CN111647654A

CN111647654A - Primer composition, kit and method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation

Info

Publication number: CN111647654A
Application number: CN202010528919.3A
Authority: CN
Inventors: 黄坚; 宋燚; 贾思雨
Original assignee: Beijing Friendship Hospital
Current assignee: Beijing Friendship Hospital
Priority date: 2020-06-11
Filing date: 2020-06-11
Publication date: 2020-09-11
Anticipated expiration: 2040-06-11
Also published as: CN111647654B

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 containing the primer composition and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation. The detection method of the hemochromatosis and hepatolenticular degeneration susceptibility gene mutation integrates the gene variation and copy number variation information of the whole exon and intron splicing region of the susceptibility gene of the hemochromatosis and hepatolenticular degeneration in Chinese population, and has the following advantages: the detection flux is high; high specificity and sensitivity; sequencing coverage 100%, depth >30 ×; the interpretation of the gene detection result is clear and objective, and the accuracy and the repeatability are good; the cost is low, the operation is simple and convenient, and the popularization is easy; not only can detect known high-incidence mutation, but also can discover new mutation sites.

Description

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

Technical Field

The present invention relates to the field of gene detection. In particular to a primer composition, a kit and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation. More particularly, relates to a primer composition, a kit and a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation of Chinese population based on high-throughput sequencing.

Background

Hemochromatosis (HC) and hepatolenticular degeneration (HLD) refer to metabolic disorders of the liver caused by genetic mutations, with congenital, lifelong and familial characteristics. Because early clinical symptoms and laboratory examinations do not have specific indications, missed diagnosis or misdiagnosis often occurs, and diagnosis and differential diagnosis of the hereditary metabolic liver disease are greatly hindered.

The hemochromatosis is also called hereditary hemochromatosis (HHC, HH), belongs to common chronic iron overload diseases, is an autosomal recessive genetic disease, and known that the main pathogenic gene mutation of European and American populations is HFE C282Y pure and mutation, and the main pathogenic gene mutation of Chinese populations is HJV, SLC40A1, SUGP2 and DENND3 genes, wherein SUGP2R63 639Q and DENND 3L 708V are newly discovered pathogenic genes and hot spot mutations which are specific to the Chinese populations; excessive iron is stored in parenchymal cells of the liver, heart and pancreas due to inappropriate increases in intestinal iron absorption, resulting in tissue organ degeneration and diffuse fibrosis, metabolism and malfunction. The main clinical characteristics are skin pigmentation, liver cirrhosis and secondary diabetes. These complications are difficult to reverse and early diagnosis is important to prevent serious complications, especially the occurrence of liver cancer.

The hemochromatosis currently has no most effective method for early diagnosis. In cases without secondary infection and with concurrent liver cancer, the simplest and practical screening assay is Serum Iron (SI), serum ferritin, total iron binding and transferrin saturation determination.

Hepatolenticular degeneration, also known as Wilson Disease (WD), is an autosomal recessive hereditary Disease. The pathogenic gene ATP7B maps to chromosome 13q14.3 and encodes a 1465 amino acid copper transport P-type ATPase. The ATP7B gene mutation causes the decrease or disappearance of atpase function, which leads to the decrease of serum Ceruloplast (CP) synthesis and biliary copper excretion disorder, and the deposition of copper ions accumulated in the body at the liver, brain, kidney, cornea, etc., causing progressive exacerbation of cirrhosis, extrapyramidal symptoms, mental symptoms, renal damage, and the keratechromenic ring (Kayser-fleischer ring, K.F ring), etc. The ATP7B gene has many variation sites, and there are more than 800 sites described in the human genome database.

The current commonly used auxiliary detection methods for hepatolenticular degeneration comprise biochemical determination of metabolites, hematuria routine, liver and kidney function and imaging detection and the like; and detection methods for genes such as first-generation sequencing and the like.

Early symptoms of hemochromatosis and hepatolenticular degeneration are not obvious, and the disease is usually aggravated progressively, if the early symptoms are not recognized, diagnosed, intervened and treated, disability or death is often caused, and the cause of the disease cannot be confirmed. Therefore, the development of early screening of the hereditary metabolic liver disease is the key to prevention and treatment.

