JPH1057065A - Primer set for amplifying sarcoglycan gene product and amplification of the same gene prodect - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject primer set, capable of screening a base mutation readily causing diseases and useful for amplification, etc., by hybridizing thereof with a part of an upstream region, etc., adjacent to a translated region of a specific sarcoglycan gene product. SOLUTION: This primer set is capable of respcrctively hybridizing with at least a part of an upstream and a downstream regions adjacent to a translated region of a sarcoglycan gene product containing a base sequence, etc., represented by the formula. Furthermore, the oligonucleotide primer set comprises at least one set of CCCCTGTCTC TGTCACTCAC C, GGCTGGACCT GGAACCACTG G, etc., and the primer set and a polymerase are used to provide 21 kit for amplifying a sarcoglycan gene product. The resultant kit is preferably used to carry out a polymerase chain reaction and amplify the sarcoglycan gene product.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a primer set for amplifying a sarcoglycan gene product and a method for amplifying the gene product.

[0002]

2. Description of the Related Art Muscular dystrophy is a general term for a group of progressive hereditary diseases that cause primary degeneration of skeletal muscle. Disease types such as Duchenne muscular dystrophy, limb girdle muscular dystrophy, myotonic dystrophy, facial scapulohumeral type, congenital, distal type, ocular muscle type, etc. according to the age of onset, affected muscle analysis, clinical course, etc. are categorized.
Those whose symptoms worsen with age are called progressive.

[0003] Since these muscular dystrophy diseases can be caused by different causative genes even though clinical symptoms are very similar (genetic polymorphism), especially in order to enable future gene therapy, molecular biology is required. And genetic analysis is required. Of progressive muscular dystrophy,
Previously, the causative gene was identified only in Duchenne muscular dystrophy linked to the sex (X) chromosome (Koenig, M. et al., Cell 53, 219-228 (198
8)).

However, recently, a causative gene of limb-girdle muscular dystrophy linked to an autosome was identified (Roberd
s, SL et al., Cell 78 (4) 625-633 (1994); Bonnenman
n, CG et al., Nature Genet. 11 (3) 266-273 (1995);
Noguchi, S. et al., Science 270, 819-822 (1995)). It has been found that Duchenne muscular dystrophy is caused by an abnormality in a cell membrane lining protein called dystrophin, whereas limb-girdle muscular dystrophy is caused by an abnormality in a protein that binds to dystrophin called sarcoglycan.

There are three types of sarcoglycans, α-sarcoglycan, β-sarcoglycan, and γ-sarcoglycan, and any of the sarcoglycan-encoding genes is mutated (substitution, deletion, insertion, etc. of the base sequence). ) Will result in limb girdle muscular dystrophy. Therefore, if any of the three sarcoglycan-encoding genes or a cDNA (gene product) corresponding to the gene can be detected, the detection method can be performed using limb-girdle muscular dystrophy using the presence or absence of gene mutation as an index. It can be used for diagnosis and the like. For that purpose, it is necessary to amplify the above genes and the like to such an extent that they can be detected.

[0006] Conventionally, a candidate gene product is divided into several mutually overlapping fragments on the order of several hundred bases,
It was amplified by R. However, the reaction conditions of PCR generally differ for each PCR, so that it was necessary to perform the PCR many times. Therefore, labor and time were required for molecular diagnosis.
In particular, when there are a plurality of candidate genes, such as muscular dystrophy, the analysis is troublesome because the corresponding overlapping gene fragments must be amplified.

[0007]

SUMMARY OF THE INVENTION The present invention provides a primer set for amplifying a gene product which specifically amplifies a candidate gene product of a human genetic disease, particularly a sarcoglycan gene product at once, and a method for amplifying the gene product. The purpose is to provide.

[0008]

Means for Solving the Problems As a result of intensive studies based on the above-mentioned problems, the present inventors have identified a causal candidate gene product by performing a polymerase chain reaction using an oligonucleotide primer having a specific sequence. The present inventors have found that the present invention can be amplified, and have completed the present invention. That is, the present invention provides an oligo that can hybridize with at least a part of the upstream and downstream regions adjacent to the translation region of the sarcoglycan gene product containing the nucleotide sequence represented by SEQ ID NO: 7, 8, 9, or 10, respectively. It is a nucleotide primer set. Examples of the oligonucleotide primer set include those containing at least one set of those represented by SEQ ID NOS: 1 and 2, SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6.

Further, the present invention is a kit for amplifying a sarcoglycan gene product comprising the oligonucleotide primer set and a polymerase. Further, the present invention is a method for amplifying a sarcoglycan gene product, comprising performing a polymerase chain reaction using the kit for amplifying a sarcoglycan gene product. The polymerase chain reaction is performed, for example, at 94 ° C for 1 minute, at 45 to 65 ° C for 45 seconds and at 72 ° C.
The reaction is performed for 25 to 35 cycles, with the reaction being performed for 1 to 5 minutes as one cycle, and the reaction is further performed at 72 ° C. for 10 minutes.
Hereinafter, the present invention will be described in detail.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a set of oligonucleotide primers that enables specific amplification of a gene causing a human genetic disease, particularly a sarcoglycan gene product. In the present invention, the “gene product” refers to cDNA synthesized by reverse transcriptase from mRNA obtained by splicing a transcript of genomic DNA (hereinafter, the gene product may be referred to as cDNA).

[0011] The cDNA that can be amplified using the primer set of the present invention is one that causes the development of progressive muscular dystrophy when a mutation (deletion, substitution, insertion, etc.) occurs in the cDNA. Examples of such cDNA include cDNA encoding sarcoglycan (hereinafter, referred to as cDNA).
“Sarcoglycan cDNA”), for example, α-sarcoglycan cDNA represented by SEQ ID NO: 7 or 8, β-sarcoglycan cDNA represented by SEQ ID NO: 9, and γ-sarcoglycan represented by SEQ ID NO: 10 There are three types of cDNA. Therefore, in the present invention, α-,
Primers are designed using β- and / or γ-sarcoglycan cDNA as a gene product to be amplified.

