CN107419030A - Detect genetic chip, kit and its application method of Duchenne muscular dystrophy - Google Patents

Abstract

The invention discloses a gene chip for detecting pseudohypertrophic muscular dystrophy, a kit and a use method thereof. The gene chip includes a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide The nucleotide probe is the nucleotide sequence shown in SEQ ID NO.1‑345; the gene chip is included in the kit. The method of using this kit includes the following steps: (a) preparing sample DNA fragments; (b) fluorescently labeling DNA fragments; (c) purifying gDNA labeled products; (d) hybridizing gDNA labeled products with gene chips; (e) washing The hybridized gene chip scans the hybridization signal of the chip and obtains the result. The gene chip and kit of the present invention have low cost, simple detection operation steps, high specificity and good stability, and can be completed within two days from sample extraction to scanning results, and are suitable for clinical patients with one or more genes in the DMD gene. Detection and prenatal diagnosis of exon deletion or duplication mutation.

Description

Gene chip, kit and method for detecting pseudohypertrophic muscular dystrophy

technical field

The invention belongs to the field of biotechnology, in particular to a gene chip for detecting pseudohypertrophic muscular dystrophy, a kit and a use method thereof.

Background technique

Pseudohypertrophic muscular dystrophy is a genetic disorder characterized by progressive muscle weakness and atrophy, and is the most common X-linked recessive, hereditary muscle wasting disorder, including Duchenne progressive muscular dystrophy (DMD) and Bayer's progressive muscular dystrophy Muscular dystrophy BMD, the overall incidence of the two is about 1/3500-1/5000 live-born male babies, both caused by DMD gene mutations. The dystrophin protein encoded by the DMD gene is mainly located in skeletal muscle and cardiac muscle, and has the function of protecting and stabilizing muscle fibers, and also has the function of intracellular chemical signal transduction.

DMD patients generally have no obvious abnormalities after birth, and most children learn to walk late, usually starting to walk at one and a half years old. Usually insidious onset at the age of 3-5 years old, progressively aggravated, manifested as slow walking, toes on the ground, duck gait, easy to fall, unable to jump, difficult to go upstairs and squat to stand, weakness in upper limbs, Gower's sign positive. When they were 9 to 12 years old, they could not walk around and needed to sit in a wheelchair. In the late stage, their muscles were obviously atrophied and contracted. Most of them died of respiratory tract infection and heart failure at the age of 20 to 30. 90% of the children have muscle pseudohypertrophy and toughness to touch, especially the gastrocnemius muscle; 30% of the children have different degrees of mental retardation; most of the children are accompanied by myocardial damage, and a few can develop congestive heart failure. The clinical manifestations of BMD are similar to those of DMD, but the age of onset is later, the progression is slow, and the condition is mild. It can still walk at the age of 12, the heart is rarely involved, the intelligence is normal, and the survival period is long, which is close to the normal life span.

Auxiliary examinations for DMD include determination of serum creatine kinase and lactate dehydrogenase, electromyography, electrocardiogram, muscle MRI examination, and dystrophin immunohistochemical examination. The DMD gene mutation is mainly the deletion or duplication mutation of one or more exons within the gene, accounting for 70%-80% of the total mutation types. This type of mutation is generally detected by multiple ligation probe amplification (MLPA) technology to test. However, MLPA technology has certain limitations, such as the need to accurately measure the concentration of DNA, the sample is easily contaminated, and there is a certain possibility of false positives, especially for the case of a single exon deletion mutation, further verification experiments need to be performed using PCR. Currently there is no rapid, low-cost, high-throughput, and automated method for detecting deletion or duplication mutations of one or more exons within the DMD gene.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a gene chip, a kit and a use method thereof for detecting pseudohypertrophic muscular dystrophy. In the kit for detecting pseudohypertrophic muscular dystrophy of the present invention, the number of oligonucleotide probes contained in the gene chip is small, the cost is low, and the operation steps are simple, the detection specificity is high, and the stability is good.

The technical scheme that the present invention takes is as follows:

A gene chip and a kit for detecting pseudohypertrophic muscular dystrophy. The gene chip includes a solid phase carrier and an oligonucleotide probe immobilized on the solid phase carrier, wherein the oligonucleotide probe is SEQ ID NO.1- The nucleotide sequence shown in 345; the solid phase carrier is one of glass slide, silicon wafer, nitrocellulose membrane, nylon membrane or polystyrene. The gene chip is included in the kit. In addition, the kit also includes enzyme cutting reagents, markers, purification columns for labeled products, hybridization solution and washing solution.

The method of use includes the following steps:

(a) Preparation of sample DNA fragments: extract the whole genome DNA (gDNA) from the patient's blood, prepare enzyme digestion reaction solution and digest the gDNA;

(b) Fluorescent labeling of DNA fragments: prepare a labeling reaction solution, and use fluorescein Cy3-dUTP and Cy5-dUTP to label the gDNA of the control sample and the patient sample, respectively;

(c) Purify the gDNA-labeled product: centrifuge the gDNA-labeled product and purify it with a purification column;

(d) Hybridize the gDNA labeled product with the gene chip: pretreat the sample first, then assemble and fix it in the hybridization chamber according to the structure of "gasket-hybridization sample-chip", and fix the assembled hybridization chamber on a rotating Put it into a hybridization oven at 67°C and rotate at a speed of 20rpm, and hybridize for 24 hours;

(e) Wash the hybridized gene chip, scan the hybridization signal of the chip, and obtain the result.

The beneficial effects of the present invention are:

The gene chip of the present invention is based on many years of clinical experience of relevant professionals of the applicant, and after consulting a large number of relevant documents, it is summarized, excluding nucleotide sequences that will not cause DMD, and reducing the number of probes in a targeted manner. Finally, only 345 oligonucleotide probes can detect whether there is a deletion or duplication mutation of one or more exons in the DMD gene, which reduces the cost of making probes, improves detection efficiency, and shortens the detection time. Time, from sample extraction to scan results can be completed within two days.

There are many existing detection methods for pseudohypertrophic muscular dystrophy, but each has its own shortcomings. The present invention synthesizes some better detection methods, and even refers to a breakthrough technology—Comparative Genomic Hybridization (CGH) chip analysis technology, which can quickly Moreover, the difference in the copy number of genomic DNA between the experimental sample and the control sample is visually detected, suggesting that there is a duplication or deletion mutation in the sample DNA at a specific position on the chromosome. The gene chip and the kit of the present invention have simple detection operation steps, high specificity, and good stability, and the chip has high repeatability in multiple tests, and is suitable for one or more exon deletions or repeated mutations in the DMD gene of clinical patients detection and prenatal diagnosis.

detailed description

In order to introduce the present invention in more detail, the present invention will be further described below in conjunction with the embodiments. The experimental methods for which specific conditions are not indicated in the examples are usually implemented according to conventional conditions or according to the conditions suggested by the manufacturer.

Embodiment, content is as follows:

The gene chip comprises a solid phase carrier and oligonucleotide probes immobilized on the solid phase carrier, wherein the oligonucleotide probe is the nucleotide sequence shown in SEQ ID NO.1-345; the solid phase carrier is the Glass slides, silicon wafers, nitrocellulose, nylon, or polystyrene. The gene chip is included in the kit. In addition, the kit also includes enzyme cutting reagents, markers, labeled product purification columns, hybridization solution, and washing solution. The oligonucleotide probes in this example were produced by Agilent Technologies Co., Ltd., and the gene chip was also produced by Agilent Technologies Co., Ltd. according to conventional methods.

How to use, the steps are as follows:

(1) Preparation of sample DNA fragments:

The patient's blood was taken, and the whole genome DNA (gDNA) was extracted by conventional methods. Thaw 10×Restriction EnzymeBuffer and BSA (see table below), flick the bottom of the tube and click off. For each reaction, add patient sample and same-sex control sample gDNA to appropriate nuclease-free PCR tubes and bring up the volume to 10.1 μL with nuclease-free deionized water.

Prepare digestion master mix on ice according to the table below:

Add 2.9 μL of enzyme digestion reaction solution into the gDNA reaction tube to make the volume reach 13 μL, and gently pipette up and down with a pipette to mix well. Run the following reaction program on the PCR machine: incubate at 37°C for 2h, and incubate at 65°C for 20min. Immediately after the reaction, the reaction tube was transferred to ice, and 2 μL of digested products were electrophoresed on a 0.8% agarose gel. SYBR Gold staining was used to check whether the digested products were complete. Most of the digested products were between 200bp and 500bp in length.

(2) Fluorescent labeling of DNA:

Add 2.5 μL of random primers to each reaction tube to bring the volume to 13.5 μL, and mix by gently pipetting up and down with a pipette. Run the following reaction program on the PCR instrument: denaturation at 95°C for 3 minutes, and immediately transfer the reaction tube to ice for 5 minutes after the reaction.

