JP2006129807A - Long chain polymerase chain reaction (pcr) method for amplifying high gc content repeated sequence and method for diagnosing facioscapulohumeral muscular dystrophy therewith - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a long chain polymerase chain reaction method by which a high GC (guanine and cytosine) content repeated sequence can be amplified, and to provide a method for simply diagnosing facioscapulohumeral muscular dystrophy (FSHD) in high reliability. <P>SOLUTION: This long chain polymerase chain reaction method is characterized by using a buffer for amplifying a high GC content DNA, using a dNTP containing 7-deaza dGT adjusted to the optimal concentration, setting the concentration of magnesium chloride to a concentration giving the best amplification efficiency, performing a thermal denaturation at high temperature for a short time, controlling an annealing/elongation temperature to a common temperature, controlling a reaction time to ≥100 times a thermal denaturation time, and further elongating the reaction time after 11 cycles. The method for diagnosing facioscapulohumeral muscular dystrophy (FSHD) comprises amplifying a genome region characteristic to the high GC content repeated sequence with a primer set specific to the fourth chromosome 4q35 by the PCR method, and then confirming the repeating number by an electrophoresis. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a long-chain polymerase chain reaction (PCR) method, a chromosome 4 specific primer set, and a rapid diagnosis method of facial scapulohumeral muscular dystrophy using them.

  Fasocapulohumeral muscular dystrophy (FSHD) is the third most common genetic neuromuscular disease after Duchenne muscular dystrophy and myotonic dystrophy. It has an autosomal dominant form of inheritance, and the morbidity is said to be 2-5 per 100,000 population. The age of onset is as wide as 0 to 65 years, but it often develops by puberty. Muscular atrophy is strong on the face cheeks, shoulders, and upper arms, and is characterized by pterygium. The muscular strength of the waistband and the lower limbs is often relatively maintained at an early stage, but the disability spreads as it progresses, and a wheelchair is often required around the age of 40. Retinopathy and neurological hearing loss are also present in about 50%, and in younger cases, central nervous symptoms such as mental retardation and convulsions are often accompanied.

The FSHD locus is present in the subtelomeric region of chromosome 4 long arm 4q35. In this part, a 3.3 kb repeat sequence (D4Z4) that is digested with the restriction enzyme Kpn I is sandwiched between Eco RI cleavage sites, and normally this repeat number is about 10 to 100. On the other hand, the number of FSHD patients decreased to 7 or less (FIGS. 1 and 2). However, no causative gene was found in this deletion region, and the onset mechanism is still unknown.

Therefore, when carrying out genetic diagnosis, it is necessary to examine the length of a fragment containing the D4Z4 repeat sequence therein by Southern blot analysis using genomic DNA digested with the restriction enzyme Eco RI. What is more complicated is that a 3.3 kb Kpn I repeat sequence having 98% or more homologous sequence with this D4Z4 repeat sequence is also present in the chromosome 10 subtelomere region 10q26 and reacts with the probe p13E-11 used in Southern blot analysis. is there. Therefore, in order to determine whether the detected fragment is derived from chromosome 4 or 10, Eco RI / Bln I duplex by another restriction enzyme ( Blin I) capable of cleaving only the D4Z4 sequence of chromosome 10. Southern blot analysis using the cleaved fragment must also be used in combination.

The length of the Eco RI digested fragment containing the D4Z4 repeat sequence may be as high as 300 kb or more in normal cases, but is 28 kb or less in FSHD (FIG. 3). Also, between 29 kb and less than 50 kb, the judgment differs depending on the reporter and is currently considered as an indeterminate region. Therefore, in order to diagnose FSHD, it is necessary to extract DNA having a length of at least 50 kb. For this reason, when DNA is extracted from human tissues such as blood, it must be carefully performed over several days. The extracted genomic DNA is digested with restriction enzymes such as Eco RI and Bln I, and then electrophoresed in an electrophoresis tank with a circulation device for 68 hours using an extremely thin agarose gel of 0.3%, and then subjected to Southern blot analysis. In this case, detection is difficult unless a probe labeled with a radioisotope is used. Therefore, when this method is used, it takes 3 weeks to 1 month at the earliest to detect the length of the restriction enzyme cleavage fragment.