[0012] The primer of the present invention comprises
It is designed so that it can hybridize with at least a part of the upstream and downstream regions adjacent to the protein translation region of DNA, respectively. Where "adjacent"
This means that the upstream (5 'side) and downstream (3' side) primers of the sarcoglycan cDNA translation region are located one base upstream and one base downstream from the protein translation region, respectively. It means that a part may overlap with the translation region and may be separated by about 300 bases. For example, if a certain cDNA fragment is 300
Assuming that the translation region is located at positions 101 to 200, the 5′-side primer is, for example, 76 to
100th (one base upstream from the 5 'end of the translation region),
Alternatively, the primer should be hybridized to the 41st to 75th region (25 bases upstream from the 5 'end of the translation region), and the 3' primer should be hybridized to the 201st to 220th region (1 base from the 3 'end of the translation region). It is designed to be able to hybridize to the region downstream (downstream) or to the region 211 to 235 (downstream from the 3 'end of the translation region by 10 bases) (FIG. 1). In addition, "at least a part" means that the polymerase chain reaction described below can be started,
The length is not particularly limited, but is 20 to 35, preferably 21 to 24 nucleotides in the present invention.

[0013] The primer set of the present invention designed as described above includes, for example, 2 primers represented by SEQ ID NOS: 1 and 2.
A set of two types of oligonucleotide primers, a set of two types of oligonucleotide primers represented by SEQ ID NOS: 3 and 4, and a set of two types of oligonucleotide primers represented by SEQ ID NOS: 5 and 6 And

The set of two types of oligonucleotides represented by SEQ ID NOs: 1 and 2 is α-sarcoglycan cDN
A (SEQ ID NOS: 7 and 8) and an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 1 (hereinafter referred to as "primer 1") An oligonucleotide (hereinafter, referred to as “primer 2”) that hybridizes to the 21st sequence (located 23 bases upstream when viewed from the 5 ′ end of the protein translation region) and has the base sequence represented by SEQ ID NO: 2
In the base sequence represented by SEQ ID NO: 7, the sequence at positions 1216 to 1236 (located 12 bases downstream when viewed from the 3 ′ end of the protein translation region), and in the base sequence represented by SEQ ID NO: 8
Hybridizes with the 844th to 864th sequence (located 12 bases downstream from the 3 'end of the protein translation region).

A set of two kinds of oligonucleotides represented by SEQ ID NOs: 3 and 4 is β-sarcoglycan cDN
A (SEQ ID NO: 9), an oligonucleotide having a nucleotide sequence represented by SEQ ID NO: 3 among the nucleotide sequences represented by SEQ ID NO: 9 (hereinafter, referred to as “primer 3”)
Is an oligonucleotide having a base sequence represented by SEQ ID NO: 4 (hereinafter referred to as “primer 4”) hybridizes with the 26th to 46th sequences (four bases on the 5 ′ side of the protein translation region overlap). ) Hybridizes with the 1001-1021 sequence (located 5 bases downstream from the 3 'end of the protein translation region).

The set of two types of oligonucleotides represented by SEQ ID NOS: 5 and 6 is γ-sarcoglycan cDN
A (SEQ ID NO: 10), an oligonucleotide having the nucleotide sequence represented by SEQ ID NO: 5 among the nucleotide sequences represented by SEQ ID NO: 10 (hereinafter referred to as “primer 5”)
Hybridizes with the 92nd to 115th sequence (located 10 bases upstream from the 5 'end of the protein translation region) and has an oligonucleotide having the base sequence represented by SEQ ID NO: 6 (hereinafter referred to as "primer 6") ) Is 1068-10
Sequence 91 (from the 3 'end of the protein translation region
(Located 71 bases downstream).

Each of the above primers can be obtained by ordinary chemical synthesis, for example, chemical synthesis using an automatic DNA synthesizer manufactured by Applied Biosystems. At least one of the above-mentioned primer sets is used together with a polymerase as a kit for cDNA amplification. Therefore, one set may be used alone, or two or more sets may be used in combination. For example, a set of primers 1 and 2 is used for the purpose of detecting only α-sarcoglycan cDNA, and a set of primers 1 and 2 for detecting all of α-, β- and γ-sarcoglycan cDNA. , A set of primers 3 and 4 and a set of primers 5 and 6 are used. Then, the gene is amplified using the primer set and the polymerase prepared as described above.

The cDNA serving as a template is α-, β- or γ
-Sarcoglycan cDNA. Preparation of cDNA
It can be performed by a known method. For example, tissues such as myocardium, skeletal muscle, and liver are treated by acidic guanidine-phenol-chloroform method (using Isogen (NipponGene)) to prepare total RNA. As the mRNA, commercially available mRNA (for example, Poly (A) ⁺ RNA from Clonetech) can also be used.

Next, a reverse transcriptase (eg, SuperScript II;
CDNA is synthesized from total RNA using GIBCO BRL). Each of the above-mentioned sarcoglycan cDs is contained in the synthesized cDNA.
Since NA is included, any cDNA is amplified depending on the primer used.

The amplification is performed by the polymerase chain reaction (PCR).
Performed by LATaq polymerase as polymerase
(Takara), AmpliTaq (Perkin Elmer), Expand High Fi
delity (Boehringer Mannheim), etc.
Taq polymerase (Takara) is preferred. The polymerase chain reaction using LATaq polymerase (Takara) is called “Long PCR”. The use of this polymerase makes it possible to amplify a candidate gene product at one time.

The amplification conditions are as follows: a reaction at 94 ° C. for 1 minute, a reaction at X ° C. for 45 seconds, and a reaction at Y at 72 ° C. are performed as one cycle, and the Z cycle is performed. Here, X, Y and Z can be appropriately changed depending on the sarcoglycan cDNA to be amplified. In the present invention, for example, X is 45-65 ° C., Y is 1-5 minutes, and Z is 25-35.
The cycle is appropriate, but X is 55-62 ° C, Y is 1-1.5
More preferably, Z is 30 cycles.