Prepare the labeling mix according to the table below:

Add 11.5 μL of labeling reaction solution to each reaction tube to make the volume reach 25 μL, and mix by gently pipetting up and down with a pipette gun. Run the following reaction program on the PCR instrument: incubate at 37°C for 2h, incubate at 65°C for 10min, and transfer the reaction tube to ice immediately after the reaction. Control sample gDNA was labeled with Cy3-dUTP, and patient sample gDNA was labeled with Cy5-dUTP.

(3) Purification of gDNA labeled products:

Centrifuge the gDNA-labeled product at 6000 g for 1 min, and add 430 μL of 1×TE (pH 8.0) to each reaction tube. Place the labeled purification column (Agilent p/n 5190-3391) in a 2mL collection tube, add the gDNA labeled product into the purification column, close the cap, centrifuge at 14000g for 10min at room temperature, and discard the filtrate. Add 480 μL of 1×TE (pH 8.0) to each purification column, close the cap, centrifuge at 14,000 g for 10 min at room temperature, and discard the filtrate.

Invert the purification column into a new 2mL collection tube, centrifuge at 1000g at room temperature for 1min to collect the purified sample. The final volume of the sample was brought to 9.5 μL by supplementing with 1×TE (pH 8.0) or concentrating with a concentrator. Take 1.5 μL of purified product from each sample tube, and use NanoDrop 2000 UV-VIS to detect the yield and specific activity. Mix 8 μL Cy5-labeled patient gDNA purified labeling product and 8 μL Cy3-labeled control gDNA purified labeling product in a nuclease-free PCR tube to a volume of 16 μL.

(4) Sample pretreatment

Add 1350 μL of nuclease-free deionized water to a vial of 10× aCGH Blocking Agent lyophilized powder. Before use or storage, place at room temperature for 60 minutes, and vortex to mix.

Prepare the Hybridization Mix according to the table below:

Add 29 μL of hybridization reaction solution into each reaction tube to make the volume reach 45 μL, and gently blow up and down with a pipette to mix well and spot off. Run the following reaction program on the PCR instrument: incubate at 98°C for 3 minutes, incubate at 37°C for 30 minutes, keep the sample at 37°C, and prepare for hybridization.

(5) Chip hybridization:

Place a clean gasket on the bottom of the hybridization chamber (Agilent p/n G2534) with the gasket logo facing up and aligned with the rectangular area on the bottom of the hybridization chamber. Add 40 μL of Hybridization Sample Mixture slowly in a circular motion to the inner area of the spacer gasket. Place the chip active side (the side with the "Agilent" logo) face down on the spacer, making sure they are aligned with each other. Cover the hybridization chamber lid on the "spacer-hybridization sample-chip" sandwich structure, slide the clamp to fix it, and manually tighten the clamp of the hybridization chamber. Wet the chip by rotating the assembled hybridization chamber vertically to see if air bubbles can flow freely. If stationary air bubbles are found, place the hybridization chamber on the table and gently tap the chip or spacer to move it. The assembled hybridization chamber was fixed on a rotator, put into a hybridization oven at 67° C. and rotated at 20 rpm, and hybridized for 24 hours.

(6) Chip washing and scanning:

Fill the entire staining jar 1# with Wash Buffer 1 (Agilent Oligo aCGH Wash Buffer 1, Agilent p/n 5188-5221) at room temperature. Put a chip rack and a magnetic stirrer in the staining jar 2#, put enough Wash Buffer 1 into the staining jar 2# at room temperature to cover the chip rack, and put the staining jar 2# on the magnetic stirrer device. Put 3/4 volume of Wash Buffer 2 (Agilent Oligo aCGH Wash Buffer 1, Agilent p/n 5188-5222) and a magnetic stirrer into dyeing vat 3#, and preheat it in a circulating water bath at 37°C for more than 12 hours , put the preheated dyeing vat 3# on a magnetic stirrer with a heating element, turn on the heating element, keep the temperature of Wash Buffer 2 at 37°C, and monitor the temperature with a thermometer.

Remove a hybridization chamber from the hybridization oven and record whether air bubbles form during hybridization and whether all air bubbles can rotate freely. Disassemble the hybridization chamber, place the hybridization chamber on a horizontal surface, turn the screw counterclockwise, loosen the hybridization chamber clamp and remove. Quickly transfer the sandwich structure to staining jar 1#, and keep the chip barcode face up. Pry the sandwich structure from one end of the barcode. Insert the clean tweezers between the two slides, gently rotate the tweezers, gently separate the two slides, and let the gasket fall into the bottom of staining jar 1#. Quickly move the chip to the chip rack of staining jar 2# (Wash Buffer 1). Also avoid exposing the chip to air. Turn on the magnetic stirrer for stirring at a speed of 350 rpm for 5 min, and adjust the speed to achieve good but not excessive stirring.

Transfer the chip rack with chips to staining vat 3# with Wash Buffer 2 preheated to 37°C, turn on the magnetic stirrer and stir for at least 1 min, but not more than 2 min. Slowly move the chip holder out of the liquid surface within 5-10s, and minimize the droplets on the chip. Discard used Wash Buffer 1 and Wash Buffer 2. Scan the chip immediately to reduce the influence of oxidizing agents in the environment on the signal intensity.

The gene chip and the test kit of the present invention have been applied in clinical detection, and are compared with existing conventional detection methods through actual tests, the contents are as follows:

Randomly selected 3 samples of DMD clinical patients who came to the applicant for treatment, used the gene chip and kit of the present invention and the current DMD clinical routine detection method (ie MLPA) to conduct a parallel double-blind experiment, and after analyzing the results, the two methods were compared. The results are compared, and the results show that the detection results of the two methods are completely consistent, and the detection results are shown in the following table:

In addition, the gene chip and the kit of the present invention have simple detection steps, high specificity, and good stability, and the chip has high repeatability in multiple tests; it can be completed within two days from sample extraction to scan results, Short time, high efficiency and high accuracy.

It is easy for those skilled in the art to understand that the above embodiments are only used to further illustrate the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention fall within the scope of the present invention. Within the scope of protection required by the present invention.

