CN111647654B - Primer composition, kit and method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation - Google Patents

Abstract

The invention discloses a primer composition, a kit and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation. The invention firstly discloses a primer composition for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation. The invention further discloses a kit comprising the above-mentioned primer composition and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation. The detection method for hemochromatosis and hepatolenticular degeneration susceptibility gene mutation of the present invention integrates the whole exon and intron splicing region gene variation of the susceptibility gene for hemochromatosis and hepatolenticular degeneration in the Chinese population. Copy number variation information has the following advantages: high detection throughput; high specificity and sensitivity; 100% sequencing coverage, depth >30×; clear and objective interpretation of genetic testing results, with good accuracy and repeatability; low cost, It is easy to operate and easy to popularize; it can not only detect known high-frequency mutations, but also discover new mutation sites.

Description

Primer combination for detection of hemochromatosis and hepatolenticular degeneration susceptibility gene mutations Substances, kits and methods

technical field

The present invention relates to the field of gene detection. Specifically, it relates to a primer composition, a kit and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutations. More specifically, it relates to a primer composition, kit and method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutations in Chinese population based on high-throughput sequencing.

Background technique

Hemochromatosis (HC) and hepatolenticular degeneration (HLD) are hepatic metabolic disorders caused by gene mutation, with congenital, lifelong and familial characteristics. Because early clinical symptoms and laboratory tests often have no specific indications, missed or misdiagnosed cases often occur, which greatly hinders the diagnosis and differential diagnosis of inherited metabolic liver diseases.

Hemochromatosis, also known as hereditary hemochromatosis (HHC, HH), is a common chronic iron overload disease. It is an autosomal recessive genetic disease. It is known that the main pathogenic gene mutation in European and American populations is HFE C282Y pure and mutation. Chinese population The main pathogenic gene mutations in the Chinese population are HJV, SLC40A1, SUGP2 and DENND3 genes, of which SUGP2R639Q and DENND3 L708V are newly discovered pathogenic genes and hotspot mutations specific to the Chinese population; due to the inappropriate increase in intestinal iron absorption, excessive Iron is stored in parenchymal cells such as liver, heart and pancreas, leading to degeneration and diffuse fibrosis of tissues and organs, metabolic and dysfunctional disorders. The main clinical features are skin pigmentation, liver cirrhosis, and secondary diabetes. These complications are irreversible, and early diagnosis is important to prevent serious complications, especially liver cancer.

There is currently no most effective way to make an early diagnosis of hemochromatosis. In cases without secondary infection and concurrent liver cancer, the most simple and practical screening test is serum iron (SI), serum ferritin, total iron binding capacity and transferrin saturation determination.

Hepatolenticular degeneration, also known as Wilson disease (Wilson Disease, WD), is an autosomal recessive genetic disease. The pathogenic gene ATP7B is located on chromosome 13q14.3, encoding a copper-transporting P-type ATPase consisting of 1465 amino acids. ATP7B gene mutation leads to the weakening or disappearance of ATPase function, which leads to the reduction of serum ceruloplasmin (Ceruloplas minutes, CP) synthesis and the obstruction of biliary copper excretion. The copper ions accumulated in the body are deposited in the liver, brain, kidney, cornea, etc. Sexually exacerbated cirrhosis, extrapyramidal symptoms, psychiatric symptoms, renal damage and corneal pigment ring (Kayser-Fleischerring, K.F ring). There are many mutation sites of ATP7B gene, and more than 800 sites have been recorded in the human genome database.

Currently, commonly used auxiliary examination methods for hepatolenticular degeneration include biochemical determination of metabolites, routine blood and urine, liver and kidney function, and imaging examinations; as well as gene-based detection methods such as next-generation sequencing.

The symptoms of hemochromatosis and hepatolenticular degeneration are often inconspicuous in the early stage, and often progressively aggravate after the onset. Failure to identify, diagnose, intervene and treat early can often lead to disability or death, and the cause is often unidentified. Therefore, early screening of inherited metabolic liver diseases is the key to prevention and treatment.

At present, the biggest limitations of the diagnostic techniques and methods for hemochromatosis and hepatolenticular degeneration are generally low throughput and high price, which cannot satisfy the increasing number of patients with these two inherited metabolic liver diseases. The test results suggest indirect evidence, and there are disadvantages such as difficult interpretation of results, poor repeatability, and many false positives and false negatives.

Currently, there is an urgent need for a high-throughput and low-cost method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutations.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a primer composition and a kit for detecting hemochromatosis and hepatolenticular degeneration gene mutations.

Another object of the present invention is to provide a method for detecting hemochromatosis and hepatolenticular degeneration based on high-throughput sequencing. The method has high detection throughput, high sensitivity and strong specificity, and can cover hemochromatosis in one detection. Gene variation and copy number variation of all exons and intron splice regions of five susceptibility genes for hepatolenticular degeneration.

