KR20100123353A - Method of determining myotonic dystrophy type 1 using semi-quantitative pcr - Google Patents

Abstract

PURPOSE: A method for diagnosing type 1 myotonic dystrophy by semiquantitative multiplex PCR is provided to quickly and simply diagnose the disease and to predict the risk of disease. CONSTITUTION: A method for providing information needed for diagnosing type 1 myotonic dystrophy comprises: a step of measuring DMPK(dystrophia myotonica protein kinase) gene (GenBank accession No. NM_004409.3) level of DNA sample from an individual; and a step of comparing the gene level with that of healthy person. The gene level is measure by semiquantitative multiplex PCR.

Description

Method of Determining Myotonic Dystrophy Type 1 Using Semi-quantitative PCR}

The present invention relates to a technique for diagnosing type 1 myotonic dystrophy by measuring abnormal amplification of the DMPK gene specific to type 1 myotonic dystrophy using semiquantitative multiplex polymerase chain reaction.

Myotonic dystrophy type 1 (DM1) is a hereditary muscle disease with muscular symptoms, muscle degeneration atrophy, and various systemic clinical symptoms such as cataracts, dyspnea, arrhythmia, and the like. The DMPK (dystrophia myotonica protein kinase) gene 3 Gene mutations due to unstable amplification of tribasic repeat sequences (CTGs) located in non-transcriptional areas are a major cause.

The existing molecular genetic diagnostic method of DM1 is to identify the presence of tribasic repeat sequences increased more than 50 times. To this end, genomic DNA is isolated from peripheral blood and polymerase chain reaction is performed using specific primers for the surrounding nucleotide sequences including the tribasic repeat sequence in the DMPK gene. For the result, the peak size according to the sequence number is obtained by using a GeneScan Analysis program of ABI Prism 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). From the test result, the number of 146 reference sequences is subtracted, divided by three, and the number of reference tribase repeats is added five times to obtain the number of repeats of the tribase of each allele.

Normal humans can identify peaks for both alleles, but in DM1 patients, alleles with abnormally increased trinucleotide repeat sequences are significantly increased in gene size, which is detected only by gene scan tests through general polymerase chain reaction. In most cases, only one peak for one normal allele can be identified. In this case, the Southern Blot Method can be used to directly identify a 9 kb or 10 kb band resulting from an abnormal increase in the number of tribasic repeat sequences.

Currently, if a single peak is identified as a result of a gene scan test, it cannot be distinguished whether it is a result of a normal homozygote allele or a normal heterozygote allele of a DM1 patient.

For this reason, the diagnosis of DM1 patients must perform a step of identifying a band of 9 kb or 10 kb or more by a sudden blot test. The sudden blot method is not only very complicated but also requires a long time of 2-3 weeks. Diagnosis is a difficult way. Therefore, the development of an accurate, faster and easier diagnostic method is required.

The present invention, by developing a test method for providing an objective basis for effectively distinguishing between homozygotes and heterozygotes, by complementing the conventional diagnosis of type 1 myotonic dystrophy as described above, patients and carriers are selected more quickly and easily. It was designed to do so.

Accordingly, one example of the present invention is to provide a technique for quickly, easily and accurately diagnosing type 1 myotonic dystrophy by measuring whether or not abnormal amplification of the DMPK gene using a semi-quantitative multiple polymerase chain reaction method. It is done.

As described above, type 1 myotonic dystrophy 1 (DM1) is a genetic disease caused by abnormal amplification of the CTG tribasic repeat sequence in the DMPK gene. Diagnosis of patients with type 1 myotonic dystrophy and carriers can be made by checking the size of the tribasic repeat sequence, followed by polymerase chain reaction test to confirm normal and 50-100 CTG repeat sequences, and sudden blot test. The only test is to identify abnormally amplified CTG repeats. The polymerase chain reaction can be tested relatively easily and quickly, but only alleles of the normal CTG repeat sequence range and some carriers can be diagnosed, and the CTG repeat homozygotes and heterozygotes of abnormal alleles cannot be distinguished. Therefore, most patients and carriers can be diagnosed only by Sudden Blot. However, the Sudden blot method is very complicated and difficult, and the test is very difficult because it takes 2-3 weeks.