sequence listing

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

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<212>DNA

<213> Artificial sequence

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<210> 59

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<210> 60

<211> 60

<212>DNA

<213> Artificial sequence

<400> 60

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<210> 61

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<213> Artificial sequence

<400> 61

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<210> 62

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<212>DNA

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<210> 63

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<212>DNA

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<210> 64

<211> 60

<212>DNA

<213> Artificial sequence

<400> 64

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<210> 65

<211> 60

<212>DNA

<213> Artificial sequence

<400> 65

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<210> 66

<211> 60

<212>DNA

<213> Artificial sequence

<400> 66

aaaacattac cactttaatc taatctgaat cctcaagtat catctccata ccaagcaaat 60

<210> 67

<211> 60

<212>DNA

<213> Artificial sequence

<400> 67

gaaccatacc aagaaatgac ttgacacatc agcataaaca tagttcatcc atgttccttt 60

<210> 68

<211> 60

<212>DNA

<213> Artificial sequence

<400> 68

aaggcatctg tgagatattt agtttcagag cactgatagg aatggtcaac aactgaataa 60

<210> 69

<211> 60

<212>DNA

<213> Artificial sequence

<400> 69

agtttgactt cactgtaaca atatcctctt ttaaccaaat aaccttcctt aaaccttgga 60

<210> 70

<211> 60

<212>DNA

<213> Artificial sequence

<400> 70

aaaactatcc tccactagaa tctcctcatt ttatgcattt tatgcaaggt tgcttagtgg 60

<210> 71

<211> 60

<212>DNA

<213> Artificial sequence

<400> 71

cttctgatgt ctaaaagatg cttatcacta ctcactagac tctttttcac ttgctttgca 60

<210> 72

<211> 60

<212>DNA

<213> Artificial sequence

<400> 72

tgccttcttt cataaaacac atcacctagt tttcacgttt ctctgaatat cctatgttgt 60

<210> 73

<211> 60

<212>DNA

<213> Artificial sequence

<400> 73

atcattagcg tcatttcctt ataggtcacc tctaacctaa ccacttcaca gtagttaaat 60

<210> 74

<211> 60

<212>DNA

<213> Artificial sequence

<400> 74

aactcctttg caacttatat tacggatagg gacatttcaa aatagacact cgaacagaca 60

<210> 75

<211> 60

<212>DNA

<213> Artificial sequence

<400> 75

acctctatct ttcccattta ctcagacaac aacttttaca atgttaacat gtgttcaaca 60

<210> 76

<211> 60

<212>DNA

<213> Artificial sequence

<400> 76

aatgattctt gttcactaga tgccagtaaa attttctcat ggaacgcaat agattcctgc 60

<210> 77

<211> 60

<212>DNA

<213> Artificial sequence

<400> 77

ataatgcacc accagaaata agagatagaa ggccctttaa cctattttga ctatgagaca 60

<210> 78

<211> 60

<212>DNA

<213> Artificial sequence

<400> 78

aaaaacactg caacatcaga ctacagtttt gtgggaatta tcacttaagg ctaaaagcag 60

<210> 79

<211> 60

<212>DNA

<213> Artificial sequence

<400> 79

agtgttgata atctaatttt gtttgtgctc caaatgattc ttccaatgca aaaaggtccc 60

<210> 80

<211> 60

<212>DNA

<213> Artificial sequence

<400> 80

agcactacac aaatggttgt tacataacat acttgaaatg aaattagagt tacctacgca 60

<210> 81

<211> 60

<212>DNA

<213> Artificial sequence

<400> 81

ttgggaagca attaaacacc tcacatggac aaaaatcata tcggtttctt actatttggg 60

<210> 82

<211> 60

<212>DNA

<213> Artificial sequence

<400> 82

tctggagtta cttacttgaa gagcatatac tatatgggaa ttataagaca acactgcccc 60

<210> 83

<211> 60

<212>DNA

<213> Artificial sequence

<400> 83

tgctttctca ctatcaagaa acagacattc ttccatctag actaatatga tctccagtgg 60

<210> 84

<211> 60

<212>DNA

<213> Artificial sequence

<400> 84

atagcaagaa gacaatcatg tgagtcttga gtattcctga gcatttatagt cagaggttct 60

<210> 85

<211> 60

<212>DNA

<213> Artificial sequence

<400> 85

aatcaatagt ttcagtagtt caccgtttta ccatgacatc tccgagtctt tcatcaaaag 60

<210> 86

<211> 60

<212>DNA

<213> Artificial sequence

<400> 86

tgccccacgt tatacagtta tacattacag ttactcaaaa acattagttc cttctaccca 60

<210> 87

<211> 60

<212>DNA

<213> Artificial sequence

<400> 87

ccatcttcat ccgtcatcta tgagatgtgt tacaataaat actgaacttt gaactcgagg 60

<210> 88

<211> 60

<212>DNA

<213> Artificial sequence

<400> 88

caaggacatt tttgtgcatt ttgtttcctc aattatgata cctttctatg cagatcaggg 60

<210> 89

<211> 60

<212>DNA

<213> Artificial sequence

<400> 89

aagaaaactc ccactctaga tgatgagtat gtgaagatca ggatgttata ccatacaacc 60

<210> 90

<211> 60

<212>DNA

<213> Artificial sequence

<400> 90

aacagaaata aggaaatgaa atctcatgac agtgtcatca taaagtcatc agccagatgg 60

<210> 91

<211> 60

<212>DNA

<213> Artificial sequence

<400> 91

aaggatacaa ggcttagttt gaggagatac tgtctataag cgctgttaat ttccattcat 60

<210> 92

<211> 60

<212>DNA

<213> Artificial sequence

<400> 92

cctctcacca ttaacttcaa gactcatcta ttaaacctct gacattacca aacaactctg 60

<210> 93

<211> 60

<212>DNA

<213> Artificial sequence

<400> 93

gatggccgtt aatcattagt tttacttgtc ttgcaactgt agcactttaa tctctaactg 60

<210> 94

<211> 60

<212>DNA

<213> Artificial sequence

<400> 94

agtagtgatg acacattcga tgtaaagacc agataaccat tttatttgct ccctaccttt 60

<210> 95

<211> 60

<212>DNA

<213> Artificial sequence

<400> 95

ccccactttc tctttataat gatataaaaa tgccatttct aaagttaacc tttgcccagc 60

<210> 96

<211> 60

<212>DNA

<213> Artificial sequence

<400> 96

taagttgtga tgttagaaac ataaaatgtg ggaggaggga gagtcaaagt gttgagtata 60

<210> 97

<211> 60

<212>DNA

<213> Artificial sequence

<400> 97

cagtgtggta gtctgacatc atatagacct ggtaattgtc cataactgag catcaattta 60

<210> 98

<211> 60

<212>DNA

<213> Artificial sequence

<400> 98

aaagactcag aagaagtaga gaacaagaaa gtagggaggtt gagtaaaata tcaggtagcc 60

<210> 99

<211> 60

<212>DNA

<213> Artificial sequence

<400> 99

aaaatcagac ttgtgaagtt tcaaagcatt acagccttct gataattact tggcatgagg 60

<210> 100

<211> 60

<212>DNA

<213> Artificial sequence

<400> 100

gccactgaag taatatagtt tattacccttc aactaacaag agtcctaatg gttacccctc 60

<210> 101

<211> 60

<212>DNA

<213> Artificial sequence

<400> 101

acatctccaa cacatgtaaa agacaatcct ttgattagac actcaaatac acagaagtca 60

<210> 102

<211> 60

<212>DNA

<213> Artificial sequence

<400> 102

aaacagtggt taaaagaatt tcttgctaat aagaacacaa gcatatggtg agagtaacga 60

<210> 103

<211> 60

<212>DNA

<213> Artificial sequence

<400> 103

ttaacagata cccatacgta aaaagtgttg attgaccaaa tcactgacta caatgaaggg 60

<210> 104

<211> 60

<212>DNA

<213> Artificial sequence

<400> 104

atattcccca ttactatcta ctcaaatgtt ctgctacact ttctcgtaaa ggtcagtgac 60

<210> 105

<211> 60

<212>DNA

<213> Artificial sequence

<400> 105

tcatttttcc taagcaaatc catttcaatc tccactgttt ttatttgcca aacatgtcct 60

<210> 106

<211> 60

<212>DNA

<213> Artificial sequence

<400> 106

cacattttga attatgaaga aagaaacaca cctattcagt taaaacactc aatcctccca 60

<210> 107

<211> 60

<212>DNA

<213> Artificial sequence

<400> 107

cctcctacaa atctccttta tttgctctat cctccaactt attctacatg ttcttgactg 60

<210> 108

<211> 60

<212>DNA

<213> Artificial sequence

<400> 108