In order to achieve the above object, the present invention firstly provides a primer composition for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutations that are unique or common in Chinese population, including the ability to specifically amplify hemochromatosis and hepatolenticular degeneration. A primer set for gene variation and/or copy number variation of all exons and intron splice regions of susceptibility genes, which are HJV, SLC40A1, SUGP2 and DENND3 genes associated with hemochromatosis and The ATP7B gene associated with hepatolenticular degeneration.

In the present invention, the HJV and SLC40A1 genes are the common hemochromatosis-related genes in the Chinese population, and the SUGP2 and DENND3 genes are the unique hemochromatosis-related genes in the Chinese population.

Further, the primer set includes primer set I, primer set II and primer set III, the primer set I includes the upstream and downstream primer sequences shown in SEQ ID NO.1-122, and the primer set II includes SEQ ID NO.123 The upstream and downstream primer sequences shown in -248, the primer set III includes the upstream and downstream primer sequences shown in SEQ ID NO. 249-252, as shown in Table 1.

The sequence and detection conditions of the primer composition of the present invention are obtained through repeated optimization of a large number of experiments, the construction of a sequencing library is carried out through multiple PCR reactions, and then a massively parallel sequencing (second-generation sequencing) analysis is carried out. Methods such as splicing and alignment, as well as bioinformatics analysis, finally obtain gene variation and copies of all exons and intron splicing regions of the above-mentioned susceptibility genes for hemochromatosis and hepatolenticular degeneration of the sample to be tested information on the variation of numbers.

The information on gene variation and copy number variation of all exons and intron splicing regions of the above susceptible genes is shown in Tables 8 to 12.

The present invention further provides a kit comprising the above primer composition.

In the present invention, in addition to the above-mentioned primer composition, the kit also includes reagents such as ligase, amplifying Taq enzyme, linker, purified magnetic beads and the like.

The application of the above primer composition or kit in the preparation of a product for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation also falls within the protection scope of the present invention.

The present invention further provides a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation.

The method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation of the present invention comprises the following steps:

Obtain the DNA sample of the sample to be tested;

Synthetic primer sets are designed for the gene variation and/or copy number variation of all exons and intron splicing regions of the susceptibility genes for hemochromatosis and hepatolenticular degeneration. The related HJV, SLC40A1, SUGP2 and DENND3 genes and the ATP7B gene associated with hepatolenticular degeneration;

Using the primer set to perform multiple PCR on the DNA sample of the sample to be tested and construct a sequencing library;

Process the data of the sequencing library to obtain information on the gene variation and copy number variation of all exons and intron splice regions of the susceptibility genes for hemochromatosis and hepatolenticular degeneration of the sample to be tested , so as to determine the detection results of the hemochromatosis and hepatolenticular degeneration susceptibility gene mutations in the sample to be tested.

In the present invention, the HJV and SLC40A1 genes are the common hemochromatosis-related genes in the Chinese population, and the SUGP2 and DENND3 genes are the unique hemochromatosis-related genes in the Chinese population.

Further, the primer set includes primer set I, primer set II and primer set III, the primer set I includes the upstream and downstream primer sequences shown in SEQ ID NO.1-122, and the primer set II includes SEQ ID NO.123 The upstream and downstream primer sequences shown in -248, the primer set III includes the upstream and downstream primer sequences shown in SEQ ID NO. 249-252, as shown in Table 1.

In the present invention, the HJV and SLC40A1 genes are the common hemochromatosis-related genes in the Chinese population, and the SUGP2 and DENND3 genes are the unique hemochromatosis-related genes in the Chinese population.

In the present invention, the genetic variation is a single nucleotide variation (SNP) and an insertion deletion marker (InDel), and the information on the gene variation and copy number variation of all exons and intron splicing regions of the above-mentioned susceptibility gene As shown in Table 8 to Table 12.

Further, the DNA sample of the sample to be tested is human genomic DNA, which can be blood genomic DNA, peripheral blood cell genomic DNA, cell genomic DNA, tumor tissue genomic DNA or body fluid DNA, etc.; preferably, the sample to be tested is a Chinese population .

In the present invention, the library construction and the data processing of the sequencing library are all performed by conventional methods in the art. In a specific embodiment of the present invention, the sequencing library construction process includes performing multiple PCR reactions three times and combining the products and purifying the combined products. , linker sequence PCR reaction and purification, library quantification and quality inspection; the data of the sequencing library includes using Trimmomatic to perform quality control on the data of the sequencing library, using the BWA algorithm to map the quality-controlled reads to the human hg19 reference genome, using VarScan software detects and analyzes the above-mentioned susceptibility gene mutations on the Clean data obtained after mapping.