Accordingly, the present invention is a novel test for screening patients and carriers of type 1 myotonic dystrophy, using semi-quantitative multiple polymerase chain reaction, and the normal and carrier using only polymerase chain reaction without Sudden Blot. / Has the advantage of screening patients. The main feature of the present invention is a normal heterozygote by simultaneously amplifying a reference gene such as a DMPK gene and amelogenin by a semi-quantitative multiple polymerase chain reaction method, and then analyzing the number and size of the amplified alleles. And normal homozygotes, heterozygous carriers and patients at high risk can be selected quickly and easily.

Hereinafter, the present invention will be described in more detail.

One example of the present invention

1) measuring the level of dystrophia myotonica protein kinase (DMPK) gene (GenBank accession No. NM — 004409.3) of the DNA sample obtained from the sample; And

Comparing the obtained result with the level of a gene other than the DMPK gene (hereinafter referred to as 'reference gene') in the same DNA sample, or the level of the DMPK gene of a normal person

It relates to a method for providing information necessary for the diagnosis of type 1 myotonic dystrophy comprising.

All of these gene levels were measured by semiquantitative multiplex polymerase chain reaction, where two alleles are homozygous without Sudden Blot and one allele is normal and the other is abnormally amplified DMPK gene. The heterozygote can be distinguished from one another, and thus the presence of the DMPK gene can be confirmed more simply and accurately.

In more detail, the general polymerase chain reaction method uses a pair of primers to exponentially amplify a nucleotide sequence of a desired position on a gene. The maximum amplification value of the amplification product can be obtained regardless of. In this case, when the amount of DNA used for the reaction is increased, the maximum amplification value is reached within a faster reaction time, and when a small amount of DNA is amplified, the reaction time for reaching the maximum amplification value is longer.

Semi-quantitative polypolymerase chain reaction is a method that reverses this characteristic, using a very small amount of DNA in the reaction and appropriately adjusting the reaction time and conditions to stop the reaction before reaching the maximum amplification value. It is a method to infer the amount of genes used in the reaction by checking.

As can be seen in Figure 1, generally, when the maximum amplification value of the polymerase chain reaction method is reached, the amount of amplified gene is saturated, and the amount of gene amplified from a pair of homozygous alleles (blue ① ) And the amount of genes amplified from alleles of one of the heterozygous alleles (marked in red) become the same, making it impossible to distinguish between them. On the other hand, if the polymerase chain reaction is stopped before the maximum amplification level, the amplified gene amount of a pair of homozygous alleles is about twice the amount of the amplified gene of one allele of the heterozygote. It is possible to distinguish between and heterozygotes.

In diagnosis of type 1 myotonic dystrophy, amplification of the abnormally amplified DMPK gene by polymerase chain reaction increases the amount of the gene to 10 kb or more (see FIG. 3). The gene scan test did not identify a peak. Therefore, the results of polymerase chain reaction of heterozygotes containing DMPK genes with abnormally amplified CTG repeat sequences show only normal allele results with one peak in the gene scan test. In addition, since a pair of homozygous alleles have the same CTG repeat sequence, the polymerase chain reaction result is observed only as one peak in the gene scan test.

Therefore, if only one peak of the DMPK gene is observed as a result of polymerase chain reaction, the general polymerase chain reaction method that performs the reaction up to the maximum amplification value shows the amount of gene amplified from the normal homozygous allele. It is difficult to distinguish whether the result shows the amount of the gene amplified from the normal allele of the heterozygous containing, and thus, a complex identification process such as a sudden blot should be additionally performed. However, when using a semiquantitative multiplex polymerase chain reaction as in the present invention, the amplified gene amount of a pair of homozygous alleles is about 2 of the amplified gene amount of a normal heterozygous allele of the carrier or patient. As a result, the presence of abnormal heterozygous alleles, including abnormal amplification of the DMPK gene, can be determined without performing further identification.

For the relative quantification of the gene amount of DMPK measured through semi-quantitative multiplex polymerase chain reaction, it is possible to compare the amount of DMPK gene amplified from normal homozygous as a control, or to reference genes other than DMPK gene in the same DNA sample. have. In a more preferred embodiment, by amplifying a reference gene present at a constant level simultaneously with the DMPK gene irrespective of the development of type 1 myotonic dystrophy in the DNA sample, by quantifying the amount of the DMPK gene relative to the reference gene The accuracy can be improved.