atttctcaca caatggttat tggctcctca tgttctacat tcgtatggga ttttatgctc 60

<210> 109

<211> 60

<212>DNA

<213> Artificial sequence

<400> 109

ccaacctgac tttaaacata tccttccttc actaacttga atagagaaat aatcattgcc 60

<210> 110

<211> 60

<212>DNA

<213> Artificial sequence

<400> 110

atcagagttg ctgtttctta tgtgaaatat gcacacttcc tgtaactaaa agacccatga 60

<210> 111

<211> 60

<212>DNA

<213> Artificial sequence

<400> 111

aggcactttt tctaaatcct tcagttatgc ttcaccgatt aaattattaa gaacctctga 60

<210> 112

<211> 60

<212>DNA

<213> Artificial sequence

<400> 112

tagttcacag tatctggtat aaggatgatg aaatggaaag ggtcacacta tacaagatcc 60

<210> 113

<211> 60

<212>DNA

<213> Artificial sequence

<400> 113

aaatgtatca tttgttcagttattctgcta cctccaattt attccttgag tgtgcatccc 60

<210> 114

<211> 60

<212>DNA

<213> Artificial sequence

<400> 114

aacagtagaa gtttcacatt ctatcactgt cttggatgtt aacaatgatg ccactgtagt 60

<210> 115

<211> 60

<212>DNA

<213> Artificial sequence

<400> 115

caaagtcctg tctacctaaa tgattgtgaa agtctctaga ggaacttatg gcttttcttc 60

<210> 116

<211> 60

<212>DNA

<213> Artificial sequence

<400> 116

gggcttcttt taatccaagc actatttatg ccttctaatg aattatgatt gtgctacccc 60

<210> 117

<211> 60

<212>DNA

<213> Artificial sequence

<400> 117

ttctaaaacc acagagtaga tggaagtgtt tagagaagaa gagccatatg atacgattcg 60

<210> 118

<211> 60

<212>DNA

<213> Artificial sequence

<400> 118

ctctcctcaa gtttatgcat tgggattagc atttattcaa cctcttatac tccaagactt 60

<210> 119

<211> 60

<212>DNA

<213> Artificial sequence

<400> 119

aacattaact gagaaaccaa gactgatcaa taaacagcca tgattacagg atctcaggaa 60

<210> 120

<211> 60

<212>DNA

<213> Artificial sequence

<400> 120

tgactagcat ccttctctat ccactccata ttcttcaatc acccataaat agttccaaaa 60

<210> 121

<211> 60

<212>DNA

<213> Artificial sequence

<400> 121

agacagagat gctatctatc cacactttct ctacttgtca ttcacaaaag ttccttcaaa 60

<210> 122

<211> 60

<212>DNA

<213> Artificial sequence

<400> 122

acagggataa atttaccccc aactttgtaa taattaaccc agtatgacat cactgataca 60

<210> 123

<211> 60

<212>DNA

<213> Artificial sequence

<400> 123

ggtagttttt agtgtggatt tgacattatg ggcaattatg ctccttatac tgcaaggaag 60

<210> 124

<211> 60

<212>DNA

<213> Artificial sequence

<400> 124

agcacaatta aaacagggat agaaaatgaa tcacagtaac aaatatgatg caggcattct 60

<210> 125

<211> 60

<212>DNA

<213> Artificial sequence

<400> 125

cccaccatca taacatttac ctcatgctat ttaactttct atttacattg ctccttctca 60

<210> 126

<211> 60

<212>DNA

<213> Artificial sequence

<400> 126

agcaccaagt taactgatta gtcatttgaa aaacatgaac cttgactctc cacatcatac 60

<210> 127

<211> 60

<212>DNA

<213> Artificial sequence

<400> 127

agcctgcctt tatctaaaaa atggttctta acaatctgac gctatcttcc attcttcaaa 60

<210> 128

<211> 60

<212>DNA

<213> Artificial sequence

<400> 128

gtgtttccta gactcattga aaagaagcaa cctacgtat gttaatccca tgtggttaat 60

<210> 129

<211> 60

<212>DNA

<213> Artificial sequence

<400> 129

ggctgataca gaagaatagc gaataacagg ggtttagttt ttatgcatca tttcttcagg 60

<210> 130

<211> 60

<212>DNA

<213> Artificial sequence

<400> 130

aaagcattta aagaccaaaa gaaatccaag aattacatcc cgtataatcc tagttgtgga 60

<210> 131

<211> 60

<212>DNA

<213> Artificial sequence

<400> 131

tttctgtcac ttacagcatt aagagaacca atagacgcat taagtcagtt acttacaggc 60

<210> 132

<211> 60

<212>DNA

<213> Artificial sequence

<400> 132

tgctctcagg aacaaagaat gaccttatta catttaccta tatcatcact acccctttca 60

<210> 133

<211> 60

<212>DNA

<213> Artificial sequence

<400> 133

atgacacaca acctagtaat aaacctcatg tcagtgtgaa attaggag gcaaagtcaa 60

<210> 134

<211> 60

<212>DNA

<213> Artificial sequence

<400> 134

attcatgtca cagtaactat gacccactag ttccaattat atcacccact ctaacctttg 60

<210> 135

<211> 60

<212>DNA

<213> Artificial sequence

<400> 135

aaattgcttg gaatagagat aataatccct gttgctttag agtaaaccct atggggaagt 60

<210> 136

<211> 60

<212>DNA

<213> Artificial sequence

<400> 136

aaagccatta ccatccgata gaaaatgcaa ttaagagggt atgacttcaa accgtttttt 60

<210> 137

<211> 60

<212>DNA

<213> Artificial sequence

<400> 137

actccttttt tacaaacttt cactttcaga actttcatag gtatgttgct cctaaaggca 60

<210> 138

<211> 60

<212>DNA

<213> Artificial sequence

<400> 138

agatactttc cttttacaca atcgtcattt ttccttaagt attctccaaa ttccacacca 60

<210> 139

<211> 60

<212>DNA

<213> Artificial sequence

<400> 139

aaaacagggt tggaaagata ggtcagaaca gattctatgt tcaattcttt tgctactacg 60

<210> 140

<211> 60

<212>DNA

<213> Artificial sequence

<400> 140

aatgtcctac aaatcaatta gttggaaacc ttttcaagag caaattcgat ttcttgtccc 60

<210> 141

<211> 60

<212>DNA

<213> Artificial sequence

<400> 141

aacaagatag atcaatgaat ttggcgaaat ggtaaggaaa agaaacagac aagttgaggt 60

<210> 142

<211> 60

<212>DNA

<213> Artificial sequence

<400> 142

aagtatctac ctactcatct tttctggcat gatcttctgt taaaacaagg aaaggcagtt 60

<210> 143

<211> 60

<212>DNA

<213> Artificial sequence

<400> 143

accacccgtcc atttactgct tctatgagat cgatgttttt agattgtgca tatgaatgag 60

<210> 144

<211> 60

<212>DNA

<213> Artificial sequence

<400> 144

gcaagaggta gtaaagtaag tccacagagt gaaaaatgag taggcaaatt tgaaagtcac 60

<210> 145

<211> 60

<212>DNA

<213> Artificial sequence

<400> 145

aaatttcatc cttgactttt agagtgacta aaccagatga ttactaccga gaaacaacaa 60

<210> 146

<211> 60

<212>DNA

<213> Artificial sequence

<400> 146

atgcactcta aaagttccat taaaatctag ggcatcatca ctcattctta tctgcagcag 60

<210> 147

<211> 60

<212>DNA

<213> Artificial sequence

<400> 147

ctttgaagca cccttcatta caatcagtgc gtttttctgt aacacacaac gttatcataa 60

<210> 148

<211> 60

<212>DNA

<213> Artificial sequence

<400> 148

aaacatcatt gatttccttc tcacccataa acctacactc tgctaaaatg tgttaactgc 60

<210> 149

<211> 60

<212>DNA

<213> Artificial sequence

<400> 149

gcctacgtgg acaagtatac aaaatttgag catagcaacg atacaattta aggattttgc 60

<210> 150

<211> 60

<212>DNA

<213> Artificial sequence

<400> 150

ccaaaccagt ttacatgaag aacaagtgag aaagtgaata gaaaaaccct tcgtaaactc 60

<210> 151

<211> 60

<212>DNA

<213> Artificial sequence

<400> 151

tcccccatta caaataatca cattgacatt gtgctcatta tccactagtt tgaacctatg 60

<210> 152

<211> 60

<212>DNA

<213> Artificial sequence

<400> 152

aaaatcactc aatttagact ctagtaagga cacaagaatt accaaaacag ttacttctca 60

<210> 153

<211> 60

<212>DNA

<213> Artificial sequence

<400> 153

aaaggaacat gggtatactt caatgattat gtaagggtcc aggctaatca aatcatcact 60

<210> 154

<211> 60

<212>DNA

<213> Artificial sequence

<400> 154

ttggtatctt cacacttttg ctgactatat taaaacagat aattgaact gagtggtccc 60

<210> 155

<211> 60

<212>DNA

<213> Artificial sequence

<400> 155

agacttcgag tgacccatat tttttgccat ttgtattttt ctttaatttc ctagcccact 60

<210> 156

<211> 60

<212>DNA

<213> Artificial sequence

<400> 156

aaagtccaag gcaacaatat aagatgtagc ataagtgacc tcgtaatgaa ctgaaataga 60

<210> 157

<211> 60

<212>DNA

<213> Artificial sequence

<400> 157

gtttcattta tttccacact ccaaaaagtc tagatccaca aaatgcaaac ctaggattcc 60

<210> 158

<211> 60