The beneficial effects of the present invention are as follows:

The detection method for hemochromatosis and hepatolenticular degeneration susceptibility gene mutation of the present invention integrates the whole exons and introns of 5 susceptibility genes with detection significance for hemochromatosis and hepatolenticular degeneration in the Chinese population. The information of gene variation and copy number variation in the cut region has the following advantages: (1) The detection throughput is high, and the gene variation and/or copy of all exons and intron cut regions of five susceptible genes can be detected in one reaction (2) High specificity and sensitivity; 100% sequencing coverage, depth > 30×; (3) The interpretation of genetic testing results is clear and objective, with good accuracy and repeatability, which can assist in the etiology of clinical liver disease patients Diagnostic function; (4) low cost, more than 600 cases can be detected at one time; easy to operate and easy to popularize. The detection method of the invention is suitable for: (1) differential diagnosis of patients with recurrent liver function abnormality of unknown clinical reasons; (2) susceptible populations with hemochromatosis and hepatolenticular degeneration, especially for patients with hemochromatosis and hepatolenticular degeneration It has certain auxiliary diagnosis and prompting functions for the population with the family history of the disease; (3) the general population can be easily and efficiently screened for hemochromatosis and hepatolenticular degeneration.

Description of drawings

The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

Figure 1 is a map of the quality distribution of the sequenced bases in the sample H25, wherein the quality of the sequencing data is all distributed above Q30 (bases with a quality value >=30 in the original data).

Figure 2 is the accumulation map of the sequencing depth of the sample H25, in which, the cumulative proportion of different sequencing depths (normalized processing, the average depth is 1) of the target region, for example, when the depth is 0.2, the coordinate is 97.83, which means that 97.83% of the target regions have a sequencing depth greater than Equal to 20% of the average depth.

Detailed ways

In order to illustrate the present invention more clearly, the present invention will be further described below with reference to the preferred embodiments and accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. Those skilled in the art should understand that the content specifically described below is illustrative rather than restrictive, and should not limit the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. All primers in the following examples are of electrophoresis grade (PAGE) or HPLC grade, free of impurity bands.

Example 1 Hemochromatosis and Hepatolenticular Degeneration Susceptibility Gene Mutation Detection

The clinical blood samples involved in this example were obtained from Beijing Friendship Hospital affiliated to Capital Medical University and Pediatric Research Institute of Beijing Children's Hospital affiliated to Capital Medical University. After blood samples were collected, they were immediately stored in a -80°C refrigerator.

1. Genomic DNA extraction

Genomic DNA (gDNA) from blood samples was extracted using the QIAamp DNA Blood Mini kit (Qiagen, USA).

DNA concentration was quantified using the Qubit dsDNA HS assay kit with a Qubit Fluorometer (Life Technologies, USA) machine.

DNA purity (A260/280 and A260/230) was identified using Nanodrop-2000 (Thermo Fisher Scientific, USA).

2. Construction of sequencing library

AI-Mito-Multi Panel(AIMT)(北京艾吉泰康生物科技有限公司)制备。试剂均于冰上解冻，并在冰盒上进行加样操作。Sequencing library via

AI-Mito-Multi Panel (AIMT) (Beijing Aiji Taikang Biotechnology Co., Ltd.) was prepared. Reagents were thawed on ice and loaded on ice boxes.

2.1 Multiplex PCR reaction

For HJV, SLC40A1, SUGP2, DENND3 and ATP7B genes, multiple primer pairs were designed, and the primer pairs were divided into 3 groups according to their sequence characteristics and Tm values, as shown in Table 1, namely primer group I, primer group II and primer set III, the primer set I includes the upstream and downstream primer sequences shown in SEQ ID NO. 1-122, the primer set II includes the upstream and downstream primer sequences shown in SEQ ID NO. 123-248, the primers Group III includes the upstream and downstream primer sequences shown in SEQ ID NO. 249-252.

Table 1 Primer pair for detection of hemochromatosis and hepatolenticular degeneration susceptibility gene mutation

The initial amount of gDNA in blood samples was 50 ng/reaction tube; the initial concentration of DNA was quantified using Qubit (Life Technologies). Primer groups I and II were prepared in PCR tubes according to Table 2, and PCR reaction amplification was carried out according to Table 4. The reaction system of primer set III was prepared according to Table 3, and PCR amplification was carried out according to Table 5. Three reactions were performed for each sample with primer sets I, II, and III, and the concentration of each primer was 100 uM.

The reaction system of table 2 primer sets I and II

The reaction system of table 3 primer set III

The PCR reaction amplification program of table 4 primer group I, II

Table 5 PCR reaction amplification program of primer set III

2.2 Purification of multiplex PCR products

Take out the AMPure XP magnetic beads stored at 4°C, vortex and vortex, and then place at room temperature for 25-30 minutes to equilibrate the magnetic beads. Before use, vortex to resuspend the magnetic beads to ensure that the magnetic beads are uniform and the concentration is consistent.