The reference gene to be compared is a gene having a constant gene quantity without overlapping, deletion, or similar sequence while having a sequence of conditions that can be amplified simultaneously with an allele by a polymerase chain reaction regardless of whether type 1 myotonic dystrophy is developed. Any one can be used, and in an embodiment of the present invention, the amelogenin gene (GenBank accession No. NM_001143.1), the GAPDH gene (GenBank accession No. NM_002046.3), and the ABL1 gene (GenBank accession No. One or more genes selected from the group consisting of reference genes such as NM_007313.2) and HPRT1 gene (GenBank accession No. NM_000194.2) may be used, but is not limited thereto. For example, amylogenin is a specific gene present in the X and Y chromosomes, which is characterized by gender, and has the advantage of preventing some of the specimen inversion by using the same. In a preferred embodiment of the present invention, amylo Zenin was selected and used as a representative reference gene.

2A to 2C, for example, in the case of FIG. 2B, since two peaks corresponding to the DMPK gene are observed in a normal range, both alleles may be determined as heterozygotes that are normal. However, since only one peak corresponding to the DMPK gene is observed in FIGS. 2A and 2C, it is necessary to distinguish whether the result is a normal homozygosity or a result of one normal allele of the patient. While the corresponding peak height is almost equal to the sum of the peak heights of the amelogenin gene as the reference gene, it can be judged as a normal homozygous, while in FIG. 2C, the peak corresponding to the DMPK gene is the reference gene. About half of the sum of the peak heights corresponding to the amelogenin gene is considered to be a normal allele of a heterozygous patient with an abnormal allele that does not become a peak in the gene scan test.

If the level of the DMPK gene in the DNA sample obtained from the sample is less than or equal to about 60% of the level of the reference gene in the same DNA sample, or the level of the DMPK gene in normal individuals, the DMPK gene is determined to contain abnormally amplified abnormal alleles. As such, the sample may be determined to be a patient with type 1 myotonic dystrophy type 1 (DM1).

 In the present invention, the conditions of the semi-quantitative multiple polymerase chain reaction method is a method of quantifying by terminating the reaction before the reaction reaches the maximum amplification value, and conditions may vary depending on the DNA sample used. The reaction time and temperature of the polymerase chain reaction vary depending on the properties of the primer and / or DNA used in each experiment, and most of the experiments determine the temperature, time, and the number of reactions that are considered most suitable. Can be used. In addition, when the polymerase chain reaction proceeds under the same conditions, the higher the DNA concentration used, the faster the reaction equilibrium (plateau) is reached. It is not a problem to use a high DNA concentration when performing a conventional polymerase chain reaction.However, when using a semiquantitative method for comparative quantification as in the present invention, the equilibrium state should not be reached, so that the minimum amount of DNA is required. Use in concentration is easy to control the reaction.

In the present invention, the amount of all DNA samples that can be used in the semiquantitative polymerase chain reaction method for measuring the level of the DMPK gene is 0.5 ng to 5 ng, preferably 0.7 to 3 ng, most preferably 1 ng. In the semi-quantitative polypolymerase chain reaction method, the annealing reaction temperature range is about 52 ° C to 62 ° C, preferably about 60 ° C, and the total number of cycles is 26 to 30 times, preferably about 28 times. The annealing reaction time may be 30 seconds to 90 seconds, preferably 50 seconds to 70 seconds, but is not limited thereto.

For example, if the DNA sample concentration is about 1 ng, the conditions of the semiquantitative multiplex polymerase chain reaction are as follows:

95 ° C., 5 minutes (once),

94 ° C, 1 minute-60 ° C, 1 minute-72 ° C, 1 minute (28 times),

60 ° C., 60 minutes.

In the present invention, the level of the DMPK gene is measured to determine whether the amplification of the tribasic (CTG) repeat sequence of the DMPK gene is abnormal. , DMPK gene may be a fragment comprising a tribasic (CTG) sequence region. Therefore, the primers that can be used in the semi-quantitative polymerase chain reaction method for measuring the level of the DMPK gene in the present invention include all 10 to 50 bp designed to be amplified, including the tribasic (CTG) sequence region of the DMPK gene, Preferably it may be a polynucleotide of size 15 to 30 bp, for example, the base sequence of SEQ ID NO.1 (5'-GAAGGGTCCTTGTAGCCGGGAA-3 ') and SEQ ID NO.2 (5'-GGAGGATGGAACACGGACGG-3') Primer pairs can be used.