<212>DNA

<213> Artificial sequence

<400> 158

aaaactgcct gctaaatcta aacttggaaa ctgcctactt aattctataa tgagaagggt 60

<210> 159

<211> 59

<212>DNA

<213> Artificial sequence

<400> 159

gaaggagatg ttacctctag tgggaaattt tagatgactt catgacaatg ttgtgctct 59

<210> 160

<211> 60

<212>DNA

<213> Artificial sequence

<400> 160

tgccaagtat gacttgtgtt tatctaataa tggactttgt tacttatagc cacatggaat 60

<210> 161

<211> 60

<212>DNA

<213> Artificial sequence

<400> 161

aaaccatcaa gtcaaaacga gtgatctttc taatacatac aaagactcag gatatgccat 60

<210> 162

<211> 60

<212>DNA

<213> Artificial sequence

<400> 162

tcgctctcta attctaaaac ttctaaccat gagataagtt tctaagttac cctcacacac 60

<210> 163

<211> 60

<212>DNA

<213> Artificial sequence

<400> 163

acagccattc aattatgctc agtgatttcc taatagtttc cagaattcta gccatgagaa 60

<210> 164

<211> 60

<212>DNA

<213> Artificial sequence

<400> 164

aaactaatct ggctcacaga caatttcagt ttatcctcat ttttatcacc atgtgacctc 60

<210> 165

<211> 60

<212>DNA

<213> Artificial sequence

<400> 165

tggcttcaga tgtatgtata aggaaggcct attatattcg taaacactgc taagttgaga 60

<210> 166

<211> 60

<212>DNA

<213> Artificial sequence

<400> 166

aaaaagaaag tgagcatctc ctagaatatt acagtggaag tatccaacat tgaaggcaga 60

<210> 167

<211> 60

<212>DNA

<213> Artificial sequence

<400> 167

accatctgat caagtcataa tcttctcaga tccttcataa atcttcttac tgccatgctc 60

<210> 168

<211> 60

<212>DNA

<213> Artificial sequence

<400> 168

aacgtgaata tgaggggggta agagaaaata agttgatta atggtgaact agggacaaag 60

<210> 169

<211> 60

<212>DNA

<213> Artificial sequence

<400> 169

ttctcaccaa taatcattgc atcctaaaag ccaaagaaaa actgctataa gggacttctc 60

<210> 170

<211> 60

<212>DNA

<213> Artificial sequence

<400> 170

aggagacaag cataaattac aaggtcagag gaataccttc aagaatctta aacccatttg 60

<210> 171

<211> 60

<212>DNA

<213> Artificial sequence

<400> 171

atgccattgc taaagttttc ctcctcatgt taatctttac ccgtgtaaaa gaaactatcc 60

<210> 172

<211> 60

<212>DNA

<213> Artificial sequence

<400> 172

aagaagcaat tttctcacca tctttattgc taccatggta ttaggtaaga acctcaggat 60

<210> 173

<211> 60

<212>DNA

<213> Artificial sequence

<400> 173

ctcagtctgt atacacat ctccaagtta tgaacatcaa cttcatgcat atcctcagaa 60

<210> 174

<211> 60

<212>DNA

<213> Artificial sequence

<400> 174

ctaccatcta acttgatcct tctaccattt tctcaattct attcacctta tccaccttgg 60

<210> 175

<211> 60

<212>DNA

<213> Artificial sequence

<400> 175

aattacacaa aatatgtttc agtccctacc aaatttccta aagccatagt tcgtctgtcc 60

<210> 176

<211> 60

<212>DNA

<213> Artificial sequence

<400> 176

aaacatttgg ccctaagaca agacatgttg gaaacacata aacaagcaaa taaaattggc 60

<210> 177

<211> 60

<212>DNA

<213> Artificial sequence

<400> 177

acacgattct tggaccatat ttctttgtaa actgttttct acatattctt agcccttcca 60

<210> 178

<211> 60

<212>DNA

<213> Artificial sequence

<400> 178

tgcatcatgg tcttaattcc aatttcatg tagcagaatg tatcccttca ttttgagatc 60

<210> 179

<211> 60

<212>DNA

<213> Artificial sequence

<400> 179

cttgcctatg atcaggtatc ataagtgcta gaaaacacac tattaattga ggcaccagtt 60

<210> 180

<211> 60

<212>DNA

<213> Artificial sequence

<400> 180

gagtcaggtt cattacactg cattctcaca ctataaatga aagagtagca agtattggct 60

<210> 181

<211> 60

<212>DNA

<213> Artificial sequence

<400> 181

tccgccaaat ctatatatca aacagagatt accatgagag taacaatgta attgaaggca 60

<210> 182

<211> 60

<212>DNA

<213> Artificial sequence

<400> 182

atttactcca gtgatttttc catcaaagtt tctatcagca tttgaatctc agcagtgtgg 60

<210> 183

<211> 60

<212>DNA

<213> Artificial sequence

<400> 183

aatgttcatc agtttcaact aaaccacact gtcaagaaat gaggacatt agacattctc 60

<210> 184

<211> 60

<212>DNA

<213> Artificial sequence

<400> 184

gtttctcaat cactcatcat attggtaatt gccatcctct atcaaattca acaaatcccc 60

<210> 185

<211> 60

<212>DNA

<213> Artificial sequence

<400> 185

aaaactccta ataacttttc ctcagcatat tccaactcat ctctccctat tttcggtttc 60

<210> 186

<211> 60

<212>DNA

<213> Artificial sequence

<400> 186

aaggctaatt ttagtttgaa tctactgaca ccataaacat taagaggatt tcacccccac 60

<210> 187

<211> 60

<212>DNA

<213> Artificial sequence

<400> 187

accaccttta acattaccct ctgatgactt tttaacagtt aacttcaatg tctagcacgt 60

<210> 188

<211> 60

<212>DNA

<213> Artificial sequence

<400> 188

agccagttag attcagagac aatttggtc aaactttgtc cttgacatat aaatagtgtt 60

<210> 189

<211> 60

<212>DNA

<213> Artificial sequence

<400> 189

atacacaagg gctttctttt agaatttcga attccaag atctctacca acatctcaag 60

<210> 190

<211> 60

<212>DNA

<213> Artificial sequence

<400> 190

aatgcaatag cactaatgcc tcaaaagaac aacctacaaa tgtctaccca cagaagttat 60

<210> 191

<211> 60

<212>DNA

<213> Artificial sequence

<400> 191

tcacttaact tcacttatct catttttctg tgttcttcaa ccttaaacta catccagcct 60

<210> 192

<211> 60

<212>DNA

<213> Artificial sequence

<400> 192

aaaatgttca attackttga gaaaaagact ggggatattg atctatggct aactctgaga 60

<210> 193

<211> 60

<212>DNA

<213> Artificial sequence

<400> 193

tgtatgtgct tatcatgtat cttcccaact acatatggca ttgtgacaaa tagaagaggc 60

<210> 194

<211> 60

<212>DNA

<213> Artificial sequence

<400> 194

aactagagtc actttctgtt agttgtaact cagaaccttg aatggtctat agaccttgac 60

<210> 195

<211> 60

<212>DNA

<213> Artificial sequence

<400> 195

tcaaacttaa tatgacgcaa acaaaattct gaactgcatt tcctaatcct tctcatccca 60

<210> 196

<211> 60

<212>DNA

<213> Artificial sequence

<400> 196

caaacaaggg acctatttgc caactaatca gtttaagata aaggtatgaa gcaaccctta 60

<210> 197

<211> 60

<212>DNA

<213> Artificial sequence

<400> 197

tcctcacagc aacttagata tctaagaaaa aagtccttta ttcagaatta cacctgccca 60

<210> 198

<211> 60

<212>DNA

<213> Artificial sequence

<400> 198

tggtgcctct tgctattatc taattctcct aataccagaa tagaaaagca gtcactttgc 60

<210> 199

<211> 60

<212>DNA

<213> Artificial sequence

<400> 199

ctaagaggta tggagacaga gttattcaat gggcaactta ataagatcag ggtcactact 60

<210> 200

<211> 60

<212>DNA

<213> Artificial sequence

<400> 200

gggtagaaag atgttgaata atcaatctaa tgagtctaag catattggtt atgaccgtgc 60

<210> 201

<211> 60

<212>DNA

<213> Artificial sequence

<400> 201

gcaggcagtt aaaaaaacac aactttagga actcgactaa aagatggaga ataacctaca 60

<210> 202

<211> 60

<212>DNA

<213> Artificial sequence

<400> 202

aggaaacctg cttgacaaat tttcaccttt ttaggctatc ataaggataa gagataccct 60

<210> 203

<211> 60

<212>DNA

<213> Artificial sequence

<400> 203

tgtctttcct tagtatgcct acttctagaa gtaaccgtaa gagaaactga tgttctgaac 60

<210> 204

<211> 60

<212>DNA

<213> Artificial sequence

<400> 204

ataccactga tgagaaattt ctagagcctt ttttcttctt tgagacctca aatcctgttc 60

<210> 205

<211> 60

<212>DNA

<213> Artificial sequence

<400> 205

accgtaagtg ctcctatatt accataccat agtaattcag catttgtaat aaaaggccaa 60

<210> 206

<211> 60

<212>DNA

<213> Artificial sequence

<400> 206

tagatgacca caaaagaact ggaatcacaa ttaaatcaca caatgttaga cagtgcacac 60

<210> 207

<211> 60

<212>DNA

<213> Artificial sequence

<400> 207

agaaaagcca tattctaacc taatattatc ctgtataacc cctactataa agaaacctga 60