Add 27 μl of AMPure XP magnetic beads equilibrated at room temperature to 30 μl of PCR combined product, gently pipette and mix 20 times; after 5 minutes of incubation at room temperature, place the PCR tube on the DynaMag-96Side magnetic stand for 3 minutes; thoroughly Remove the supernatant, remove the PCR tube from the magnetic frame, add 40 μl of YF buffer B to the tube, gently pipette and mix 20 times; after 5 minutes of incubation at room temperature, place the PCR tube on the DynaMag-96Side magnetic frame on for 3 minutes; remove the supernatant, continue to place the PCR tube on the magnetic frame, add 180 μl of 80% ethanol solution to the tube, and let stand for 30 seconds; remove the supernatant, continue to place the PCR tube on the magnetic frame, and add 180 μl to the tube 80% ethanol solution, let stand for 30 seconds and then completely remove the supernatant; let stand for 4 minutes at room temperature to completely evaporate the residual ethanol; remove the PCR tube from the magnetic stand, add 24 μl Nuclease-free water, and gently use a pipette Aspirate and mix 20 times, resuspend the magnetic beads to avoid air bubbles, and let stand for 3 minutes at room temperature; place the PCR tube back on the magnetic stand and let stand for 3 minutes; use a pipette to aspirate 17 μl of the supernatant and transfer it to a new The supernatant in the tube is the purified multiplex PCR product.

2.3 Adapter sequence PCR reaction

The system of the linker sequence PCR reaction is as follows in Table 6:

Table 6 Linker sequence PCR reaction system

Run the PCR program and set the parameters of the PCR instrument as follows: heated lid at 105°C; 95°C for 3 minutes and 30 seconds; 9 cycles of 98°C for 20 seconds, 58°C for 1 minute, and 72°C for 30 seconds; 72°C for 5 minutes.

2.4 Magnetic beads purify the PCR reaction product obtained in step 2.3

Add 23 μl of AMPure XP magnetic beads equilibrated at room temperature to 25 μl of PCR reaction product, gently pipette and mix 20 times; after 5 minutes of incubation at room temperature, place the PCR tube on the DynaMag-96Side magnetic stand for 3 minutes; Completely remove the supernatant, remove the PCR tube from the magnetic stand, add 40 μl of YF buffer B to the tube, gently pipette and mix 20 times; after 5 minutes of incubation at room temperature, place the PCR tube in DynaMag-96Side On the magnetic frame for 3 minutes; remove the supernatant, continue to place the PCR tube on the magnetic frame, add 180 μl of 80% ethanol solution to the tube, and let it stand for 30 seconds; remove the supernatant, continue to place the PCR tube on the magnetic frame, and add 180 μl of 80% ethanol solution to the tube. Add 180 μl of 80% ethanol solution, let stand for 30 seconds and then completely remove the supernatant; let stand for 4 minutes at room temperature to completely evaporate the residual ethanol; remove the centrifuge tube from the magnetic stand, add 24 μl Nuclease-free water or 1×TE buffer (pH 8.0), gently pipette and mix 20 times, resuspend the magnetic beads to avoid air bubbles, and let stand for 3 minutes at room temperature; put the PCR tube back on the magnetic stand and let stand for 3 minutes; Pipette 20 μl of the supernatant and transfer it to a new PCR tube. The supernatant in the tube is the prepared multiplex PCR library, which is the sequencing library.

2.5 Library quantification

3.0Fluorometer(Qubit dsDNA HS Assay Kit)进行测序文库浓度测定，记录文库浓度，最终得到血液的gDNA的测序文库的浓度范围为34ng/μl-50ng/μl。Take 2 μl of sequencing library to use

3.0 Fluorometer (Qubit dsDNA HS Assay Kit) was used to measure the concentration of the sequencing library, and the concentration of the library was recorded. The final concentration range of the gDNA sequencing library of blood was 34ng/μl-50ng/μl.

2.6 Library Quality Inspection

Take 1 μl of the sequencing library and use the Agilent 2100Bioanalyzer system (High Sensitivity DNAKit) or use the Qsep400 automatic nucleic acid and protein analysis system (Hosawa Bio) to measure the length and purity of the library fragment, and the target fragment distribution range of the obtained sequencing library is between 300bp and 450bp. , the main peak is around 417bp.

3. Data processing of sequencing library

Using Trimmomatic to perform quality control on the raw data (in FASTQ file format) obtained by sequencing, it is necessary to preliminarily filter the Raw reads to obtain Clean reads. The main contents of the data filter are as follows: (1) Cut out the 8bp sliding window. Sequences with an average base quality value less than 20; (2) Remove the linker sequence at the end of the sequence; (3) If the first base or tail of the sequence is less than 20, the base will be cut directly; (4) Usually Next, if the remaining sequence length is less than 40 (double-ended) after removal, the pair of sequences is discarded.

BWA software was used to compare the Clean reads with the reference genome to obtain the bam result file (the human hg19 reference genome is the human (Homo sapiens) standard reference genome (GRCh37/hg19)). Based on the bam result file aligned with the genome reference sequence, and then using VarScan software for mutation analysis, the gene variation of all exons and intron splice regions of 5 genes (HJV, SLC40A1, SUGP2, DENND3, ATP7B) was obtained. and copy number variation information, and then use ANNOVAR software to annotate gene variation and copy number variation sites, determine the gene information, functional information, harmfulness, etc. corresponding to the mutation sites, and clarify the hemochromatosis and hepatolenticular nucleus of blood samples. The results of testing for mutations in transgender susceptibility genes.