As described above, the sarcoglycan cDNA can be efficiently amplified by a single PCR using the primer set of the present invention. Amplifying sarcoglycan cDNA using the primer set of the present invention also indicates that physiologically expressed products of three types of sarcoglycan cDNA have been isolated. “Physiological” means that these genes are naturally expressed and not leaked genes (Illegitimate Transcription). “Leaked” means mRN that should be expressed physiologically only in a specific tissue (eg, brain).
A means that A leaks out of the tissue (for example, in blood).

The amplified product is subjected to agarose gel electrophoresis, and mRNN originally expressed in vivo is expressed.
If a band corresponding to the band obtained by RNA blot analysis for detecting A can be obtained, it can be determined that the amplified gene product is a physiologically expressed gene product. From the results of the RNA blot analysis already performed, it is known that the tissues expressing the three sarcoglycan molecules are quite characteristic. That is, α- and γ-sarcoglycans are restricted to heart and skeletal muscle,
Sarcoglycans are widely expressed throughout the body. Also, α
-In the case of sarcoglycan, it has also been confirmed that there is a 1.4 kb band and a 1.1 kb band in which a part of the protein translation region is shortened.

Therefore, cDN derived from skeletal muscle and cardiac muscle
From A, two for α-sarcoglycan cDNA,
One band is expected to be amplified for β- and γ-sarcoglycan cDNA, and one band is expected to be amplified for β-sarcoglycan cDNA only from cDNA derived from other tissues (for example, liver). .

[0025]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention does not limit the technical scope to these examples. Example 1 1 μg of human heart-derived poly (A) ⁺ RNA (Clone
tech) from Rando as a primer for cDNA synthesis.
m Hexamer (Pharmacia) and SuperScript II (GIBCO B
(RL) cDNA was synthesized using reverse transcriptase (at 37 ° C).
60 minutes reaction). The reaction mixture is as follows.

H ₂ O 9.5 μl 10 × LA PCR Buffer (Takara) 1.25 μl dNTP (2.5 mM, each) (Takara) 1.25 μl 5 ′ primer for cDNA synthesis (10 mM) 0.125 μl 3 ′ primer for cDNA synthesis ( (10 mM) 0.125 μl cDNA aqueous solution 1.0 μl PerfectMach (Stratagene) 0.125 μl Total volume 12.375 μl

Next, 0.125 μl of LAT was added to the above reaction mixture.
Add aq polymerase (Takara) (total volume 12.5 μl) and add Th
Long P using ermal Cycler 480 (Perkin Elmer Cetus)
Performed CR. (1) Amplification of α-sarcoglycan cDNA 5 ′ primer: Primer 1 (SEQ ID NO: 1) 3 ′ primer: Primer 2 (SEQ ID NO: 2) The reaction was carried out at 94 ° C. for 1 minute, at 55 ° C. for 45 seconds, and at 72 ° C. The reaction is repeated 30 times, with the reaction at 1.5 ° C for 1.5 minutes as one cycle.
For 10 minutes and one cycle. After completion of the reaction, the amplification product was subjected to 1% agarose gel electrophoresis.

As a result, it was found that the cDNA encoding α-sarcoglycan was amplified (FIG. 2 (1)).
). In FIG. 2, "M" indicates a molecular weight marker (kbp).
This amplified cDNA is 1 to 12 in SEQ ID NO: 7.
It had a base sequence of 1236 bp corresponding to the 36th sequence, and had a base sequence of 864 bp corresponding to the 1st to 864th sequence in SEQ ID NO: 8.

It is known that genomic DNA encoding α-sarcoglycan undergoes so-called “alternative splicing” during the transcription process to produce two gene products (EMMcNally, P
roc.Natl.Acad.Sci.USA.91,9690-9694 (1994)). Alternative splicing refers to the mechanism by which multiple isoform mRNAs are generated from one gene, and refers to the generation of a mature transcript lacking a certain exon in the process of processing a gene transcript. 1236bp and 864bp two types of cD
NA occurred because this alternative splicing occurred. The base sequence represented by SEQ ID NO: 8 is a sequence shortened by subjecting the 623th to 994th sequences of the base sequence represented by SEQ ID NO: 7 to alternative splicing.

(2) Amplification of β-sarcoglycan cDNA 5 ′ primer: Primer 3 (SEQ ID NO: 3) 3 ′ primer: Primer 4 (SEQ ID NO: 4) The reaction was performed at 94 ° C. for 1 minute and at 62 ° C. for 45 minutes. The reaction was repeated 30 times at 72 ° C for 1 minute as one cycle.
One cycle of a 10 minute reaction was performed. After completion of the reaction, the amplification product was subjected to 1% agarose gel electrophoresis. As a result, 996 corresponding to the 26th to 1021st sequence of SEQ ID NO: 9
A bp amplification product was obtained (FIG. 2 (2)).

(3) Amplification of γ-sarcoglycan cDNA 5 ′ primer: Primer 5 (SEQ ID NO: 5) 3 ′ primer: Primer 6 (SEQ ID NO: 6) The reaction was carried out at 94 ° C. for 1 minute and at 62 ° C. for 45 minutes. The reaction was repeated 30 times at 72 ° C for 1 minute as one cycle.
One cycle of a 10 minute reaction was performed. After completion of the reaction, the amplification product was subjected to 1% agarose gel electrophoresis. As a result, 1000 corresponding to the 92nd to 1091st sequence of SEQ ID NO: 10
A bp amplification product was obtained (FIG. 2 (3)). (1) to (3) above
The bands obtained in Step 1 were excised from the gel, and directly with the primers used for amplification, or the plasmid vector pBlues
It was cloned into criptSK (-) (Stratagene) and sequenced (using 373A from Applied Biosystems). As a result, it was confirmed that the amplified DNA was indeed α-, β- and γ-sarcoglycan cDNA.

It was also confirmed that the 1236 bp and 864 bp products of the α-sarcoglycan cDNA corresponded to the 1.4 kb and 1.1 kb bands in the RNA blot analysis. Therefore, according to the present invention, the full-length c including all the regions (open reading frames)
Not only could DNA be amplified by a single PCR, but also a physiologically expressed gene product could be amplified.