As mentioned above, the present invention is designed to predict the risk of disease morbidity by identifying the presence of abnormal alleles including tribasic repeat sequences that are increased more quickly and easily in carrier selection and diagnosis of patients. The present invention has been completed by providing a semi-quantitative multiple polymerase chain reaction test that can effectively distinguish between the homozygote allele and one allele of the heterozygote when only one peak is identified.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example  1: DNA Sample Preparation

DNA was obtained from peripheral blood of normal subjects and patients using the Roche High Pure PCR Template Preparation Kit. The normal person was a healthy person clinically showing no type 1 myotonic dystrophy and other neuromuscular symptoms. In addition, the patients were diagnosed with type 1 myotonic dystrophy through molecular genetic tests such as Sudden Blot, showing clinical symptoms appropriate to the disease. 200ul of Binding Buffer (Roche Diagnostics, Mannheim, Germany) and 40ul of Proteinase K (Roche Diagnostics) were mixed with 200ul of whole blood, and then incubated at 70 ° C for 10 minutes. Here, 100ul of isopropanol was added and mixed well. A filter tube (High Filter tube, Roche Diagnostics) was put in a collection tube, the prepared sample sample was transferred thereto, and centrifuged at 8000 g for 1 minute.

After centrifugation, the filter tube was removed from the collection tube and the filtered liquid was discarded and the removed filter tube was placed in a new collection tube. 500 ul of Wash Buffer (Roche Diagnostics) was added thereto, and centrifuged at 8000 g for 1 minute. The Wash Buffer addition and centrifugation were repeated once more. Rotate for 10 seconds at maximum centrifugal force to remove remaining Wash Buffer. The filter tube was placed in a new microtube and 200 ul of prewarmed Elution Buffer (Roche Diagnostics) was added and centrifuged at 8000 g for 1 minute. The DNA thus extracted was stored frozen until used for testing.

Example  2: Tribasic  Check the number of repetitive sequences

The number of repeat sequences was measured using a polymerase chain reaction method to selectively amplify the tribasic repeat sequences in the DMPK gene.At this time, a fluorescent material was attached to a primer to be used, and a capillary electrophoresis method using a fluorescent standard was used. Including the step of determining the size (amplification) of the amplification product using the following steps were carried out.

The polymerase chain reaction method was performed using the DNA sample prepared in Example 1. The base sequence of the primer is as follows:

Sense: FAM-GAA GGG TCC TTG TAG CCG GGA A (SEQ ID NO.1)

Antisense: GGA GGA TGG AAC ACG GAC GG (SEQ ID NO.2)

Add 1ul (> 500ng) of DNA sample, 33ul of sterile distilled water, 5ul of Buffer (Roche Diagnostics), 4ul of dNTP, 2.5ul of primer (DM1a, DM2), 1.5ul of formamide, and 0.5ul of Expanded High Fidelity (Roche Diagnostics) The polymerase chain reaction was performed under the conditions.

<Polymerase Chain Reaction Conditions>

95 ° C., 5 minutes (once),

94 ° C, 1 minute-60 ° C, 1 minute-72 ° C, 1 minute (28 times),

60 ° C., 60 minutes.

The amplification products of the DMPK gene obtained by the polymerase chain reaction were analyzed by capillary electrophoresis. 1 ul of amplified product diluted 10-fold, 10 ul of formimide, and 0.3 ul of ROX standard were mixed. The mixture was heated at 95 ° C. for 2 minutes, immediately cooled on ice, mounted in ABI 3130xl Genetic Analyze (Applied Biosystems, Foster City, CA, USA) and subjected to capillary electrophoresis. Amplification products were sized by comparing the mixed standards using the GeneScan Analysis program.

Example  3: Tribasic  When checking the number of repeat sequences homozygote  to distinguish between allele and heterozygote allele Semiquantity  Multiple Polymerase Chain Reaction

If one peak is identified as a result of the current gene scan test, it is not possible to distinguish whether the result is from a normal homozygous allele or a normal heterozygous allele of a DM1 patient. Therefore, the sudden blot test must be performed to check the bands of 9 kb and 10 kb or more, so that the DM1 patient can be judged. In order to solve this inconvenience, in this embodiment, a method of separating homozygous alleles and heterozygote alleles using a semiquantitative method of polymerase chain reaction in a gene scan test for checking trinucleotide repeat sequences It was.