<210> 208

<211> 60

<212>DNA

<213> Artificial sequence

<400> 208

acaatgtagt gtctataagg tatatttgcg tattacacct ctcataccct tttatcggaa 60

<210> 209

<211> 60

<212>DNA

<213> Artificial sequence

<400> 209

aaattattgg aaaaatcatc tggtcctata ggaacacctt tacaaacatt tcccttcagg 60

<210> 210

<211> 60

<212>DNA

<213> Artificial sequence

<400> 210

tacttcatgc cccctataaa acattctcac ttcacctttt aaatgccatc atcaagtaag 60

<210> 211

<211> 60

<212>DNA

<213> Artificial sequence

<400> 211

acctagaatt ctcaactgta tcctcgttac atctttaaag tgtctcacat ctgctcaaaa 60

<210> 212

<211> 60

<212>DNA

<213> Artificial sequence

<400> 212

aaaaaaagca ttttgtaaag gctggacaat tatctgatca atgctacatg tatatttcca 60

<210> 213

<211> 60

<212>DNA

<213> Artificial sequence

<400> 213

tactggatac gcaaggatat aggagttgta aaaacaaaag catttctggt tgaagagaga 60

<210> 214

<211> 60

<212>DNA

<213> Artificial sequence

<400> 214

acatctaggg ggatatggat agtaagcatt tgaagaaaac aaataagcca accacataca 60

<210> 215

<211> 60

<212>DNA

<213> Artificial sequence

<400> 215

ctgtccccta aaatgattaa gagccatatg caagttaatt gacatgttaa caaatggggt 60

<210> 216

<211> 60

<212>DNA

<213> Artificial sequence

<400> 216

gcgtgaccag tatatgacag aaaatctgag caggtatttc aaagaggact taatatggta 60

<210> 217

<211> 60

<212>DNA

<213> Artificial sequence

<400> 217

aaaaaattag caaaatcatc tcagtcattg caatctgtct ttctcaggtc gaatgttcct 60

<210> 218

<211> 60

<212>DNA

<213> Artificial sequence

<400> 218

aagtgtagca tgtctaagaa actctggaag agaaaactct gcatgtgaaa taaaaaaccg 60

<210> 219

<211> 60

<212>DNA

<213> Artificial sequence

<400> 219

gaacttgaca tcagatagcc atcactttca agtaaagcat tgagcattga tagaatggtc 60

<210> 220

<211> 60

<212>DNA

<213> Artificial sequence

<400> 220

aacctggaga tgctaatcaa agataacgca aagaaaaatc ccttcgcttt tgattttcta 60

<210> 221

<211> 60

<212>DNA

<213> Artificial sequence

<400> 221

gcataaagtc gacatttaat cactatctct accctctttc taaataaaag ggcttgtggc 60

<210> 222

<211> 60

<212>DNA

<213> Artificial sequence

<400> 222

tgactattca ataagtcatt agtcaatggt ttcatgcata ctaccctagt tactaccccc 60

<210> 223

<211> 60

<212>DNA

<213> Artificial sequence

<400> 223

tcctcagaga aactgtttaa catggataac aagactgtta caacaatgat catttcagcc 60

<210> 224

<211> 60

<212>DNA

<213> Artificial sequence

<400> 224

cacatatgac catgtattga catatcattg acaaagacca agaaaagcaa ctgacttcca 60

<210> 225

<211> 60

<212>DNA

<213> Artificial sequence

<400> 225

aaaggctacg ggttcaaata tttagtgttt ccaaattctc agaaatgcct tcaaactacc 60

<210> 226

<211> 60

<212>DNA

<213> Artificial sequence

<400> 226

aaagggttct tattgctaca ttcagagtgt aagcaagact tcaaacaaga cattgttgtg 60

<210> 227

<211> 60

<212>DNA

<213> Artificial sequence

<400> 227

accaaatgtg gttttatgat gttactctcg agaacattaa atcatatccc cctctagaga 60

<210> 228

<211> 60

<212>DNA

<213> Artificial sequence

<400> 228

aaacagttat tctttcccct ataatgtgac ctatatcaac ccaaaatttg tcaaatatct 60

<210> 229

<211> 60

<212>DNA

<213> Artificial sequence

<400> 229

caacatttta gcccaatttc ctatcaaaat caatattcta tcctccagtc aaaatatgcc 60

<210> 230

<211> 60

<212>DNA

<213> Artificial sequence

<400> 230

tacccttcac aaatatctga taagaaacat ccatgacagt atagacaagc ctctatcacc 60

<210> 231

<211> 60

<212>DNA

<213> Artificial sequence

<400> 231

acaaaatgca tctcaatgta gaaattcctt agatgtgtgt atttgcctaa caagtcaagt 60

<210> 232

<211> 60

<212>DNA

<213> Artificial sequence

<400> 232

actgccactt aaagtgaagt attcctcatc aagtatattc atcattagcc atgaagccag 60

<210> 233

<211> 60

<212>DNA

<213> Artificial sequence

<400> 233

tcagatggat tattattttc acaccaaaat taccaacgtt attacctcaa cgactgcctt 60

<210> 234

<211> 60

<212>DNA

<213> Artificial sequence

<400> 234

cctttccaat tagcaggtaa aacttctgag ttgtcttgaa attcagtttc acaaagacag 60

<210> 235

<211> 60

<212>DNA

<213> Artificial sequence

<400> 235

ggaaggatgg attaaatcaa agtattctag aaactgcata ttagttaggg agttgggcac 60

<210> 236

<211> 60

<212>DNA

<213> Artificial sequence

<400> 236

ctcccgctat tgataaaatg caaatcgttc ataggtaacc acatgataac aggaactttt 60

<210> 237

<211> 60

<212>DNA

<213> Artificial sequence

<400> 237

gtcaagacct agttcattac caatcatctt ctactctact atctctcacc agaaaacaca 60

<210> 238

<211> 60

<212>DNA

<213> Artificial sequence

<400> 238

gttagcagtg aagcgacaat ttttttctcc tttacaaata tactcatcag aaacccctga 60

<210> 239

<211> 60

<212>DNA

<213> Artificial sequence

<400> 239

ctaccttttc aacctcattc cttacttcta aacttccttt acttcatact tctgctggag 60

<210> 240

<211> 60

<212>DNA

<213> Artificial sequence

<400> 240

attctctgat cccatagcat tctctagaat tcacattact atattcttca aaacactgct 60

<210> 241

<211> 60

<212>DNA

<213> Artificial sequence

<400> 241

tcctaaatgt ttgccttttt gctatcttcg ttcagtctaa atttatcaat caagtgtcgt 60

<210> 242

<211> 60

<212>DNA

<213> Artificial sequence

<400> 242

tgtcactcaa tgcattagaa tgaggaaaac ttcaagcatg ataagaataa cgtagtgcct 60

<210> 243

<211> 60

<212>DNA

<213> Artificial sequence

<400> 243

ggctcttgac atgctatatt gaaatctgtg caaaattagt caaaatacac tacacacaca 60

<210> 244

<211> 60

<212>DNA

<213> Artificial sequence

<400> 244

agggatgcac gtcaaaataa ctttctttat caaataatgg caatctttct taacacggca 60

<210> 245

<211> 60

<212>DNA

<213> Artificial sequence

<400> 245

aagagaagag agtttagcac agtgtatcta tacctagaca tgtagtgaaa actggtgaca 60

<210> 246

<211> 60

<212>DNA

<213> Artificial sequence

<400> 246

gagtcacgtt tcttaaattg tttaatctat caccacttaa aaaaaaatac acgcatgggt 60

<210> 247

<211> 60

<212>DNA

<213> Artificial sequence

<400> 247

tgcaggttaa gaccctataa tacacttctc tgctaaatga attttatgtg taatgagccc 60

<210> 248

<211> 60

<212>DNA

<213> Artificial sequence

<400> 248

aaaacagatt gagtaatcca gacattgtgttctttcaaat ctacctaaca ccctattggc 60

<210> 249

<211> 60

<212>DNA

<213> Artificial sequence

<400> 249

cttaaagaaa actttcctca ggaatcatct gttgcgttca aagttcagtc taagtcagag 60

<210> 250

<211> 60

<212>DNA

<213> Artificial sequence

<400> 250

ccctagata aaaccagaat aattcaaata ctgcctttct aacttacaaa tgtggctctg 60

<210> 251

<211> 60

<212>DNA

<213> Artificial sequence

<400> 251

aaactgcaat gacctaacct aatactaatc tattttaccc tcatctaata cattctgact 60

<210> 252

<211> 60

<212>DNA

<213> Artificial sequence

<400> 252

attgcatttc tctttttcac tgcctatttt cttaattacc aatctcaagt cccaattgcc 60

<210> 253

<211> 60

<212>DNA

<213> Artificial sequence

<400> 253

aaaggcagat ttcgaaggta tgaggtaaag gagcagatat tcctattcaa agccataaat 60

<210> 254

<211> 60

<212>DNA

<213> Artificial sequence

<400> 254

accactgcta gtctacttaa ggtatgtgtt cgatctatta gttaactactg ctaaaactca 60

<210> 255

<211> 54

<212>DNA

<213> Artificial sequence

<400> 255

gtttttcact tcagggtagc aatttttccc tggtgggttt gcaagatcct atat 54

<210> 256

<211> 60

<212>DNA

<213> Artificial sequence

<400> 256

ggcaaggtct gacaaaatta tacagttttt atccccctaa aactaatgtg ttcattctcc 60

<210> 257

<211> 60

<212>DNA

<213> Artificial sequence

<400> 257