[Example 2] In the same manner as in Example 1, cDNA was replaced with 1 µg of human liver-derived poly (A) ⁺ RNA (Clo
netech) and PC under the same conditions as in Example 1.
R was performed. In addition, poly (A) ⁺ RNA derived from cardiac muscle and skeletal muscle
Similarly, cDNA was prepared for 1 μg (Clonetech) and PCR was performed. The results are shown in FIG. In FIG. 3, lane 1 is a molecular weight marker (kbp), lanes 2 to 5 are those obtained by amplifying α-sarcoglycan cDNA, lane 6
Nos. 9 are amplifications of β-sarcoglycan cDNA, and lanes 10 to 13 are amplifications of γ-sarcoglycan cDNA.

FIG. 3 shows that β-sarcoglycan c
Only DNA was amplified (lane 8) and α- and γ-sarcoglycan cDNA was not amplified (lanes 4 and
12). From this, the result of this example was consistent with the result of RNA blot analysis. Then, it was confirmed that, under the PCR conditions of the present invention, not only cDNA can be amplified at one time, but also a gene product which is physiologically expressed can be amplified. In other words, it was confirmed that a non-physiologically minimally expressed gene product was not non-specifically amplified by PCR.

[0035]

According to the present invention, a kit for cDNA amplification and a method for amplifying cDNA are provided. By using the kit of the present invention, it is possible to amplify a cDNA covering the entire protein translation region of the above three types of human sarcoglycan molecules. In addition, since the 5 ′ and 3 ′ protein untranslated regions are only several tens of bases, the target cDN
After amplification of A, there is no need to perform unnecessary analysis on regions other than the translation region, and it is possible to easily screen for a base mutation that can cause a disease. For this reason, the kit of the present invention is useful for rapid mutation detection of the causative gene of non-Duchenne muscular dystrophy.

[0036]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: CCCCTGTCTC TGTCACTCAC C 21

SEQ ID NO: 2 Sequence length: 21 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGCTGGACCT GGAACCACTG G 21

SEQ ID NO: 3 Sequence length: 21 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GCGGCGGGCG CGGGAAGATG G 21

SEQ ID NO: 4 Sequence length: 21 Sequence type: number of nucleic acid strands: single strand Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GTTGGTGACC TCTGGGGTTC T 21

SEQ ID NO: 5 Sequence length: 24 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: TCATTCGCCA GTGTGCTTTT CTTA 24

SEQ ID NO: 6 Sequence length: 24 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: other nucleic acid (synthetic DNA) Sequence: GCTGCCTCAG TCTTTCACGA TGTG 24