This method requires a large amount of genomic DNA of at least 40 μg, but it is not easy to prepare a large amount of genomic DNA from a small amount of tissue such as biopsy skeletal muscle directly. Furthermore, the Kpn I cleaved fragment (D4Z4) has an extremely high GC content of 73% or more, and is a repetitive sequence, so that it was difficult to amplify by PCR using genomic DNA.

Yamanaka G, Goto K, Ishihara T, Oya Y, Miyajima T, Hoshika A, Hayashi YK, et al. FSHD-like patients without 4q35 deletion. J Neurol Sci 2004; 219 (1-2): 89-93. Goto K, Nishino I, Hayashi YK.Very low penetrance in 85 Japanese families with facioscapulohumeral muscular dystrophy 1A. J Med Genet 2004; 41 (1): E12. Winokur ST, Chen YW, Masny PS, Martin JH, Ehmsen JT, Tapscott SJ, Hayashi YK, et al.Expression profiling of FSHD muscle supports a defect in specific stages of myogenic differentiation.Hum Mol Genet 2003; 12 (22): 2895 -2907. Matsumura T, Goto K, Yamanaka G, Lee JH, Zhang C, Hayashi YK, et al. Chromosome 4q; 10q translocations; comparison with different ethnic populations and FSHD patients.BMC Neurol 2002; 2 (1): 7. Yamanaka G, Goto K, Hayashi YK, Miyajima T, Hoshika A, Arahata K. [Clinical and genetical features of Japanese early-onset facioscapulohumeral muscular dystrophy]. No To Hattatsu 2002; 34 (4): 318-324. Yamanaka G, Goto K, Matsumura T, Funakoshi M, Komori T, Hayashi YK, et al. Tongue atrophy in facioscapulohumeral muscular dystrophy. Neurology 2001; 57 (4): 733-735. Goto K, Lee JH, Matsuda C, Hirabayashi K, Kojo T, Nakamura A, et al. DNA rearrangements in Japanese facioscapulohumeral muscular dystrophy patients: clinical correlations. Neuromuscul Disord 1995; 5 (3): 201-208 Goto K, Song MD, Lee JH, Arahata K. [Genetic analysis of facioscapulohumeral muscular dystrophy (FSHD)]. Rinsho Shinkeigaku 1995; 35 (12): 1416-1418. Lee JH, Goto K, Sahashi K, Nonaka I, Matsuda C, Arahata K. Cloning and mapping of a very short (10-kb) EcoRI fragment associated with facioscapulohumeral muscular dystrophy (FSHD). Muscle Nerve 1995; 2: S27-31 . Lee JH, Goto K, Matsuda C, Arahata K. Characterization of a tandemly repeated 3.3-kb KpnI unit in the facioscapulohumeral muscular dystrophy (FSHD) gene region on chromosome 4q35. Muscle Nerve 1995; 2: S6-13.

  The present invention provides a simple, rapid, and highly accurate FSHD gene diagnostic method that can replace the above-described complicated and skillful FSHD gene analysis method.

The feature of the present invention is that, using genomic DNA as a template, a very long region having a high GC content and having a repetitive sequence can be amplified by PCR. The Kpn I cleaved fragment (D4Z4) has an extremely high GC content of 73% or more, and it has been difficult to amplify by PCR using genomic DNA. However, the method we have developed this time makes it possible to amplify such a difficult-to-amplify repetitive sequence to a length of 18.4 kb, and in the future, the same high GC-containing genomic region (for example, Huntington repeat) ) Can be said to be applicable to PCR amplification.