At present, the diagnosis technology and method for hemochromatosis and hepatolenticular degeneration have the biggest limitation that the general flux is low, the price is high, and the current situation that the two types of hereditary metabolic liver disease patients increase year by year cannot be met; meanwhile, the detection result of the enzyme activity or the metabolite suggests indirect evidence, and the defects of difficult interpretation of the result, poor repeatability and more false positive and false negative exist.

Currently, a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation with high throughput and low cost is needed.

Disclosure of Invention

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

The invention also aims to provide a method for detecting hemochromatosis and hepatolenticular degeneration based on high-throughput sequencing, which has the advantages of high detection throughput, high sensitivity and strong specificity, and can detect the gene variation and copy number variation of all exon and intron splicing regions of 5 susceptible genes covering hemochromatosis and hepatolenticular degeneration in one time.

In order to achieve the above objects, the present invention provides a primer composition for detecting hemochromatosis and hepatolenticular degeneration susceptible gene mutation, which is specific or common to chinese population, comprising a primer set capable of specifically amplifying genetic variation and/or copy number variation of all exons and intron splicing regions of hemochromatosis and hepatolenticular degeneration susceptible genes, wherein the susceptible genes are HJV, SLC40a1, SUGP2 and DENND3 genes related to hemochromatosis and ATP7B gene related to hepatolenticular degeneration, respectively.

In the invention, the HJV and SLC40A1 genes are common genes related to hemochromatosis of Chinese people, and the SUGP2 and DENND3 genes are specific genes related to hemochromatosis of Chinese people.

Further, the primer group comprises a primer group I, a primer group II and a primer group III, wherein the primer group I comprises upstream and downstream primer sequences shown by SEQ ID NO.1-122, the primer group II comprises upstream and downstream primer sequences shown by SEQ ID NO.123-248, and the primer group III comprises upstream and downstream primer sequences shown by SEQ ID NO.249-252, which are specifically shown in Table 1.

The sequence and the detection condition of the primer composition are obtained by repeated optimization of a large number of experiments, a sequencing library is constructed through multiple PCR reactions, then large-scale parallel sequencing (second-generation sequencing) analysis is carried out, data obtained by the sequencing analysis are analyzed through methods such as sequence screening, splicing and comparison and bioinformatics, and finally the information of the gene variation and copy number variation of all exons and intron splicing regions of the susceptible gene of the hemochromatosis and hepatolenticular degeneration of the sample to be detected is obtained.

The information on the gene variation and copy number variation of all exon and intron splicing regions of the above-mentioned susceptibility genes is shown in tables 8 to 12.

The invention further provides a kit containing the primer composition.

In the present invention, the kit comprises reagents such as ligase, amplified Taq enzyme, adaptor, purified magnetic beads, etc. in addition to the above primer composition.

The application of the primer composition or the kit in the preparation of products for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation is also within the protection scope of the invention.

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

The invention discloses a method for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation, which comprises the following steps:

obtaining a DNA sample of a sample to be detected;

respectively designing and synthesizing primer sets aiming at gene variation and/or copy number variation of all exons and intron splicing regions of susceptible genes of hemochromatosis and hepatolenticular degeneration, wherein the susceptible genes are HJV, SLC40A1, SUGP2 and DENND3 genes related to hemochromatosis and ATP7B genes related to hepatolenticular degeneration;

performing multiplex PCR on the DNA sample of the sample to be detected by using the primer group and constructing a sequencing library;

and processing the data of the sequencing library to obtain the information of the gene variation and copy number variation of all exons and intron shearing regions of the susceptibility genes of the hemochromatosis and the hepatolenticular degeneration of the sample to be detected, thereby determining the detection result of the hemochromatosis and the hepatolenticular degeneration susceptibility gene mutation of the sample to be detected.

In the invention, the HJV and SLC40A1 genes and common hemochromatosis related genes of Chinese people, and the SUGP2 and DENND3 genes and specific hemochromatosis related genes of Chinese people are disclosed.