SEQ ID NO: 7 Sequence length: 1436 Sequence type: Nucleic acid number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: CCCCTGTCTC TGTCACTCAC CGGGCGGGCC AGGCCGGGCA GCC ATG GCT GAG ACA 55 Met Ala Glu Thr 1 CTC TTC TGG ACT CCT CTC CTC GTG GTT CTC CTG GCA GGG CTG GGG GAC 103 Leu Phe Trp Thr Pro Leu Leu Val Val Leu Leu Ala Gly Leu Gly Asp 5 10 15 20 ACC GAG GCC CAG CAG ACC ACG CTA CAC CCA CTT GTG GGC CGT GTC TTT 151 Thr Glu Ala Gln Gln Thr Thr Leu His Pro Leu Val Gly Arg Val Phe 25 30 35 GTG CAC ACC TTG GAC CAT GAG ACG TTT CTG AGC CTT CCT GAG CAT GTC 199 Val His Thr Leu Asp His Glu Thr Phe Leu Ser Leu Pro Glu His Val 40 45 50 GCT GTC CCA CCC GCT GTC CAC ATC ACC TAC CAC GCC CAC CTC CAG GGA 247 Ala Val Pro Pro Ala Val His Ile Thr Tyr His Ala His Leu Gln Gly 55 60 65 CAC CCA GAC CTG CCC CGG TGG CTC CGC TAC ACC CAG CGC AGC CCC CAC 295 His Pro Asp Leu Pro Arg Trp Leu Arg Tyr Thr Gln Arg Ser Pro His 70 75 80 CAC CCT GGC TTC CTC TAC GGC TCT GCC ACC CCA GAA GAT CGT GGG CTC 343 His Pro Gly Phe Leu Tyr Gly Ser Ala Thr Pro Glu Asp Arg Gly Leu 85 90 95 100 CAG GTC ATT GAG GTC ACA GCC TAC AAT CGG GAC AGC TTT GAT ACC ACT 391 Gln Val Ile Glu Val Thr Ala Tyr Asn Arg Asp Ser Phe Asp Thr Thr 105 110 115 CGG CAG AGG CTG GTG CTG GAG ATT GGG GAC CCA GAA GGC CCC CTG CTG 439 Arg Gln Arg Leu Val Leu Glu Ile Gly Asp Pro Glu Gly Pro Leu Leu 120 125 130 CCA TAC CAA GCC GAG TTC CTG GTG CGC AGC CAC GAT GCG GAG GAG GTG 487 Pro Tyr Gln Ala Glu Phe Leu Val Arg Ser His Asp Ala Glu Glu Val 135 140 145 CTG CCC TCA ACA CCT GCC AGC CGC TTC CTC TCA GCC TTG GGG GGA CTC 535 Leu Pro Ser Thr Pro Ala Ser Arg Phe Leu Ser Ala Leu Gly Gly Leu 150 155 160 TGG GAG CCC GGA GAG CTT CAG CTG CTC AAC GTC ACC TCT GCC TTG GAC 583 Trp Glu Pro Gly Glu Leu Gln Leu Leu Leu Asn Val Thr Ser Ala Leu Asp 165 170 175 180 CGT GGG GGC CGT GTC CCC CTT CCC ATT GAG GGC CGA AAA GAA GGG GTA 631 Arg Gly Gly Arg Val Pro Leu Pro Ile Glu Gly Arg Lys Glu Gly Val 185 190 195 TAC ATT AAG GT G GGT TCT GCC TCA CCT TTT TCT ACT TGC CTG AAG ATG 679 Tyr Ile Lys Val Gly Ser Ala Ser Pro Phe Ser Thr Cys Leu Lys Met 200 205 210 GTG GCA TCC CCC GAT AGC CAC GCC CGC TGT GCC CAG GGC CAG CCT CCA 727 Val Ala Ser Pro Asp Ser His Ala Arg Cys Ala Gln Gly Gln Pro Pro 215 220 225 CTT CTG TCT TGC TAC GAC ACC TTG GCA CCC CAC TTC CGC GTT GAC TGG 775 Leu Leu Ser Cys Tyr Asp Thr Leu Ala Pro His Phe Arg Val Asp Trp 230 235 240 TGC AAT GTG ACC CTG GTG GAT AAG TCA GTG CCG GAG CCT GCA GAT GAG 823 Cys Asn Val Thr Leu Val Asp Lys Ser Val Pro Glu Pro Ala Asp Glu 245 250 255 260 GTG CCC ACC CCA GGT GAT GGG ATC CTG GAG CAT GAC CCG TTC TTC TGC 871 Val Pro Thr Pro Gly Asp Gly Ile Leu Glu His Asp Pro Phe Phe Cys 265 270 275 CCA CCC ACT GAG GCC CCA GAC CGT GAC TTC TTG GTG GAT GCT CTG GTC 919 Pro Pro Thr Glu Ala Pro Asp Arg Asp Phe Leu Val Asp Ala Leu Val 280 285 290 ACC CTC CTG GTG CCC CTG CTG GTG GCC CTG CTT CTC ACC TTG CTG CTG 967 Thr Leu Leu Val Pro Leu Leu Val Ala Leu Leu Leu Thr Leu Leu Leu 295 300 305 GC C TAT GTC ATG TGC TGC CGG CGG GAG GGA AGG CTG AAG AGA GAC CTG 1015 Ala Tyr Val Met Cys Cys Arg Arg Glu Gly Arg Leu Lys Arg Asp Leu 310 315 320 GCT ACC TCC GAC ATC CAG ATG GTC CAC CAC TGC ACC ATC CAC GGG AAC 1063 Ala Thr Ser Asp Ile Gln Met Val His His Cys Thr Ile His Gly Asn 325 330 335 340 ACA GAG GAG CTG CGG CAG ATG GCG GCC AGC CGC GAG GTG CCC CGG CCA 1111 Thr Glu Glu Leu Arg Gln Met Ala Ala Ser Arg Glu Val Pro Arg Pro 345 350 355 CTC TCC ACC CTG CCC ATG TTC AAT GTG CAC ACA GGT GAG CGG CTG CCT 1159 Leu Ser Thr Leu Pro Met Phe Asn Val His Thr Gly Glu Arg Leu Pro 360 365 370 CCC CGC GTG GAC AGC GCC CAG GTG CCC CTC ATT CTG GAC CAG CAC TGA 1207 Pro Arg Val Asp Ser Ala Gln Val Pro Leu Ile Leu Asp Gln His 375 380 385 CAGCCCAGCC AGTGGTTCCA GGTCCAGCCC TGACTTCATC CTCCCTTCTC TGTCCACACC 1267 ACGAGTGGCA CATCCCACCT GCTGATTCCA GCTCCTGGCC CTCCTGGAAC CCAGGCTCTA 1327 AACAAGCAGG GAGAGGGGGT GGGGTGGGGT GAGAGTGTGT GGAGTAAGGA CATTCAGAAT 1387 AAATATCTGC TGCTCTGCTC ACCAATTGCT GCTGGCAGCC TCTCCCGTC 143
6