  Furthermore, what is epoch-making in the present invention is a highly specific primer set designed with a repeat portion interposed so as to recognize only the D4Z4 repeat sequence of chromosome 4. By using this primer set, it is possible to reliably identify the D4Z4 repeat sequence derived from chromosome 4 without amplifying homologous sequences on other chromosomes.

By combining this primer set with a long chain PCR method, can be detected by amplifying the corresponding fragment in the Southern blotting technique in recognized Eco RI cleavage fragments up 25 kb (corresponding D4Z4 repeat sequences within 5 or less) It became possible. This covers 95% or more of FSHD having a deletion at 4q35 (see FIG. 2), and is extremely useful as a diagnostic method. Using this method, the time required after extracting the DNA is only 15 hours, and the required genomic DNA can be analyzed in an amount of 0.4 μg, which is 1/100 of the conventional amount.

Table 1 shows a comparison between the method of the present invention and the conventional method.

So far, we have estimated the number of D4Z4 repeats based on the length of the Eco RI-cut fragment obtained by Southern blotting. By using this method, we can identify a certain number of repeats, clinical course, It becomes possible to examine the correlation between the degree of gene and the size of gene deletion in more detail, and it is considered that it will bring about a great progress in elucidating the pathological mechanism.

First, a long-chain PCR method capable of amplifying a template having a high GC content according to the present invention will be described.
Next, the chromosome 4 specific primer set will be described.
Finally, analysis of FSHD patients using the above primer set and long PCR will be described.

<Long PCR method for high GC content DNA>
As a result of detailed examination of the PCR method for amplifying a region having a GC content as high as 73%, the present inventors have made heat denaturation short-time and high-temperature using a DNA polymerase for heat-resistant hot start, and have a high PCR buffer. It has been found that it is preferable to use a GC buffer in which 7-deaza dGTP is further added to dNTPs. It has also been found that the concentrations of 7-deaza dGTP and magnesium chloride have a significant effect on amplification. As a result of further examination of PCR conditions, the annealing / elongation temperature is set to a common temperature, the reaction time is set to 100 times or more of the heat denaturation time, and the reaction time is further extended after 11 cycles so that there is a high GC content and repetition. It was found that the sequence can be amplified efficiently. Specifically, the 7-deaza dGTP concentration in the reaction solution is preferably 0.27 mM to 0.33 mM. The MgCl ₂ concentration is preferably 2.6 mM to 2.8 mM. The annealing / elongation temperature is preferably 63 ° C to 68 ° C. The reaction time after 11 cycles is preferably increased by about 15 to 25 seconds. A preferable example is that 7-deaza dGTP in the reaction solution is 0.3 mM, the MgCl ₂ concentration is 2.75 mM, the annealing / elongation temperature is 64 ° C., and the reaction time after 11 cycles is increased by about 20 seconds. .

<Primer design>
A primer set that specifically amplifies D4Z4 repeats in the 4q35 region and does not amplify repeats in 10q26 is selected, and a primer set with good amplification efficiency is selected. One example of the forward primer sequence is GGCCAGAGTT TGAATATACT GTGGTCATCT CTGCTCCAG (SEQ ID NO: 1), and one example of the reverse primer sequence is CAGGGGATAT TTGGACATAT CTCTGCACTC ATC (SEQ ID NO: 2). This is the one with the highest amplification efficiency among the primer sets prepared by the inventors selecting six locations. The positional relationship of these primers is shown in FIG. This forward primer 1 partially overlaps with the probe p13E-11 (about 800 bp fragment) used in the conventional Southern blot. In this region, the sequences of chromosomes 4 and 10 are very similar, and p13E-11 recognizes both chromosomes in Southern blot analysis. However, this PCR primer differs from the sequence of chromosome 10 only by one base, but selectively amplifies only chromosome 4. The primer is not limited to the above primer as long as it can specifically amplify D4Z4 repeat in the 4q35 region.

<Primer synthesis>
Primers may be synthesized by any of the currently known methods. Used after desalting and purification.