Based on the theory that all genes are amplified at a constant rate within the same polymerase reaction under appropriate conditions, using the Amelogenin gene (GenBank accession No. NM_001143.1) as a reference gene for the quantification of DMPK genes. Multiple polymerase chain reaction was performed. Amelogenin gene is a gene having a constant amount and can be used as a suitable standard for relatively comparative quantification of a DMPK gene having a homozygous or heterozygous value.

The components, concentrations and reaction conditions of the semiquantitative multiplex polymerase chain reaction were as follows:

<Primer>

DMPK-GS-F: 5'-FAM-GAAGGGTCCTTGTAGCCGGGAA-3 '(SEQ ID NO. 1)

DMPK-GS-R: 5'-GGAGGATGGAACACGGACGG-3 '(SEQ ID NO. 2)

Amelogenin-GS-F: 5'-FAM-CCCTGGGCTCTGTAAAGAATAGTG-3 '(SEQ ID NO. 3)

Amelogenin-GS-R: 5'-ATCAGAGCTTAAACTGGGAAGCTG-3 '(SEQ ID NO. 4)

<Composition of polymerase reaction solution>

dNTP: 200mM

MgCl ₂ : 1.25mM

Expanded high fidelity buffer (Roche Diagnostic): 2.5ul

Expanded high fidelity (Roche Diagnostic): 0.75ul

DMSO (dimethyl sulfoxide): 5%

Primer: 400nM each

DNA sample: 1 ng

<Reaction Condition>

95 ℃, 5min (once)

94 ℃, 1min-60 ℃, 1min-72 ℃, 1min (28 times)

60 ℃, 60min

For gene scanning using amplification products, RODi standards were prepared by mixing 30ul of HiDi formamide (Applied Biosystems, Foster City, CA, USA) and 30ul of 500 ROX size standard Applied Biosystems.

The mixture obtained by mixing 10 ul of ROX standard with 1 ul of the amplified product was heated at 95 ° C. for 2 minutes, cooled immediately on ice, mounted on ABI 3130xl Genetic Analyze (Applied Biosystems), and subjected to capillary electrophoresis. The size and amount of amplification products were determined by comparing the mixed standards using the GeneScan Analysis program (Applied Biosystems).

The relative quantitative value of the DMPK gene for the amelogenin used as the reference gene is calculated and compared to the quantitative value of a normal sample that is known to be homozygous. It can be distinguished whether the normal heterozygous allele is the result of amplification.

The results obtained above are shown in FIGS. 2 to 5.

2a to 2c exemplarily show a result of a gene scan test in which a polymerase chain reaction was performed with an amelogenin gene-specific primer and a DMPK gene-specific primer as reference markers in the test. Figure 2a is a 5 times three salt-based box homozygotes, the ratio of the gene scan test results of the amelogenin gene and DMPK gene was 93.6%, it can be determined as a normal homozygotes, this case is determined as the control group of the test Corrected to 100%. Figure 2b is a heterozygosity of five times the three salt-based boxer sequence and 14 times the three salt-based boxer sequence, showing two peak results, the ratio of each allele to the amylogenin gene is 51.4%, 43.5%, when corrected with a control As 54.9% and 46.5%, respectively, it can be judged as a normal heterozygote. Figure 2c shows only one peak result and only five tris salt-based boxers are observed. If this is a normal homozygotes, the allele for the amylogenin gene should be close to 100% for the amylogenin gene. Since the ratio of 51.5% and 55.0% of the control group was found to be corrected, it can be confirmed that the heterozygote of the 5 times trisulfate-based sequence, which includes an increased tribasic repeat sequence not detected in the gene scan. The presence of the DM1 allele, which could not be confirmed by the multiple polymerase chain reaction test, was confirmed.

3 is an example of the results of the sudden blot test. Patient 1 shows normal 9kb and 10kb bands, so it can be seen that both alleles contain the normal range of tribasic repeats. Patients 2 and 3 can be confirmed as DM1 patients because the 9kb normal allele and the band resulting from the increased number of tribasic repeats of 12 kb and 11 kb, respectively, are identified.