SEQ ID NO: 8 Sequence length: 1064 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence: CCCCTGTCTC TGTCACTCAC CGGGCGGGCC AGGCCGGGCA GCC ATG GCT GAG ACA 55 Met Ala Glu Thr 1 CTC TTC TGG ACT CCT CTC CTC GTG GTT CTC CTG GCA GGG CTG GGG GAC 103 Leu Phe Trp Thr Pro Leu Leu Val Val Leu Leu Ala Gly Leu Gly Asp 5 10 15 20 ACC GAG GCC CAG CAG ACC ACG CTA CAC CCA CTT GTG GGC CGT GTC TTT 151 Thr Glu Ala Gln Gln Thr Thr Leu His Pro Leu Val Gly Arg Val Phe 25 30 35 GTG CAC ACC TTG GAC CAT GAG ACG TTT CTG AGC CTT CCT GAG CAT GTC 199 Val His Thr Leu Asp His Glu Thr Phe Leu Ser Leu Pro Glu His Val 40 45 50 GCT GTC CCA CCC GCT GTC CAC ATC ACC TAC CAC GCC CAC CTC CAG GGA 247 Ala Val Pro Pro Ala Val His Ile Thr Tyr His Ala His Leu Gln Gly 55 60 65 CAC CCA GAC CTG CCC CGG TGG CTC CGC TAC ACC CAG CGC AGC CCC CAC 295 His Pro Asp Leu Pro Arg Trp Leu Arg Tyr Thr Gln Arg Ser Pro His 70 75 80 CAC CCT GGC TTC CTC TAC GGC TCT GCC ACC CCA GAA GAT CGT GGG CTC 343 His Pro Gly Phe Leu Tyr Gly Ser Ala Thr Pro Glu Asp Arg Gly Leu 85 90 95 100 CAG GTC ATT GAG GTC ACA GCC TAC AAT CGG GAC AGC TTT GAT ACC ACT 391 Gln Val Ile Glu Val Thr Ala Tyr Asn Arg Asp Ser Phe Asp Thr Thr 105 110 115 CGG CAG AGG CTG GTG CTG GAG ATT GGG GAC CCA GAA GGC CCC CTG CTG 439 Arg Gln Arg Leu Val Leu Glu Ile Gly Asp Pro Glu Gly Pro Leu Leu 120 125 130 CCA TAC CAA GCC GAG TTC CTG GTG CGC AGC CAC GAT GCG GAG GAG GTG 487 Pro Tyr Gln Ala Glu Phe Leu Val Arg Ser His Asp Ala Glu Glu Val 135 140 145 CTG CCC TCA ACA CCT GCC AGC CGC TTC CTC TCA GCC TTG GGG GGA CTC 535 Leu Pro Ser Thr Pro Ala Ser Arg Phe Leu Ser Ala Leu Gly Gly Leu 150 155 160 TGG GAG CCC GGA GAG CTT CAG CTG CTC AAC GTC ACC TCT GCC TTG GAC 583 Trp Glu Pro Gly Glu Leu Gln Leu Leu Leu Asn Val Thr Ser Ala Leu Asp 165 170 175 180 CGT GGG GGC CGT GTC CCC CTT CCC ATT GAG GGC CGA AAA GGA AGG CTG 631 Arg Gly Gly Arg Val Pro Leu Pro Ile Glu Gly Arg Lys Gly Arg Leu 185 190 195 AAG AGA GAC C TG GCT ACC TCC GAC ATC CAG ATG GTC CAC CAC TGC ACC 679 Lys Arg Asp Leu Ala Thr Ser Asp Ile Gln Met Val His His Cys Thr 200 205 210 ATC CAC GGG AAC ACA GAG GAG CTG CGG CAG ATG GCG GCC AGC CGC GAG 727 Ile His Gly Asn Thr Glu Glu Leu Arg Gln Met Ala Ala Ser Arg Glu 215 220 225 GTG CCC CGG CCA CTC TCC ACC CTG CCC ATG TTC AAT GTG CAC ACA GGT 775 Val Pro Arg Pro Leu Ser Thr Leu Pro Met Phe Asn Val His thr Gly 230 235 240 GAG CGG CTG CCT CCC CGC GTG GAC AGC GCC CAG GTG CCC CTC ATT CTG 823 245 250 255 260 GAC CAG CAC TGA 835 Asp Gln His CAGCCCAGCC AGTGGTTCCA GGTCCAGCCC TGACTTCATC CTCCCTTCTC TGTCCACACC 895 ACGAGTGGCA CATCCCACCT GCTGATTCCA GCTCCTGGCC CTCCTGGAAC CCAGGCTCTA 955 AACAAGCAGG GAGAGGGGGT GGGGTGGGGT GAGAGTGTGT GGAGTAAGGA CATTCAGAAT 1015 AAATATCTGC TGCTCTGCTC ACCAATTGCT GCTGGCAGCC TCTCCCGTC 1064