  The DNA preparation is preferably purified from the patient's blood but can also be extracted from the tissue. In order to purify DNA from blood, genomic DNA is extracted from heparinized human peripheral blood whole blood by a conventional method, dissolved in 1 × TE buffer, and adjusted to a concentration of 0.2-0.3 μg / μl. In order to obtain high purity high molecular weight genomic DNA, avoid vigorous stirring throughout the entire process and carefully extract.

<Amplification and detection of Kpn I repeat>
By performing amplification by the above PCR method using a synthetic primer set using the above DNA preparation as a template, the D4Z4 repeat of the fourth chromosome can be amplified. The obtained amplification product is subjected to agarose electrophoresis and detected by ethidium bromide staining. The amplification products obtained are 5.2 to 18.4 kb, but these are actually 1-5 D4Z4. The fact that these amplification products are electrophoresed on an agarose gel, transferred to a nylon membrane, This can be confirmed by Southern blot analysis using the probe pMA13. In addition, the size of the purified DNA preparation can be confirmed by conventional methods (cleaving with Eco RI, electrophoresis, Southern blot using p13E-11 probe) and compared with the PCR product.

  Examples are shown below, but the present invention is not limited thereto.

  Peripheral blood was taken from 10 FSDH patients (male 6, female 4, age 7-63 years), heparin was added and gently mixed, and used in the following experiments.

1. DNA extraction from blood DNA extraction was performed according to the following procedure.
On the first day, add 10 ml of human heparin-added peripheral whole blood and 30 ml of EL buffer (10 mM KHCO ₃ , 1 mM EDTA, 155 mM NH ₄ Cl, pH 7.4) to the 50 ml centrifuge tube and mix gently.
↓
Place on ice for 15 minutes with slow mixing every 5 minutes.
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Centrifuge at 2000 rpm for 10 minutes at room temperature.
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Discard the supernatant. The process so far is repeated 2-3 times until the pellets become white.
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Add 3 ml of NL buffer (10 mM Tris-HCl, 2 mM EDTA, 400 mM NaCl, pH 8.2) and mix gently with a 2.5 ml syringe with a 21G needle so that no bubbles are generated.
↓
When the pellet is dissolved, transfer to a 15 ml centrifuge tube.
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Add 5 μl of proteinase K (10 mg / ml) (Wako Pure Chemical Industries).
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Add 150 μl of 20% SDS (sodium dodecyl sulfate).
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Mix gently.
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Centrifuge lightly to drop the liquid adhering to the tube wall.
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Incubate the centrifuge tube in a constant temperature water bath at 37 ° C. for 16 hours.
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Add 1 ml of 5M NaCl to the centrifuge tube that has been incubated on day 2.
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Mix to the extent that it is not too violent and cloudy.
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Centrifuge at 3000 rpm for 15 minutes at room temperature.
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Transfer the supernatant to a new 15 ml centrifuge tube.
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Add an equal volume of a 1: 1 phenol: chloroform solution.
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Place on rotator and mix for 15 minutes.
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Centrifuge at 2500 rpm for 10 minutes at room temperature.
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Transfer the supernatant to a new 15 ml centrifuge tube.
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Add an equal volume of chloroform solution.
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Place on rotator and mix for 15 minutes.
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Centrifuge at 2500 rpm for 10 minutes at room temperature.
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Transfer the supernatant to a new 15 ml centrifuge tube.
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Add twice the volume of 100% ethanol (stored at -20 ° C).
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Place it in a freezer refrigerator at -20 ° C for 16 hours to precipitate the DNA.
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On the third day, the centrifuge tube on which the DNA is deposited is centrifuged at 3000 rpm for 15 minutes at 4 ° C.
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Discard the supernatant.
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Wash DNA with 70% ethanol (stored at 4 ° C).
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Let the tube upside down and dry for 1-2 hours.
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Add 600 μl of 1 × TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)) and dissolve well overnight in a low-temperature room at 4 ° C. using a rotary rotator.
↓
Transfer to a 1.5 ml centrifuge tube, measure concentration, and store at 4 ° C.