ccctcttttc ataatcacgt tttttgtttc ctctcacaaa atctttcaaa aatcaggttc 60

<210> 258

<211> 60

<212>DNA

<213> Artificial sequence

<400> 258

gcatagtggc agttattgca atgacatttt catcaaatga tgcactcttt ttccagtttc 60

<210> 259

<211> 60

<212>DNA

<213> Artificial sequence

<400> 259

gtcttcatta cgttgctgag taggaaatca aatgtcaagt ctaaataatg cacgcaagtc 60

<210> 260

<211> 60

<212>DNA

<213> Artificial sequence

<400> 260

tccagtcatt ttgcttcatc gtttttcaag tcacatacct ttagatatta cactaaccct 60

<210> 261

<211> 60

<212>DNA

<213> Artificial sequence

<400> 261

aacctcacaa cataagggat gattattgtc tacattttac tatgaggaga cctgattcat 60

<210> 262

<211> 60

<212>DNA

<213> Artificial sequence

<400> 262

tgctgccata agaactcaag aactgttgaa aaaatgaatt gatgagatat atactgagga 60

<210> 263

<211> 60

<212>DNA

<213> Artificial sequence

<400> 263

gcaatgcgat tctatcactg gtcttaactg atctgttgat aataaaccct gatttccaaa 60

<210> 264

<211> 60

<212>DNA

<213> Artificial sequence

<400> 264

agtgccgaat gttctgattt caaattcaat gttcaattgc tcagttcacc aaatctagtt 60

<210> 265

<211> 60

<212>DNA

<213> Artificial sequence

<400> 265

agtgaatgaa caaaattggc gaaaaatttt atgtccgaaa gttcttaaag tctaggggaa 60

<210> 266

<211> 60

<212>DNA

<213> Artificial sequence

<400> 266

aaatttgaat gtttcagaaa gaggactaca tatcttagga agcagataat tgccactgga 60

<210> 267

<211> 60

<212>DNA

<213> Artificial sequence

<400> 267

gctgatgcct catcatatat acatctagag atctcatctg tctacacatt tttccttggc 60

<210> 268

<211> 60

<212>DNA

<213> Artificial sequence

<400> 268

agggccaaaa aactactta taagcagact gttacatcat ctcccgtttt attgttttat 60

<210> 269

<211> 60

<212>DNA

<213> Artificial sequence

<400> 269

agactgatac actgaaaact atttgagcgt tctgttaatc aataaatcct aaacctacca 60

<210> 270

<211> 60

<212>DNA

<213> Artificial sequence

<400> 270

attatctaga aaactgaatc cctcttacca ttcatgtgat ttatgttcag tgtgctcacc 60

<210> 271

<211> 60

<212>DNA

<213> Artificial sequence

<400> 271

aaagcaaaca agacaacatt actagcattt ctttgactgt ttctatctga cagagccact 60

<210> 272

<211> 60

<212>DNA

<213> Artificial sequence

<400> 272

caatcttaac tttctatgga ggcaatgaca ctactataaa ctatttatga cactgcgggg 60

<210> 273

<211> 60

<212>DNA

<213> Artificial sequence

<400> 273

aaaagttttc tcgaggtagt cataatttca aaaccctgct tatagtaatg aatcatggtt 60

<210> 274

<211> 60

<212>DNA

<213> Artificial sequence

<400> 274

gtcactgaca cttgtgaaat tatcatttga caaaccatga aataatttgg caggaatcca 60

<210> 275

<211> 60

<212>DNA

<213> Artificial sequence

<400> 275

tgtcatatag atcacttatc accatcttat cccgatacca tagaacctta gccactgttt 60

<210> 276

<211> 60

<212>DNA

<213> Artificial sequence

<400> 276

aagcacagta atggtctacc aaccttattg tccaatctat atttcaagtg tctaccttga 60

<210> 277

<211> 60

<212>DNA

<213> Artificial sequence

<400> 277

ttaaaatcta catctggcat ctgatctatt cctacaacct atccatattc tgtctgacct 60

<210> 278

<211> 60

<212>DNA

<213> Artificial sequence

<400> 278

tgcaggttta aacaaaaaag tcgtatcata ttgagtatcc agatttcact ttgtctccga 60

<210> 279

<211> 60

<212>DNA

<213> Artificial sequence

<400> 279

aacagcaggc ttagtcattt tttaaggaag aaaatgctag aagtctaaac aaatgctggg 60

<210> 280

<211> 60

<212>DNA

<213> Artificial sequence

<400> 280

aaaacacagt taactccatt ttgaagacag tgaagtaagt tttccaacta tcagcaaaat 60

<210> 281

<211> 60

<212>DNA

<213> Artificial sequence

<400> 281

atgcattatc caaaggagaa gaaacatttc aagtcaactt aatctgatcc caacttcaca 60

<210> 282

<211> 60

<212>DNA

<213> Artificial sequence

<400> 282

attgtagtgg actttgcgtt ttatcacaca acttttaact gccatgacac ttagtcaatc 60

<210> 283

<211> 60

<212>DNA

<213> Artificial sequence

<400> 283

agcaccccaa atatatttac tttcagcaca tgaatagatt actgcagatt aaagaggtca 60

<210> 284

<211> 60

<212>DNA

<213> Artificial sequence

<400> 284

attcagatca ctagggggta attaactgat tgggaattgg agatattata ttcagggaca 60

<210> 285

<211> 60

<212>DNA

<213> Artificial sequence

<400> 285

tataaaaggg atcggaattc ataaaatgta agcaaggaaa tcataagagg gaagacgagg 60

<210> 286

<211> 60

<212>DNA

<213> Artificial sequence

<400> 286

ttactgactt actccttcca aaagttaacc actctcttca attttaaacc catggaaaca 60

<210> 287

<211> 60

<212>DNA

<213> Artificial sequence

<400> 287

acactcaatt taacaaagca cacttccaat gatacatttt cacgattatc cccttttgaa 60

<210> 288

<211> 60

<212>DNA

<213> Artificial sequence

<400> 288

agggatcaat acgtttcagt agaaagtaaa gaacacaggg ataatttagc atgaccttct 60

<210> 289

<211> 60

<212>DNA

<213> Artificial sequence

<400> 289

tcctcaagcg tgctactata aaaatacaag gaaagagaca tcaaggatca aataatgtgt 60

<210> 290

<211> 60

<212>DNA

<213> Artificial sequence

<400> 290

tatagccaaa aaatcgtgtt ttatccagat ccactaagat tccattaaca cttctgcgtg 60

<210> 291

<211> 60

<212>DNA

<213> Artificial sequence

<400> 291

aagtaactca tccgaaaaga ttttctttag tggttcaacc tatacaaaca taccacccag 60

<210> 292

<211> 60

<212>DNA

<213> Artificial sequence

<400> 292

aaacctttcg ttgaaattaa ccttagacct agattcttaa aggactcaaa cttttcgcgg 60

<210> 293

<211> 60

<212>DNA

<213> Artificial sequence

<400> 293

ccttcaagca cctgaataac agcaattaca aaaatatatt tttttcatca acgactgggc 60

<210> 294

<211> 60

<212>DNA

<213> Artificial sequence

<400> 294

agcagtgatt acacccttaa tctttggctt tatggaaaag tataaaatcg ctcttcagtt 60

<210> 295

<211> 60

<212>DNA

<213> Artificial sequence

<400> 295

gggtaacgag atcactaagg gaatttttag cctagtaaag acagtttctc aacattcaag 60

<210> 296

<211> 60

<212>DNA

<213> Artificial sequence

<400> 296

aaatttactc ttttcacatt gggaaactat ccatcagagt caacaatcat tgtcctcctt 60

<210> 297

<211> 60

<212>DNA

<213> Artificial sequence

<400> 297

aattgcagga gttactttaa tcaccacatg tctacataac aatctaaact actaaacgtt 60

<210> 298

<211> 60

<212>DNA

<213> Artificial sequence

<400> 298

aatcctttcc tgtttcaaac tgattaacac tgatactgct cttttgtgaa aattgcctgt 60

<210> 299

<211> 60

<212>DNA

<213> Artificial sequence

<400> 299

atttcctctc agataagtca gtaacaagca agtattttct agcaactgga ctatacccac 60

<210> 300

<211> 60

<212>DNA

<213> Artificial sequence

<400> 300

ggaccactaa aatccccatt ttatcatgtg aagggttaag acatactttt gacactgttt 60

<210> 301

<211> 60

<212>DNA

<213> Artificial sequence

<400> 301

tcagcataac agcaaatttg acaacaatca aaaactcatc ctttctgaat atgtccctga 60

<210> 302

<211> 60

<212>DNA

<213> Artificial sequence

<400> 302

gagaagagac tgcagttaaa atcaaatatg ttcgttccta attcttgatc caagtggtga 60

<210> 303

<211> 60

<212>DNA

<213> Artificial sequence

<400> 303

ggggtaagtc tagtgttctc acgtaaaaat catcttgcag atacagacag gataaactaa 60

<210> 304

<211> 60

<212>DNA

<213> Artificial sequence

<400> 304

cagatgagac acttatgatc tcaatgctca ttctcctact actctttgga taaccttgtt 60

<210> 305

<211> 60

<212>DNA

<213> Artificial sequence

<400> 305

aatagttctt ctgggtcaaa acatatggtt cacaaaaatg acttcctttc atcttggtct 60

<210> 306

<211> 60

<212>DNA

<213> Artificial sequence

<400> 306

aagtgctttc cactgatagt ctgcaaatac acaacccttg aagaactcat tgtaactatg 60

<210> 307