SEQ ID NO: 9 Sequence length: 2037 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence: GGACAGTCGG GCGGGGAGAGCT CGGCGGCGGC GGGCGCGGGA AG ATG GCG GCA 51 Met Ala Ala 1 GCG GCG GCG GCG GCT GCA GAA CAG CAA AGT TCC AAT GGT CCT GTA AAG 99 Ala Ala Ala Ala Ala Ala Glu Gln Gln Ser Ser Asn Gly Pro Val Lys 5 10 15 AAG TCC ATG CGT GAG AAG GCT GTT GAG AGA AGG AGT GTC AAT AAA GAG 147 Lys Ser Met Arg Glu Lys Ala Val Glu Arg Arg Ser Val Asn Lys Glu 20 25 30 CAC AAC AGT AAC TTT AAA GCT GGA TAC ATT CCG ATT GAT GAA GAT CGT 195 His Asn Ser Asn Phe Lys Ala Gly Tyr Ile Pro Ile Asp Glu Asp Arg 35 40 45 CTC CAC AAA ACA GGG TTG AGA GGA AGA AAG GGC AAT TTA GCC ATC TGT 243 Leu His Lys Thr Gly Leu Arg Gly Arg Lys Gly Asn Leu Ala Ile Cys 50 55 60 65 GTG ATT ATC CTC TTG TTT ATC CTG GCT GTC ATC AAT TTA ATA ATA ACA 291 Val Ile Ile Leu Leu Phe Ile Leu Ala Val Ile Asn Leu Ile Ile Thr 70 75 80 CTT GTT ATT TGG GCC GTG ATT CGC ATT G GA CCA AAT GGC TGT GAT AGT 339 Leu Val Ile Trp Ala Val Ile Arg Ile Gly Pro Asn Gly Cys Asp Ser 85 90 95 ATG GAG TTT CAT GAA AGT GGC CTG CTT CGA TTT AAG CAA GTA TCT GAC 387 Met Glu Phe His Glu Ser Gly Leu Leu Arg Phe Lys Gln Val Ser Asp 100 105 110 ATG GGA GTG ATC CAC CCT CTT TAT AAA AGC ACA GTA GGA GGA AGG CGA 435 Met Gly Val Ile His Pro Leu Tyr Lys Ser Thr Val Gly Gly Arg Arg 115 120 125 AAT GAA AAT TTG GTC ATC ACT GGC AAC AAC CAG CCT ATT GTT TTT CAG 483 Asn Glu Asn Leu Val Ile Thr Gly Asn Asn Gln Pro Ile Val Phe Gln 130 135 140 145 CAA GGG ACA ACA AAG CTC AGT GTA GAA AAC AAC AAA ACT TCT ATT ACA 531 Gln Gly Thr Thr Lys Leu Ser Val Glu Asn Asn Lys Thr Ser Ile Thr 150 155 160 AGT GAC ATC GGC ATG CAG TTT TTT GAC CCG AGG ACT CAA AAT ATC TTA 579 Ser Asp Ile Gly Met Gln Phe Phe Asp Pro Arg Thr Gln Asn Ile Leu 165 170 175 TTC AGC ACA GAC TAT GAA ACT CAT GAG TTT CAT TTG CCA AGT GGA GTG 627 Phe Ser Thr Asp Tyr Glu Thr His Glu Phe His Leu Pro Ser Gly Val 180 185 190 AAA AGT TTG AAT GTT CAA AAG GCA TCT ACT GAA AGG ATT ACC AGC AAT 675 Lys Ser Leu Asn Val Gln Lys Ala Ser Thr Glu Arg Ile Thr Ser Asn 195 200 205 GCT ACC AGT GAT TTA AAT ATA AAA GTT GAT GGG CGT GCT ATT GTG CGT 723 Ala Thr Ser Asp Leu Asn Ile Lys Val Asp Gly Arg Ala Ile Val Arg 210 215 220 225 GGA AAT GAA GGT GTA TTC ATT ATG GGC AAA ACC ATT GAA TTT CAC ATG 771 Gly Asn Glu Gly Val Phe Ile Met Gly Lys Thr Ile Glu Plu His Met 230 235 240 GGT GGT AAT ATG GAG TTA AAG GCG GAA AAC AGT ATC ATC CTA AAT GGA 819 Gly Gly Asn Met Glu Leu Lys Ala Glu Asn Ser Ile Ile Lele Asu Gly 245 250 255 TCT GTG ATG GTC AGC ACC ACC CGC CTA CCC AGT TCC TCC AGT GGA GAC 867 Ser Val Met Val Ser Thr Thr Arg Leu Pro Ser Ser Ser Ser Gly Asp 260 265 270 CAG TTG GGT AGT GGT GAC TGG GTA CGC TAC AAG CTC TGC ATG TGT GCT 915 Gln Leu Gly Ser Gly Asp Trp Val Arg Tyr Lys Leu Cys Met Cys Ala 275 280 285 GAT GGG ACG CTC TTC AAG GTG CAA GTA ACC AGC CAG AAC ATG GGC TGC 963 Asp Gly Thr Leu Phe Lys Val Gln Val Thr Ser Gln Asn Met Gly Cys 290 295 300 305 CAA ATC TCA GAC AAC CCC TGT GGA AAC ACT CAT TAA 999 Gln Ile Ser Asp Asn Pro Cys Gly Asn Thr His 310 315 AAGAACCCCA GAGGTCACCA ACATGTTTAT ATCTTGACTT GACTTTTTTA TGCATGCAAA 1059 TCATTGTTTT TACAGAGTTT GTGATAACTC ATAATTATTT TAATGGCAGA GCACTGCTGT 1119 ATCTGTTTTA TGGTCTACAT AGTTAAAATC TTCTCAGAGA GCCTAAATTC TAATACATTT 1179 TATTAATTTA TACTAATCTT CATATTTACT GTTCTCTAAA ATAATTATGA GAAGCAAATA 1239 AAATCCAAAG TCATGTTTAA AGACGTGTTT TTAAAATTCC ACTATCCCTT TTCTAAAGGT 1299 TAAAGGTCTG AAGCAGCTGT TTAGATTCAC TGTAAGTAAA CTTTGGTAAC TCTAATGGGG 1359 ATAGACCCAC TTAAGATATT TAAAAAGGTA TGGCATCAGC GTTTCATGCT CTGCCTTTTA 1419 GCTTCTAAAA GGAAAGATGC AGATTTCTAG TGCATTAAGC CTGAGCCATA TTCTCACATG 1479 CAAGTGAAGT CATTAAAGAA CTTTACATAT GTGAGATAGA AACAATGGTT CCTTAGTTTT 1539 GCACTGGGAA GAAAATATTT TGTAAAAGAA TGTTTATTTG AAATAATGAT AACTATCAAT 1599 TGTTCACAAT GTGGTGGAAA TTAAAACACC ATCTCAGCTT TAACTTTTAA ATAATAATGA 1659 TAACTATCTT TATTGAGCAT CTTCTACATC CTAGGCATTG TCCTAGGCAT TGCATGTTTA 1719 TATCCCCAAT TCTCACCACA ACCCTGCAAG TAGGTGGTAT TATCCAAGTT TTACCCATTA 1779 AGAAACTGAA GATCAGAGAA GTTAAGAAAC TTGCTCAACA TCATATAGTA AGTAGCAGAG 1839 TTGGGATTGG AATTCCTTTT TTTTTTTTTT TAACTAGAGT AAGCATATCT GTTTTGTTTG 1899 ATTCTCATTC GTTCATTGATCAG AGTTAGTTGATCAGTTAGTTGATCTAGTATTGATC AG

