normal patients were used.

For these samples, a quantitative real-time PCR detection method using a specific sequence as a primer and a probe was performed as follows.

In all cases diagnosed as having CATCH22 syndrome, a conical artery trunk abnormal face (CTAface) was observed.
In addition, congenital heart disease (CHD), hypocalcemia, and cleft palate were recognized. The syndrome included one parent and child.

All patients with Down syndrome (22 patients) had typical symptoms such as a down-like face and developmental delay.

In all normal cases, there were no abnormal facial features or other malformations, normal development, and no CHD was observed by echocardiography. No chromosome test was performed on these normal cases. a) Preparation of genomic DNA From peripheral blood of each of the above patients, QIAampDNAMini was used.
Genomic DNA was extracted using Kit (QIAGEN), and the concentration of the extracted DNA was measured with a UV spectrometer (Shimadzu). The above-mentioned DNA concentration was adjusted to 15 to 25 ng / μl in the measurement sample used in the detection method of the present invention. Also,
The DNA concentration of a normal adult (separately prepared normal adult peripheral blood) for preparing a calibration curve was similarly adjusted to 35 ng / μl. b) Preparation of Probes and Primers In CATCH22 syndrome, 2
It has been reported that all the UFD1L genes in the gene on 2q11.2 were deleted (Pizzuti, A., et a).
l., Hum. Mol. Genet., 6 (2), 259-265 (1997): Yamagis
hi, H., et al., Science, 283 (5405), 1158-1161 (199
9)).

The present inventors focused on the nucleotide sequence of the exon 12 region of the UFD1L gene as a gene on 22q11.2, and set the following primers and probes having a specific partial nucleotide sequence present in the region. .

That is, the 18mer shown in SEQ ID NO: 2
Was used as a UFD1L forward primer, a 19-mer oligo DNA shown in SEQ ID NO: 3 was used as a UFD1L reverse primer, and a 28-mer oligo DNA shown in SEQ ID NO: 4 was used as a UFD1L probe.

In order to detect the deletion of a certain chromosome, other chromosomes must be examined and their ratios determined. The other chromosome may be any autosomal chromosome other than chromosome 22, but assuming that it also serves as a diagnostic method for trisomy 21 (Down's syndrome), which is the most frequent chromosomal disorder in humans, It was decided to quantify the S100β gene contained in the responsible region of Down syndrome.

The gene on chromosome 21 includes S1 on the long arm whose excess was determined in quantitative PCR.
Probes and primers having the following specific DNA sequence present in exon 2 of 00β were used (Chiang,
PW, et al., Genome Res., 6 , 1013-1026 (1996)).

That is, the 19-mer represented by SEQ ID NO: 6
Is used as a forward primer of S100β (corresponding to the 47-65 sequence of exon 2 of S100β), and a 21-mer oligonucleotide shown in SEQ ID NO: 7 is used as a reverse primer of S100β (119 of exon 2 of S100β). And the 28me shown in SEQ ID NO: 8.
r oligonucleotide with the S100β probe (S10
(Corresponding to the 86-111 sequence of exon 2 of Oβ).

Further, for each of the above two probes,
FAM at each 5 'end and TA at each 3' end.
Each MRA was bound by the method described in the above-mentioned literature to prepare a labeled probe. c) Quantitative real-time PCR (TaqManPCR) Quantitative real-time PCR using each primer and probe prepared in b) above was performed using ABI PRISM7.
700 Sequence DetectionSyst
em (manufactured by PE Biosystems) in accordance with the handling manual of the device, as follows.

That is, 0.2 pmol / μl of each forward and reverse primer and 0.1 pmol / μl of the probe were used, and 2 × TaqMan Universal
Using PCR Mastermix (manufactured by PE Biosystems),
The total reaction volume was adjusted to 35 μl. Then, change this to 16
μl for 2 minutes to measure UFD1L gene and S10
It was used for 0β gene measurement.

The reaction conditions were preincubation at 50 ° C. for 2 minutes, denaturation at 95 ° C. for 10 minutes, followed by 95%
40 cycles of 15 ° C. for 15 seconds and 60 ° C. for 1 minute were employed. The DNA for the calibration curve (36 ng / μl) was doubled from the stock solution by 16 times.
Five-fold dilutions were performed.

The fluorescence intensity was measured at each cycle of PCR, and the change in the fluorescence intensity was plotted on the vertical axis and the cycle number was plotted on the horizontal axis. There is a linear relationship between the common logarithm of the copy number of DNA in the specimen sample and the number of cycles exceeding the threshold at which the rapid increase in fluorescence intensity starts, and by measuring this number of cycles, the desired number of DNA can be measured quantitatively. Analysis of the results obtained by these PCR reactions
Analysis software (Sequence Detect
or).

The initial DNA amount of the desired gene in each sample is displayed as a relative value with the measured value of the stock solution of the standard curve DNA sample as 1000, and this value is used to calculate C
In the case of ATCH22 syndrome, the ratio of the UFD1L gene / S100β gene was calculated using the S100β gene as a standard gene, and in the case of Down syndrome, the ratio of the S100β gene / UFD1L gene was calculated using the UFD1L gene as a standard gene. That is, the logical value in this measurement method is 0.5 for gene deletion and 1. for normal.
0, and 1.5 for trisomy. d) Analysis results Tables 1 and 2 below show the analysis results of the reaction between the sample and each probe and primer determined according to c) above.