4. Sequencing quality analysis of clinical samples

17 clinically collected whole blood samples were tested according to the above method. The quality distribution map of the sequenced bases with sample number H25 is shown in Figure 1, and the sequence depth accumulation map is shown in Figure 2. After the sequencing is completed, Trimmomatic is used for sequencing quality analysis, where the filtering ratio (QCrate) is the total number of bases left after filtering the original data (Mb)/the total number of bases in the original data (Mb); the ratio of the target region with depth >=30X is 30X coverage, the results are shown in Table 7.

Table 7 Sequencing quality analysis results

5. Analysis of HJV, SLC40A1, SUGP2, DENND3, and ATP7B mutation sites in clinical samples. According to the HGMD database report, the gene variation and/or copy number variation of the above five genes were determined, as shown in Tables 8 to 12, respectively.

Table 8 Gene variation and/or copy number variation of HJV genes

Table 9 Gene variation and/or copy number variation of SLC40A1 gene

Table 10 Gene variation and/or copy number variation of SUJP2 gene

serial number gene location base change protein changes 1 Chr19:19121086-19121086 1916G>A Arg639Gln

Table 11 Gene variation and/or copy number variation of DENND3 gene

serial number gene location base change protein changes 1 Chr8: 142185385-142185385 2122C>G Leu708Val 2 Chr8: 142178509-142178510 1922delT Del 1bp codon 641 3 Chr8: 142170768-142170768 994G>A Ala332Thr

Table 12 Gene variation and/or copy number variation of ATP7B gene

Clinical sample detection: 14 cases of hepatoma patients and 3 cases of hemochromatosis collected from the clinic were sequenced according to the above method. The detection results of HJV, SLC40A1, DENND3 and SUJP2 gene mutations in hemochromatosis patients are shown in Table 13. Hepatolenticular degeneration human ATP7B gene The detection results of mutations are shown in Table 14.

Table 13 Detection results of HJV, SLC40A1, DENND3, SUJP2 gene mutations in hemochromatosis patients

Table 14 Detection results of hepatolenticular degeneration human ATP7B gene mutation

A total of 19 mutation types were detected in the samples of 14 liver pea patients, with an average sequencing depth of 2584×; 7 types of mutations in HJV gene, 5 types of mutations in DENND3 gene, 3 types of mutations in SUJP2 gene, and 3 types of mutations in SLC40A1 gene were detected in 3 hemochromatosis patients. 8 mutations, the average sequencing depth of these four genes is 3558×. According to the requirement of 100× sequencing depth on the market, the NextSeq ^TM 500/550Mid Output Kit v2.5 (300Cycles) kit can detect more than 600 samples at one time. The frequency of heterozygous mutation falls between 31.1% and 61.4%, and the frequency of homozygous mutation falls between 94.6% and 100%, which completely meets the requirements of the present invention for result determination. It can be seen that the detection method of the present invention has good accuracy, low cost, high throughput and good feasibility for clinical research.

Obviously, the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, rather than limiting the embodiments of the present invention. Changes or changes in other different forms cannot be exhausted here, and all obvious changes or changes derived from the technical solutions of the present invention are still within the protection scope of the present invention.

SEQUENCE LISTING

<110> Beijing Friendship Hospital Affiliated to Capital Medical University

<120> Primer composition, kit and method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation

<130> JLP20I0389

<160> 252

<170> PatentIn version 3.5

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<213> Artificial Sequence

<400> 46

gccagtcctg tgtcagctc 19

<210> 47

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 47

ctgcaatgct ggcctcga 18

<210> 48

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 48

ctatgaaggt ggtctggatg gc 22

<210> 49

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 49

aacgcgggga ggaaaatcc 19

<210> 50

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 50

tctgcgcctc ctctccc 17

<210> 51

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 51

taatcacaag ctgcagcatc ct 22

<210> 52

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 52

gagaactgtt agccaaaata gactgc 26

<210> 53

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 53

tgctccagcc aggtcgt 17

<210> 54

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 54

gtgacaccac gggttctcag 20

<210> 55

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 55

tctctctgag aaagaaaaag gtgtgat 27

<210> 56

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 56

ttataatcag cctccacaac cctg 24

<210> 57

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 57

ctggcgggca gtccttg 17

<210> 58

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 58

gggctgtccg caacctc 17

<210> 59

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 59

tttctgccaa cttcagcttg taatatttat a 31

<210> 60

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 60

gtggtcactc ttagcaggggc 20

<210> 61

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 61

taccaaagaa acacgaaaaa caacaaatag 30

<210> 62

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 62

ttccaaactt cgaagactcc actt 24

<210> 63

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 63

gacttccggc agagagagtt g 21

<210> 64

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 64

agtttttaaa catgctttta gacaaaggtg 30

<210> 65

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 65

gaggcaagca ttttagcaca gg 22

<210> 66

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 66

cctctggggc tggatctga 19

<210> 67

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 67

tttctcaatg tgacaagttt cccaac 26

<210> 68

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 68

caggctgatt gagaaagagt gtttg 25

<210> 69

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 69

cacggagcgc aaatttccag 20

<210> 70

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 70

actctagaga tgccggaaga gaa 23

<210> 71

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 71

acccctacca aaacaacaac taca 24

<210> 72

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 72

cgcccagctc caagttgt 18

<210> 73

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 73

acatgtgctc tatataatct agtaacagga tag 33

<210> 74

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 74

atggtcatcc tggctccaaa tc 22

<210> 75

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 75

acctacatta caaaaagaca ctttagttca tta 33

<210> 76

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 76

tgaagatatc cgatcaaggt tcattca 27

<210> 77

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 77

cctgtccgaa ccaaaccaca 20

<210> 78

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 78

gagatggatg ggtctcctac taca 24

<210> 79

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 79

aggtctgaac atgagaacaa aagga 25

<210> 80

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 80

taaccaacat cttagccccc atg 23

<210> 81

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 81

agcctcattt atcaccaccg attta 25

<210> 82

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 82

gtacagtgtg gtaaactgac attataactc 30

<210> 83

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 83

tgttgtttcc tttgctacag aggta 25

<210> 84

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 84

atttttctctt ttcatttaag ggagatcgg 29

<210> 85

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 85

aaattgcgca actgtgttgt aaga 24

<210> 86

<211> 35

<212> DNA

<213> Artificial Sequence

<400> 86

tgcaaagtag ttttttaataa tcatgttcta atgag 35

<210> 87

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 87

tccccagggg tctgcg 16

<210> 88

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 88

gtggcccacg tccgc 15

<210> 89

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 89

tccttttatc agtaaagagg acagtcaa 28

<210> 90

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 90

gtggccggca gcagg 15

<210> 91

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 91

ggaggtgacc atgtcgctg 19

<210> 92

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 92

aaagagctgc cccctacca 19

<210> 93

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 93

gctaacgatg acaactgcgt tc 22

<210> 94

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 94

tgtgagcatt tacattatct tcaacatctt 30

<210> 95

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 95

agcatgtact cagtgcggc 19

<210> 96

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 96

gcccatcagg aaggacgtg 19

<210> 97

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 97

tccgaaattc ttttatcaat ctttacagat ga 32

<210> 98

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 98

tactcgaagt ggcaaataca gaatct 26

<210> 99

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 99

ccaccctgca gctgctc 17

<210> 100

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 100

aagaacaaac tccctgtaac tgct 24

<210> 101

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 101

ggcacagccc agtgacc 17

<210> 102

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 102

cttgaacact gtgattccta aagagc 26

<210> 103

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 103

ttgccatgcc tgagctgg 18

<210> 104

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 104

tattctaaac atttcaaata actcagctgc ta 32

<210> 105

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 105

ctttctggcc cccgataact c 21

<210> 106

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 106

aggatctcgc tgatgagcct 20

<210> 107

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 107

gaaggacgcc agcatcatac a 21

<210> 108

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 108

aaaatactaa aattagccgg gcacg 25

<210> 109

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 109

tgtgtgtaag ttgtccagct cc 22

<210> 110

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 110

gctggtactt ggcatggct 19

<210> 111

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 111

ccggtgaact gatcttagtg attct 25

<210> 112

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 112

cagataaaat cacttaaggc gtgtaatca 29

<210> 113

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 113

ccagtcacct gctgctacag 20

<210> 114

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 114

cacgtgaatt cagaggcttc tttc 24

<210> 115

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 115

gcaacaagca gctcacagc 19

<210> 116

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 116

gttctccgca cccgtgag 18

<210> 117

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 117

ctggcgtctt atgttgccaa tg 22

<210> 118

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 118

gggcacggtg tgggc 15

<210> 119

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 119