SEQ ID NO: 10 Sequence length: 1631 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence: GTTGCTGAAG CTTCATCCTT TGCTCTCATT CTGTAAGTCA TAGAAAAGTT TGAAACATTC 60 TGTCTGTGGT AGAGCTCGGG CCAGCTGTAG TTCATTCC AGTGTGCTTT TCTTAATATC 120 TAAG ATG GTG CGT GAG CAG TAC ACT ACA GCC ACA GAA GGC ATC TGC ATA 169 Met Val Arg Glu Gln Tyr Thr Thr Ala Thr Glu Gly Ile Cys Ile 1 5 10 15 GAG AGG CCA GAG AAT CAG TAT GTC TAC AAA ATT GGC ATT TAT GGC TGG 217 Glu Arg Pro Glu Asn Gln Tyr Val Tyr Lys Ile Gly Ile Tyr Gly Trp 20 25 30 AGA AAG CGC TGT CTC TAC TTG TTT GTT CTT CTT TTA CTC ATC ATC CTC 265 Arg Lys Arg Cys Leu Tyr Leu Phe Val Leu Leu Leu Leu Ile Ile Leu 35 40 45 GTT GTG AAT TTA GCT CTT ACA ATT TGG ATT CTT AAA GTG ATG TGG TTT 313 Val Val Asn Leu Ala Leu Thr Ile Trp Ile Leu Lys Val Met Trp Phe 50 55 60 TCT CCA GCA GGA ATG GGC CAC TTG TGT GTA ACA AAA GAT GGA CTG CGC 361 Ser Pro Ala Gly Met Gly His Leu Cys Val Thr Lys Asp Gly Leu Arg 65 70 75 TTG GAA GGG GAA TCA GAA TTT TTA TTC CCA TTG TAT GCC AAA GAA ATA 409 Leu Glu Gly Glu Ser Glu Phe Leu Phe Pro Leu Tyr Ala Lys Glu Ile 80 85 90 95 CAC TCC AGA GTG GAC TCA TCT CTG CTG CTA CAA TCA ACC CAG AAT GTG 457 His Ser Arg Val Asp Ser Ser Leu Leu Leu Gln Ser Thr Gln Asn Val 100 105 110 ACT GTA AAT GCG CGC AAC TCA GAA GGG GAG GTC ACA GGC AGG TTA AAA 505 Thr Val Asn Ala Arg Asn Ser Glu Gly Glu Val Thr Gly Arg Leu Lys 115 120 125 GTC GGT CCC AAA ATG GTA GAA GTC CAG AAT CAA CAG TTT CAG ATC AAC 553 Val Gly Pro Lys Met Val Glu Val Gln Asn Gln Gln Phe Gln Ile Asn 130 135 140 TCC AAC GAC GGC AAG CCA CTA TTT ACT GTA GAT GAG AAG GAA GTT GTG 601 Ser Asn Asp Gly Lys Pro Leu Phe Thr Val Asp Glu Lys Glu Val Val 145 150 155 GTT GGT ACA GAT AAA CTT CGA GTA ACT GGG CCT GAA GGG GCT CTT TTT 649 Val Gly Thr Asp Lys Leu Arg Val Thr Gly Pro Glu Gly Ala Leu Phe 160 165 170 175 GAA CAT TCA GTG GAG ACA CCC CTT GTC AGA GCC GAC CCG TTT CAA GAC 697 Glu His Ser Val Glu Thr Pro Leu Val Ar g Ala Asp Pro Phe Gln Asp 180 185 190 CTT AGA TTA GAA TCC CCC ACT CGG AGT CTA AGC ATG GAT GCC CCA AGG 745 Leu Arg Leu Glu Ser Pro Thr Arg Ser Leu Ser Met Asp Ala Pro Arg 195 200 205 GGT GTG CAT ATT CAA GCT CAC GCT GGG AAA ATT GAG GCG CTT TCT CAA 793 Gly Val His Ile Gln Ala His Ala Gly Lys Ile Glu Ala Leu Ser Gln 210 215 220 ATG GAT ATT CTT TTT CAT AGT AGT GAT GGA ATG CTT GTG CTT GAT GCT 841 Met Asp Ile Leu Phe His Ser Ser Asp Gly Met Leu Val Leu Asp Ala 225 230 235 GAA ACT GTG TGC TTA CCC AAG CTG GTG CAG GGG ACG TGG GGT CCC TCT 889 Glu Thr Val Cys Leu Pro Lys Leu Val Gln Gly Thr Trp Gly Pro Ser 240 245 250 255 GGC AGC TCA CAG AGC CTC TAC GAA ATC TGT GTG TGT CCA GAT GGG AAG 937 Gly Ser Ser Gln Ser Leu Tyr Glu Ile Cys Val Cys Pro Asp Gly Lys 260 265 270 CTG TAC TAC CTG TCT GTG GCC GGT GTG AGC ACC ACG TGC CAG GAG CAC AGC 985 Leu Tyr Leu Ser Val Ala Gly Val Ser Thr Thr Cys Gln Glu His Ser 275 280 285 CAC ATC TGC CTC TGA 1000 His Ile Cys Leu 290 GCTGCCTGCG TCCTCTCGGT GAGCTGTGCA GTGCCGGCCC CAGA TCCTCA CACCCAGGGA 1060 GCAGCTGCAC ATCGTGAAAG ACTGAGGCAG CGTGGATGGG AAGTAAACGC TTCCAGAGGA 1120 ACTCAGAAAA AATTATGTGC CAGTGAAAGT GTTTGGACAA AAACTACATG ATCTCAAAAT 1180 GCACGTGGAT GTGAGACACA AAAGTTGACA AAATGGAAAA GCAATGTGTT TTTCCACTGG 1240 ATTAATTTTC ACCGGAACAA TTGCGAATTC TCTCTGCCTC GCCTCCCCCT ATCTTGTCCG 1300 TGTGGGCACA CACTGAGTGT TGAGTTGCCG TGTGGAGTTA ATGTATGACG CTCCACTGTG 1360 GATATCTAAT GCCCTGTTGA GAGTAGCCTT GCTCAGTACT AAAATGCCCC AAAGTTCTAT 1420 ACAGCATTTC CTTTATAGCA TTCAAACCTC ACATCCTCCC TTCAGTTTAA TGCAAGTAAG 1480 TCAGGTTTCA CAAGAAAATT TTCAAGTTTT GAAGGGAATT TGAGGTTGAT CTGGTTTTCA 1540 AGATGTAGTT AAAGGAATAA ATCACTCAAA ATTAAACTTT CTGTATATAG TCAATAAGCA 1600 ATAAAAACCT CATTTTTCAG AGTTAAAAAA C 1631

[Brief description of the drawings]

FIG. 1 is a schematic diagram of primer design.

FIG. 2 is a photograph showing the results of agarose gel electrophoresis.

FIG. 3 is a photograph showing the results of agarose gel electrophoresis.

Claims (5)

[Claims] 1. An oligonucleotide capable of hybridizing with at least a part of an upstream and downstream region adjacent to a translation region of a sarcoglycan gene product containing the nucleotide sequence represented by SEQ ID NO: 7, 8, 9, or 10, respectively. Primer set. 2. The oligonucleotide primer according to claim 1, wherein the oligonucleotide primer set comprises at least one of those represented by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, or SEQ ID NOs: 5 and 6. set. 3. A kit for amplifying a sarcoglycan gene product, comprising the oligonucleotide primer set according to claim 1 or 2 and a polymerase. 4. A method for amplifying a sarcoglycan gene product, comprising performing a polymerase chain reaction using the gene product amplification kit according to claim 3. 5. The polymerase chain reaction is performed at 94 ° C. for 1 minute,
The sarco according to claim 4, wherein the reaction is carried out for 25 to 35 cycles with a reaction of 45 to 65 ° C for 45 seconds and a reaction of 1 to 5 minutes at 72 ° C as one cycle, and further performed for one cycle at 72 ° C for 10 minutes. A method for amplifying a glycan gene product.