[0061]

[Table 1]

[0062]

[Table 2]

From Table 1, the CATCH22 syndrome and the normal group were clearly distinguished (99.7% confidence interval) without overlap even in the range of mean ± 3SD. This means that the CATC of the present invention
It has been shown that the H22 syndrome detection method can be used instead of the conventional method.

Further, from Table 2, it was proved that the diagnosis of Down syndrome was not duplicated with that of the normal group with an average of ± 3 SD.

[0065]

According to the present invention, it has become possible to measure the presence of a target gene with high sensitivity, accurately, quickly, and inexpensively with a small amount of sample in order to accurately diagnose CATCH22 syndrome.

[0066]

[Sequence List] SEQUENCE LISTING <110> Ostuka Pharmaceutical Co., ltd. <120> Detection method for CATCH22 syndrome <130> DB00JP <160> 8 <170> PatentIn Ver. 2.0 <210> 1 <211> 1156 <212> DNA <213> Exone 12 of homo sapiens ubiquitin fusion-degradation 1 like protein (UFD1L) gene <400> 1 ggcacgagga agagcggtcg gcggggtttc ttcgttgcat tgcctgagag gagcggagtc 60 tgccaggtgg tgtccatcat gttctctttc aacatgttcg accaccctat tcccagggtc 120 ttccaaaacc gcttctccac acagtaccgc tgcttctctg tgtccatgct agcatggcct 180 aatgacaggt cagatgtgga gaaaggaggg aagataatta tgccaccctc ggccctggac 240 caactcagcc gacttaacat tacctatccc atgctgttca aactgaccaa taagaattcg 300 gaccgcatga cgcattgtgg cgtgctggaa tttgtggctg atgaaggcat ctgctacctc 360 ccacactgga tgatgcagaa cttactcttg gaagaagacg gcctggtcca gttggagacc 420 gtcaaccttc aagtggccac ctactccaag agtaagttct gctacctccc acactggatg 480 atgcagaact tactcttgga agaaggcggc ctggtccagg tggagagcgt caaccttcaa 540 gtggccacct actccaaatt ccaacctcag agcgctgact tcctggacat caccaacccc 600 aaagccgtat tagaa aacgc acttaggaac tttgcctgtc tgaccaccgg ggatgtgatt 660 gccattaact acaatgaaaa gatctacgaa ctgcgtgtga tggagaccaa acccgacaag 720 gcagtgtcca ttcatgagtg tgacatgaac gtggactttg atgctcccct gggctacaaa 780 gaacccgaaa gacaagtcca gcatgaggag tcgacagaag gtgaagccga ccacagtggc 840 tatgctggag agcttggctt ccgcgctttc tctggatctg gcaatagact ggatggaaag 900 aagaaagggg tagagcccag cccctcccca atcaagcctg gagatattaa aagaggaatt 960 cccaattatg aatttaaact tggtaagata actttcatca gaaattcacg tccccttgtc 1020 aaaaaggttg aagaggatga agctggaggc agattcgtcg ctttctctgg agaaggacag 1080 tcattgcgta aaaagggaag aaagccctaa gtgaggactg ttggctgatt ggaaaataat 1140 aaaagtttca tttgca 1156 <210> 2 <211> 18 <212> DNA <213> 2 gt11c <2> ttgg11c <400> > DNA <213> Reverse primer sequence <400> 3 cagccaacag tcctcactt 19 <210> 4 <211> 28 <212> DNA <213> Prove sequence <400> 4 tctggagaag gacagtcatt gcgtaaaa 28 <210> 5 <211> 172 <212 > DNA <213> Exone 2 of Human S100 protein beta-subunit gene <400> 5 cttt gcgagc ccctaggatg tctgagctgg agaaggccat ggtggccctc atcgacgttt 60 tccaccaata ttctggaagg gagggagaca agcacaagct gaagaaatcc gaactcaagg 120 agctcatcaa caatgagctt tcccatttct tagagc tct12c <t> cct tctg tctg tctct tctg tctg tctg tctg tctg tctg tctg <211> 20 <212> DNA <213> Reverse primer sequence <400> 7 gctcattgtt gatgagctcc 20 <210> 8 <211> 26 <212> DNA <213> Probe sequence <400> 8 agacaagcac aagctgaaga aatccg 26

Claims (6)

[Claims] 1. Exon 12 of the UFD1L gene sequence represented by SEQ ID NO: 1 on chromosome 22q11.2
A method for detecting CATCH22 syndrome, comprising measuring a polynucleotide sample containing a nucleotide sequence of a region by a quantitative real-time PCR detection method to detect a defect in the nucleotide sequence. 2. The CA according to claim 1, wherein a continuous nucleotide sequence of at least 15 nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 is used as a probe or a primer.
TCH22 syndrome detection method. 3. The method according to claim 1, wherein the probe or the primer is SEQ ID NO:
3 to 15 consecutive nucleotides in the nucleotide sequence represented by 1
The method for detecting CATCH22 syndrome according to claim 2, wherein the method has a base sequence having a length of 0 bases. 4. The probe or primer according to claim 1, wherein
4. The CATC according to claim 3, which is selected from those having the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4.
H22 syndrome detection method. 5. A quantitative real-time PC comprising CATCH22 syndrome consisting of 15 to 30 consecutive nucleotides in the nucleotide sequence represented by SEQ ID NO: 1.
Oligonucleotides used as probes or primers for detection by the R detection method. 6. The oligonucleotide according to claim 5, which has the nucleotide sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4.