2. Preparation of Primers The forward primer GGCCAGAGTT TGAATACATACT GTGGTCATCT CTGCTCCAG (SEQ ID NO: 1) and the reverse primer CAGGGGATAT TGTGACATAT CTCTGCACTACT ATC (SEQ ID NO: 2) were commissioned to the supplier for desalting and purification.

3. The composition of the reaction solution per long-chain PCR test tube is as follows.
2 × GC buffer I (TaKaRa) (containing 5 mM MgCl ₂ ) 25 μl
dATP (10 mM) (Applied BioSystems) 2.5 μl
dCTP (10 mM) (Applied BioSystems) 2.5 μl
dTTP (10 mM) (Applied BioSystems) 2.5 μl
dGTP (10 mM) (Applied BioSystems) 1.0 μl
7-deaza dGTP (10 mM) (Roche) 1.5 μl
25 mM MgCl ₂ 0.5 μl
LATaqHS (5 U / μl) (TaKaRa) 0.5 μl
Forward primer (10 pmol / μl) 1 μl
Reverse primer (10 pmol / μl) 1 μl
Sterile distilled water 10 μl
48μl total

(Here, the forward primer and the reverse primer are the ssDNA prepared in 2). )

PCR was performed according to the following procedure.
Dispense 2 μl of template DNA (0.2-0.3 μg / μl) into each of eight series of 0.2 ml centrifuge tubes and place on ice ↓
Gently add 48 μl of the above reaction mixture to each tube (do not mix).
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Set to Gene Amp PCR System 9700 (PE Applied BioSystems) and perform PCR amplification under the following conditions.

PCR condition 94 ℃ 1 minute ↓
98 ° C 10 seconds → 64 ° C 20 minutes, 10 cycles ↓
98 ° C 10 seconds → 64 ° C 20 minutes + 20 seconds / cycle, 23 cycles ↓
72 ° C for 10 minutes ↓
Store at 4 ℃

4). Electrophoresis A 0.4% agarose gel (SEAKEN HGT AGAROSE FMC) having a size of 11 × 14 cm was prepared with 1 × TAE buffer. 30 μl of the PCR product was electrophoresed at 4.2 V / cm for 3 hours using an electrophoresis apparatus (trade name HORIZON11.14 (GIBCO BRL). The agarose gel after electrophoresis was stained with ethidium bromide and then irradiated with UV (VILBERLOURMAT) A photograph was taken using the lower instant film (POLAROID MD-4), which is shown in FIG.

5. Southern Blot After photography, transfer from an agarose gel to a high bond N ⁺ membrane (Amersham Bioscience) was performed as usual, and Southern blotting was performed using the probe pMA-13 labeled with ³² P radioisotope. As already mentioned, pMA-13 contains a 1.3 kb restriction enzyme Stu I cleavage fragment in D4Z4. Presence of D4Z4 repeats from 5.2 kb to 18.4 kb was confirmed.

6). Confirmation Separately, even when the amplified PCR product was digested with the restriction enzyme Bln I, it was confirmed that there was no change in the size of the amplified product. This indicates that the amplified PCR product is not a 3.3 kb Kpn I repeat (having a Bln I cleavage site) on chromosome 10. As a result, it was confirmed that the PCR amplification product from 5.2 kb to 18.4 kb corresponds to 1-5 D4Z4 repeats on chromosome 4.
For comparison with the conventional method, the results of the conventional method ( Eco RI digestion, electrophoresis, Southern blot using p13E-11 as a probe) for the DNA samples of the above 10 patients are shown in FIG. . Here, the band marked with * is a homologous sequence on the Y chromosome recognized.

Table 2 shows the comparison between the results of the PCR method of the present invention and the results of the conventional Southern blot method for 67 patients as in Example 1. The reliability of the present invention is clear from this as well.

In the PCR method, a band corresponding to 1 to 5 D4Z4 repeats (corresponding to an Eco RI fragment length of 10 to 25 kb in the conventional Southern blot method) is amplified.