gtgaagaagc aggtagggtg g 21

<210> 120

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 120

gccatccagt ttgggggaat 20

<210> 121

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 121

ctcttgggag gggcttcttt c 21

<210> 122

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 122

ctttccaaat ggcgactttc cc 22

<210> 123

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 123

gtctgaggac cgtctcacaa tc 22

<210> 124

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 124

caggtgcggg cggtg 15

<210> 125

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 125

tccctgcccc ggacc 15

<210> 126

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 126

cggtagcgtt ggcccc 16

<210> 127

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 127

atgcactgaa cccaaaatga actg 24

<210> 128

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 128

tccacatggt tcccagggt 19

<210> 129

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 129

atggccttct cagctgaaca g 21

<210> 130

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 130

catcctccag tgctgcctg 19

<210> 131

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 131

gctccactcc tttctgggc 19

<210> 132

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 132

cacagagatc cggaatgcag taa 23

<210> 133

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 133

gcttgtcgga cgtcaggg 18

<210> 134

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 134

aagccttgtt tctagaatgg ctca 24

<210> 135

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 135

cactaacccc agcaggaacc 20

<210> 136

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 136

caggctgtgg gtgctgg 17

<210> 137

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 137

tattctggag ctcctggacc tt 22

<210> 138

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 138

ggtgttgggg caggagc 17

<210> 139

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 139

ttgcgatcat cccacagagc 20

<210> 140

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 140

tgcgaagatt tcaattatat tgcttcca 28

<210> 141

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 141

accgttgcgc ctcagc 16

<210> 142

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 142

attcataccc acatgtccta cacatt 26

<210> 143

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 143

gtccgtgcag tatcccaagg 20

<210> 144

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 144

caaaggaggg agaaataatt acaagttact ag 32

<210> 145

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 145

cagcaggagc acccgc 16

<210> 146

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 146

agattgaacg acagaggatc acg 23

<210> 147

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 147

gcaatgcaca gcaccgtg 18

<210> 148

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 148

atgatggcag agcagtgtgg 20

<210> 149

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 149

gatttcccag aactcttcac ataatttcta a 31

<210> 150

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 150

cagttgtctc tttcctacgt ctagg 25

<210> 151

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 151

gacagctgct atgatatcct cctg 24

<210> 152

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 152

tattgtaaca gctggcctag aacc 24

<210> 153

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 153

tgtttactga aggagcagct cttt 24

<210> 154

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 154

tacgttcagg cctacaaatc tctg 24

<210> 155

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 155

agggccacac acagcatg 18

<210> 156

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 156

ctgcatttgc tttccaggtg g 21

<210> 157

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 157

gaggaaggca ccatgggaag 20

<210> 158

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 158

aatgcatatt ttaaccaagt accttcctc 29

<210> 159

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 159

gccatttgtc ctcgtgagtt tg 22

<210> 160

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 160

gctggaggaa cgttgagaat ct 22

<210> 161

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 161

aaccaacacg gagagaacac c 21

<210> 162

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 162

cacctaccct gggatactct gat 23

<210> 163

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 163

tgattgtggg gactttgcca a 21

<210> 164

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 164

aaccctcacc aagagccct 19

<210> 165

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 165

tcggccaaag acaccgatat tt 22

<210> 166

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 166

ccacctggga attttaaagt ttctctt 27

<210> 167

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 167

ccctaggagc tggccaatat ttt 23

<210> 168

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 168

gaagctgcca tcaagagcaa ag 22

<210> 169

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 169

ttggttgctg agtgagactt tga 23

<210> 170

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 170

gtgccactgt gaaatatgtg cc 22

<210> 171

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 171

aagtcatgcc caagatcctg ac 22

<210> 172

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 172

gcattgtttt ccattttctc agtgc 25

<210> 173

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 173

cagggaacat caactttgtt ggg 23

<210> 174

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 174

tgggctcagg tgcacaac 18

<210> 175

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 175

cccaggttct tatcggtcag c 21

<210> 176

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 176

gacctctgac ttagaagttc catcc 25

<210> 177

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 177

agcccaccaa ccaagaactg 20

<210> 178

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 178

aggggcgggc aagtct 16

<210> 179

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 179

tcttcaaact ctgcttcccc ttc 23

<210> 180

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 180

cagcaaataa aaatgttgag agattccatc 30

<210> 181

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 181

cccccaattc taaagagacc c 21

<210> 182

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 182

caaaaccatc cctgccagac 20

<210> 183

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 183

cagccccgga gccct 15

<210> 184

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 184

gtccccgttat gcttgttttg ttatct 26

<210> 185

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 185

aataccatca gagtgaccca aattttg 27

<210> 186

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 186

gcccacattc agcatatttc tcag 24

<210> 187

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 187

acctggaggg gacatccg 18

<210> 188

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 188

cctcagtaac cccctggact 20

<210> 189

<211> 31

<212> DNA

<213> Artificial Sequence

<400> 189

tcattatgta cttgtcaaaa ggctttatga t 31

<210> 190

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 190

cttttccaga ctctctttga acttgaaa 28

<210> 191

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 191

ctcatctttc ttctggggag cc 22

<210> 192

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 192

cagggcctgc tcacagc 17

<210> 193

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 193