<211> 60

<212>DNA

<213> Artificial sequence

<400> 307

tacccatagg gcaactaatt ctgaaaacgg gtgataagtg aataaaggaa gtatgcttaa 60

<210> 308

<211> 60

<212>DNA

<213> Artificial sequence

<400> 308

tcccccagtc atttctaaag ataaattaag atgcatgctt tcattaagaa taaaacaccc 60

<210> 309

<211> 60

<212>DNA

<213> Artificial sequence

<400> 309

agcaaagaca aagtatcatt ttgacgatta aataatgaca aggagcataa atcacgtctt 60

<210> 310

<211> 60

<212>DNA

<213> Artificial sequence

<400> 310

aaacagtctt cattttaaat gcttcttcca gaatgtatgc acatattata tagtgaggca 60

<210> 311

<211> 60

<212>DNA

<213> Artificial sequence

<400> 311

aaccaaagag taacttcaca catttctaat gaaaaggtct cttagtttgc actcatcacc 60

<210> 312

<211> 60

<212>DNA

<213> Artificial sequence

<400> 312

accttactga ctcaaaagtt tagtgttaat ttaccctaca tcatgcttat catgctggct 60

<210> 313

<211> 60

<212>DNA

<213> Artificial sequence

<400> 313

caaacctatt ctagcctcct tcaaatcttt actttcataa aattaatact agcccttcca 60

<210> 314

<211> 60

<212>DNA

<213> Artificial sequence

<400> 314

cattgagcac ccattagaca taaaactaac tttcaatgac ttctccccaa aatttagcct 60

<210> 315

<211> 60

<212>DNA

<213> Artificial sequence

<400> 315

ggttttcaca ggatataaaa aacatcatgc catacaagaa acaaatcact cctttctgca 60

<210> 316

<211> 60

<212>DNA

<213> Artificial sequence

<400> 316

aggcgtaaga tttaaggaaa gatactaaac tgaaggaaag atactaaact ctaccaccca 60

<210> 317

<211> 60

<212>DNA

<213> Artificial sequence

<400> 317

aaatatgtag gcattgtcta cttagcctga aacactaaaaa gagtcctttc tacagttccc 60

<210> 318

<211> 60

<212>DNA

<213> Artificial sequence

<400> 318

ggcaatggaa gttagattag atacttgtgt atccgatact gtttaaaaga ctggttctgt 60

<210> 319

<211> 60

<212>DNA

<213> Artificial sequence

<400> 319

atgacatatc tctactaaca agcagattgg aaattatggc atcaattcac catctgtgcc 60

<210> 320

<211> 60

<212>DNA

<213> Artificial sequence

<400> 320

tttgggaaaa tgagcaataa acgaagtcca tttcttagca gaaaaactca taggcaatgt 60

<210> 321

<211> 60

<212>DNA

<213> Artificial sequence

<400> 321

tgcagtatgt atgtataccc aacatcttgt atgtaatttt aagggatgca aggatcccaa 60

<210> 322

<211> 58

<212>DNA

<213> Artificial sequence

<400> 322

agaactccca aattaagggc accattttat actgcaaccc aaaaggagag aaatacta 58

<210> 323

<211> 60

<212>DNA

<213> Artificial sequence

<400> 323

caagtcagat tttccttatc ctctttactt acactatctt accaataggc tgtcagatgc 60

<210> 324

<211> 60

<212>DNA

<213> Artificial sequence

<400> 324

aaacccctaa gatactcaat catgtgagca caaagttact gtgtaatctt ccatagatgt 60

<210> 325

<211> 60

<212>DNA

<213> Artificial sequence

<400> 325

aagaatccct ttaaaaactt tccttaaagc aataacacaa gcattctaga tggttccaca 60

<210> 326

<211> 60

<212>DNA

<213> Artificial sequence

<400> 326

tttcctaaga acagaacgat aactcaaatt taagtgttcc aaactgataa gtcctcaggg 60

<210> 327

<211> 60

<212>DNA

<213> Artificial sequence

<400> 327

aaaaattatt ccagttacat ggttcagtct cttgagattt acaggtagcc aattatggcc 60

<210> 328

<211> 60

<212>DNA

<213> Artificial sequence

<400> 328

aaatgtgaga ccaaatcaaa caacaagagc gtaatgaaaa acgatagaag gaaaacaact 60

<210> 329

<211> 60

<212>DNA

<213> Artificial sequence

<400> 329

acttttactc catttatgcc aatctcctta tccacagcaa gatgctactt tgactttttg 60

<210> 330

<211> 60

<212>DNA

<213> Artificial sequence

<400> 330

tgagtgataa cttcagcact ccttagtcac ttttcattat gcagacaaaa tctgactctt 60

<210> 331

<211> 60

<212>DNA

<213> Artificial sequence

<400> 331

cccttctaat cagtaacctc agttcctcta gaaataactg tgtcgtctgc tttatatagg 60

<210> 332

<211> 60

<212>DNA

<213> Artificial sequence

<400> 332

aaaccaacag aacgaattct tccacttaaa atcagccttt gattaatggc atacccatat 60

<210> 333

<211> 60

<212>DNA

<213> Artificial sequence

<400> 333

ccacaaaaac agttcagtga gtttcattta aaccatgcat aacactaccaa aaagtgactc 60

<210> 334

<211> 60

<212>DNA

<213> Artificial sequence

<400> 334

acatcatttc aatccacaaa aataggctta gtcctcaata agtaaggaat caaggtgagg 60

<210> 335

<211> 60

<212>DNA

<213> Artificial sequence

<400> 335

gatggtgcag catttactac agacaattga ttcagactta aacttttggg ctacaaatca 60

<210> 336

<211> 60

<212>DNA

<213> Artificial sequence

<400> 336

atcacgtgtt tactatctct aacaaacaga actcatccag atgataatct tggtcttgcc 60

<210> 337

<211> 60

<212>DNA

<213> Artificial sequence

<400> 337

cctcagccta ataaactcac tgactctaat aacgaatttg aaaaacaaca tgtccttagc 60

<210> 338

<211> 60

<212>DNA

<213> Artificial sequence

<400> 338

aacgacaagg agaacaaaag acatttcaca cttctcaaag agattactac ctgttttgag 60

<210> 339

<211> 60

<212>DNA

<213> Artificial sequence

<400> 339

aaaacttgag ttaagattca tgcatattta gtaacaacct agtaacacgg atgtatgccc 60

<210> 340

<211> 60

<212>DNA

<213> Artificial sequence

<400> 340

tgctgatcca gaaggttaat atgtctaaga atgtgcattt gtgaaatttc cctatgtgct 60

<210> 341

<211> 60

<212>DNA

<213> Artificial sequence

<400> 341

tgcattttga tgaacttatg ctatgagaac actttaaaac catacctaac ctccattggc 60

<210> 342

<211> 60

<212>DNA

<213> Artificial sequence

<400> 342

aatgcatgcc agtattcata aggcttttcc aatctgaatg atgcctgagt ttcaaatatg 60

<210> 343

<211> 60

<212>DNA

<213> Artificial sequence

<400> 343

agaaaagtgg tatctcatac aacctcatct aatctcatac actagaaaaa tggatcagca 60

<210> 344

<211> 60

<212>DNA

<213> Artificial sequence

<400> 344

agcaagagac ttcgtgatat atatgtgaat tcctcattgt tgatatgccc atgtatggtt 60

<210> 345

<211> 60

<212>DNA

<213> Artificial sequence

<400> 345

atatagccct gagtatatct ccgttttttc ttaccaaact cattgtcatg ttgcataggc 60

Claims (5)

1. The gene chip and test kit for detecting pseudohypertrophic muscular dystrophy are characterized in that, the gene chip comprises a solid phase carrier and an oligonucleotide probe fixed on the solid phase carrier, wherein the oligonucleotide probe The needle is the nucleotide sequence shown in SEQ ID NO.1-345; the gene chip is included in the kit. 2. the gene chip and test kit of detecting pseudohypertrophic muscular dystrophy according to claim 1, it is characterized in that, described solid-phase carrier is glass slide, silicon chip, nitrocellulose membrane, nylon membrane or polyester One of vinyl. 3. The gene chip and kit for detecting pseudohypertrophic muscular dystrophy according to claim 1, wherein the kit also includes enzyme cutting reagents, markers, labeled product purification column, hybridization solution and washing solution . 4. A gene chip and a test kit for detecting pseudohypertrophic muscular dystrophy, characterized in that the method of use comprises the following steps: (a) preparing sample DNA fragments; (b) fluorescently labeled DNA fragments; (c) Purify the gDNA labeled product; (d) hybridizing the gDNA marker product with the gene chip; (e) Wash the hybridized gene chip, scan the hybridization signal of the chip, and obtain the result. 5 . The gene chip and kit for detecting pseudohypertrophic muscular dystrophy according to claim 4 , wherein the fluorescently labeled markers are fluorescein Cy3-dUTP and Cy5-dUTP.