From the above results, it was revealed that a region having a very high GC content and having a repetitive sequence can be easily amplified by using the long-chain PCR method of the present invention. Furthermore, by using a primer specifically designed for chromosome 4, the gene deletion of the facioscapulohumeral muscular dystrophy (FSHD) can be regarded as a rough size change by Southern blotting of the conventional Eco RI fragment. It was possible to confirm by accurately counting the actual number of D4Z4 repeats from 1 to 5. In addition, compared to the conventional method, this method can be used for simple and rapid genetic analysis, with the amount of DNA used being 1 / 100th and the required time being about 1/5, thus accounting for 95% or more of FSHD patients. This is a very useful method for screening patients with 6 or fewer D4Z4 repeats.

It is a schematic diagram showing the difference in the number of D4Z4 repeats between the FHSD patient on chromosome 4 and a normal subject, and the positional relationship between the corresponding site of the primer, the restriction enzyme cleavage site, and the probe. 214 Example Eco RI fragment of the prior art for FSHD patients - Southern blot was carried out, which is a graph showing the distribution of Eco RI fragment length. It is the result of the Southern blot by the conventional method which shows the difference in the length of the Eco RI fragment of a FHSD patient and a normal person. □ indicates the father, ● indicates the mother of FSHD, and ■ indicates the son of FSHD. The results are the results of PCR amplification of DNA samples of 10 patients by the method of the present invention, electrophoresis of the PCR products on an agarose gel, and staining with ethidium bromide. It is the result of having performed Southern blotting by the conventional method about the DNA sample of 10 patients same as FIG.

Claims (14)

A method for amplifying a human DNA region having a high GC content and a repetitive sequence by PCR,
Use DNA polymerase for heat-resistant hot start,
Using a high GC content DNA amplification buffer as a PCR buffer,
Use dNTP plus 7-deaza dGTP adjusted to the optimal concentration,
Set the concentration of magnesium chloride to the highest amplification efficiency,
Heat denaturation at high temperature for a short time,
The annealing / elongation temperature is the common temperature,
The reaction time is at least 100 times the heat denaturation time,
A method characterized by further extending the reaction time after 11 cycles.   The method according to claim 1, wherein the 7-deaza dGTP concentration in the PCR reaction solution is 0.27 mM to 0.33 mM.   The method according to claim 2, wherein the 7-deaza dGTP concentration in the PCR reaction solution is 0.3 mM. The method according to any one of claims 1 to 3, wherein the concentration of magnesium chloride in the PCR reaction solution is 2.6 mM to 2.8 mM.   The method according to claim 4, wherein the concentration of magnesium chloride in the PCR reaction solution is 2.75 mM.   7. The method according to claim 1, wherein the reaction time after 11 cycles is extended by 15 to 25 seconds.   The process according to claim 7, characterized in that the reaction time after 11 cycles is extended by 20 seconds. The method according to any one of claims 1 to 7, wherein the annealing / elongation temperature is 63C to 68C. The method of claim 8, wherein the annealing / elongation temperature is 64 ° C.   A method for diagnosing the facioscapulohumeral muscular dystrophy using the long chain PCR method using the high GC content DNA according to claim 1 as a template, wherein at least one of the primers is chromosome 4 specific, A method for diagnosing a facial scapulohumeral muscular dystrophy using a primer set characterized in that two primers sandwich a D4Z4 repeat region and genomic DNA extracted from human tissue as a template.   The method of claim 10, wherein the human tissue is blood.   The method according to claim 9 or 10, wherein the chromosome 4 specific primer is a single-stranded DNA represented by SEQ ID NO: 1 or 2.   Primer set for use in identification of facial scapulohumeral muscular dystrophy genotype, wherein the forward primer has the sequence of SEQ ID NO: 1. The primer set according to claim 13, wherein the forward primer is SEQ ID NO: 1 and the reverse primer is SEQ ID NO: 2.

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