gccctggaaa tacgtcactt ct 22

<210> 194

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 194

ctgtttgcat tctctggtct aaagtatc 28

<210> 195

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 195

cacccaccga cgacgc 16

<210> 196

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 196

ctgcgcacca ggactcg 17

<210> 197

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 197

ccacaaagga gactgaaatc aatacg 26

<210> 198

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 198

tgaaatgtat gcctgtaaac taaaatctaa tct 33

<210> 199

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 199

acccattaga catgtatatt tcagttgtaa ttt 33

<210> 200

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 200

tataactgga ataatgggaa ctgtagctt 29

<210> 201

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 201

gtcaaagccc aggacagtca tat 23

<210> 202

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 202

catgtcctcc ccaacaaaat aatgg 25

<210> 203

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 203

gactggggag ccaaatgtca t 21

<210> 204

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 204

aacaacaaat atttttccaa caaaatgtct ttc 33

<210> 205

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 205

ttcatccttt accactacca gatattcaa 29

<210> 206

<211> 28

<212> DNA

<213> Artificial Sequence

<400> 206

attatgtgtg gataagaaca gtctcact 28

<210> 207

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 207

ctgtttccat agagctctac cagaaa 26

<210> 208

<211> 26

<212> DNA

<213> Artificial Sequence

<400> 208

caagaatatt ttccattgtg ctggga 26

<210> 209

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 209

acctgggttt ccaccatatg c 21

<210> 210

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 210

ctgttgtgtt tttagaggtg ctatctc 27

<210> 211

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 211

aaaggatacc ctcgaacaag agc 23

<210> 212

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 212

ctgggcttct ctctcttctt tctc 24

<210> 213

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 213

ggtaacccag gccacttgg 19

<210> 214

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 214

gaatgttgtg ttatgtgtat ttcaccataa taaa 34

<210> 215

<211> 30

<212> DNA

<213> Artificial Sequence

<400> 215

tgaaaaatca gaaagacatc gtatagaacc 30

<210> 216

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 216

gtggctcatt ctccagcatg a 21

<210> 217

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 217

agcagctctc tgaactaatg acg 23

<210> 218

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 218

aaactcgcat cttcatcatc tctga 25

<210> 219

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 219

caccccttcg tgcccatc 18

<210> 220

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 220

cctctctgcg gcacacag 18

<210> 221

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 221

gggctgtgtg ctggtgag 18

<210> 222

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 222

gggaatgttc tctcacgtac ctg 23

<210> 223

<211> 33

<212> DNA

<213> Artificial Sequence

<400> 223

tttctctgct actaaaagtg gtatattttt cat 33

<210> 224

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 224

tacagaaaag gataatggat tgaacacac 29

<210> 225

<211> 25

<212> DNA

<213> Artificial Sequence

<400> 225

ttgtgtctat aaactaacgc gttgc 25

<210> 226

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 226

agccggagcg aggagtt 17

<210> 227

<211> 24

<212> DNA

<213> Artificial Sequence

<400> 227

tgttcatttg gaaggtctgt gcta 24

<210> 228

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 228

tgtccctgca tcagataaac gag 23

<210> 229

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 229

gtgaagagga cggtggaatc c 21

<210> 230

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 230

cctacagtca aggcctcgaa c 21

<210> 231

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 231

gtatgtggct gtaagatacc tctcttt 27

<210> 232

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 232

ttctgggtca tggcgatctt g 21

<210> 233

<211> 27

<212> DNA

<213> Artificial Sequence

<400> 233

tctctgagtg tgacattcct aactttt 27

<210> 234

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 234

aggcagccac agcaaagg 18

<210> 235

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 235

ttgaggctgc aacagtaaca ct 22

<210> 236

<211> 23

<212> DNA

<213> Artificial Sequence

<400> 236

caaacctaag cgcatcagat tgg 23

<210> 237

<211> 34

<212> DNA

<213> Artificial Sequence

<400> 237

cagtaaatgt atagaatatg acttcaccag tatc 34

<210> 238

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 238

cagagcttac tgcacctggg 20

<210> 239

<211> 22

<212> DNA

<213> Artificial Sequence

<400> 239

tgttttcagg ggcccaagtt aa 22

<210> 240

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 240

ctgctctcgt tcagcatcct t 21

<210> 241

<211> 15

<212> DNA

<213> Artificial Sequence

<400> 241

gcaggggctg tgggc 15

<210> 242

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 242

cgagacctgc cgggga 16

<210> 243

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 243

tttcatggtg tggtccaagc a 21

<210> 244

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 244

caggttagag tgccacgtcc 20

<210> 245

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 245

tcggctgagg acagatacgt 20

<210> 246

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 246

gaaggcctga gagaaggtgc 20

<210> 247

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 247

gccgccccta agcacag 17

<210> 248

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 248

gtcattggcc accggaaatt g 21

<210> 249

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 249

ggctgcaggt ggaaggac 18

<210> 250

<211> 21

<212> DNA

<213> Artificial Sequence

<400> 250

atgcctggac tctcctctct c 21

<210> 251

<211> 16

<212> DNA

<213> Artificial Sequence

<400> 251

ctggcggcct ctgtcg 16

<210> 252

<211> 17

<212> DNA

<213> Artificial Sequence

<400> 252

gggggacgac catgcag 17

Claims (3)

1. The primer composition for detecting the hemochromatosis and hepatolenticular degeneration susceptibility gene mutation is characterized in that the primer composition is a primer group capable of specifically amplifying the gene variation and/or copy number variation of all exons and intron splicing regions of the susceptibility genes of the hemochromatosis and hepatolenticular degeneration, the susceptibility genes are HJV, SLC40A1, SUGP2 and DENND3 genes which are related to the hemochromatosis which is unique or common in Chinese population and ATP7B genes which are related to the hepatolenticular degeneration respectively, wherein,

the primer composition comprises a primer group I, a primer group II and a primer group III, wherein the primer group I consists of upstream and downstream primer sequences shown by SEQ ID NO.1-122, the primer group II consists of upstream and downstream primer sequences shown by SEQ ID NO.123-248, and the primer group III consists of upstream and downstream primer sequences shown by SEQ ID NO. 249-252.

2. A kit comprising the primer composition of claim 1.

3. The primer composition of claim 1 or the kit of claim 2, wherein the primer composition is used for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation.

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