The results of the semi-quantitative polypolymerase chain reaction test of normal males and normal females confirmed to be normal homozygotes through the sudden blot test are shown in FIGS. 4A and 4B, and the tribasic repeat sequence increased in the DMPK gene using the sudden blot test. Semi-quantitative multiple polymerase chain reaction test results of male patients and female patients confirmed to have are shown in Figures 5a and 5b, respectively.

Figure 1 is a schematic diagram showing a quantitative method of semi-quantitative multiple polymerase chain reaction method.

Figures 2a to 2c shows the results of a gene scan test for multiplex polymerase chain reaction with an amelogenin gene-specific primer and a DMPK gene-specific primer as reference markers in the test, 2a is three times The results of base repeat homozygotes, 2b are the results of the heterozygote of the 5 trisulfate-based sequence and 14 times the trisulfate-based sequence, and 2c is the heterozygosity of the 5 trisulfate-based sequence, which is not confirmed by the genetic scanning test. Results are shown for patients with abnormal alleles, including increased tribasic repeats.

Figure 3 shows an example of the result of the sudden blot test.

Figures 4a and 4b shows the results of the semi-quantitative multiplex polymerase chain reaction test of the normal sample confirmed to be homozygous using the sudden blot test, 4a is the result of a normal man, 4b is the result of a normal woman.

Figures 5a and 5b shows the results of the semi-quantitative polymerase chain reaction test in the sample confirmed to have an increased tribasic repeat sequence using the Southern blot test, 5a is the result of a male patient, 5b is a female patient Is the result.

<110> SAMSUNG LIFE PUBLIC WELFARE FOUNDATION SAMSUNG MEDICAL CENTER <120> Method of Determining Myotonic Dystrophy Type 1 Using          Semi-quantitative PCR <130> DPP-2008-5297-KR <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DMPK-GS-F primer <400> 1 gaagggtcct tgtagccggg aa 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DMPK-GS-R primer <400> 2 ggaggatgga acacggacgg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin-GS-F primer <400> 3 ccctgggctc tgtaaagaat agtg 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin-GS-R primer <400> 4 atcagagctt aaactgggaa gctg 24  

Claims (5)

Measuring the level of dystrophia myotonica protein kinase (DMPK) gene (GenBank accession No. NM — 004409.3) of the DNA sample obtained from the sample; And Comparing the obtained result with the level of genes other than the DMPK gene in the same DNA sample, or the level of DMPK gene in normal persons Including, The gene level is measured by a semiquantitative multiple polymerase chain reaction method, If the level of the DMPK gene in the DNA sample obtained from the sample is less than 60% of the level of genes other than the DMPK gene in the same DNA sample, or the tribasic repeat sequence (CTG) level of the normal DMPK gene, the sample is Type 1 Myotonic dystrophy type 1 (DM1) patients characterized in that judging How to provide information needed to diagnose type 1 myotonic dystrophy. The method of claim 1, Genes other than the DMPK gene include amelogenin gene (GenBank accession No. NM_001143.1), GAPDH gene (GenBank accession No. NM_002046.3), ABL1 gene (GenBank accession No. NM_007313.2), and HPRT1 One or more selected from the group consisting of genes (GenBank accession No. NM_000194.2) How to provide information needed to diagnose type 1 myotonic dystrophy. The method according to claim 1 or 2, The semi-quantitative multiple polymerase chain reaction method is to be carried out with a DNA sample concentration of 0.5ng to 5.0ng, How to provide information needed to diagnose type 1 myotonic dystrophy. The method according to claim 1 or 2, The semi-quantitative multiple polymerase chain reaction method is carried out by the annealing reaction temperature range of 52 ℃ to 62 ℃ and the total number of cycles 26 to 30 times, How to provide information needed to diagnose type 1 myotonic dystrophy. The method according to claim 1 or 2, Semi-quantitative polypolymerase chain reaction for measuring the level of the DMPK gene is a nucleotide sequence of SEQ ID NO: 1 (5'-GAAGGGTCCTTGTAGCCGGGAA-3 ') and SEQ ID NO: 2 (5'-GGAGGATGGAACACGGACGG-3') Is performed using a pair of primers having How to provide information needed to diagnose type 1 myotonic dystrophy.

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