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

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

According to the invention, both the library construction and the data processing of the sequencing library are carried out by adopting the conventional method in the field, and in the specific embodiment of the invention, the sequencing library construction process comprises the steps of carrying out multiple PCR reactions and product combination, purifying and product combination, linker sequence PCR reaction and purification, library quantification and quality inspection in three times; and the data of the sequencing library comprises the quality control of the data of the sequencing library by using Trimmomatic, the Reads subjected to the quality control is mapped to a human hg19 reference genome by using a BWA algorithm, and the detection and analysis of the susceptibility gene mutation are carried out on the Clean data obtained after mapping by using VarScan software.

The invention has the following beneficial effects:

the detection method of the hemochromatosis and hepatolenticular degeneration susceptibility gene mutation integrates the gene variation and copy number variation information of the whole exon and intron splicing regions of 5 susceptibility genes with detection significance on the hemochromatosis and hepatolenticular degeneration in Chinese population, and has the following advantages: (1) the detection flux is high, and the gene variation and/or copy number variation of all exons and intron splicing regions of 5 susceptible genes can be detected by one reaction; (2) high specificity and sensitivity; sequencing coverage 100%, depth >30 ×; (3) the interpretation of the gene detection result is clear and objective, and the method has good accuracy and repeatability and plays an auxiliary diagnosis role in the etiology of the clinical liver disease patients; (4) the cost is low, and more than 600 cases can be detected at one time; simple operation and easy popularization. The detection method of the invention is suitable for: (1) the differential diagnosis of patients with clinical unknown reasons and repeated abnormal liver functions; (2) the kit has certain auxiliary diagnosis and prompting functions on susceptible people with hemochromatosis and hepatolenticular degeneration, particularly people with family genetic history of hemochromatosis and hepatolenticular degeneration; (3) can simply and efficiently carry out population screening on hemochromatosis and hepatolenticular degeneration of common people.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is a base mass distribution diagram of sample H25 sequencing, in which the masses of the sequencing data are distributed over Q30 (base with mass value >30 in the original data).

Fig. 2 is a cumulative plot of sequencing depths of sample H25, in which the cumulative percentage of different sequencing depths (normalized, average depth of 1) of the target region, for example, the coordinate of 97.83 when the depth is 0.2, represents that 97.83% of the sequencing depths of the target region are greater than or equal to 20% of the average depth.

Detailed Description

In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. All primers in the following examples were of electrophoretic grade (PAGE) or HPLC grade purity and contained no bands.

Example 1 detection of mutations in genes susceptible to hemochromatosis and hepatolenticular degeneration

The clinical blood samples involved in this example were from the Beijing friendship Hospital affiliated with the university of capital medical science and the Beijing Children Hospital research institute affiliated with the university of capital medical science. After the blood sample is collected, the blood sample is immediately placed into a refrigerator at the temperature of 80 ℃ below zero for storage.

1. Genomic DNA extraction

Genomic DNA (gDNA) of a Blood sample was extracted using a QIAamp DNA Blood Mini kit (Qiagen, USA).

The DNA concentration was quantified using the Qubit dsDNA HS assay kit and the Qubit Fluorometer (Life technologies, USA) machine.

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

2. Construction of sequencing libraries

Sequencing library

AI-Mito-Multi Panel (AIMT) (Beijing Aijiekang Biotech Co., Ltd.). The reagents are thawed on ice and the sample loading operation is carried out on an ice box.

2.1 multiplex PCR reactions

Aiming at HJV, SLC40A1, SUGP2, DENND3 and ATP7B genes, a plurality of primer pairs are designed, the primer pairs are divided into 3 groups according to the sequence characteristics, Tm value and other characteristics of the primers, specifically shown in Table 1, namely a primer group I, a primer group II and a primer group III, wherein the primer group I comprises upstream and downstream primer sequences shown by SEQ ID NO.1-122, the primer group II comprises upstream and downstream primer sequences shown by SEQ ID NO.123-248, and the primer group III comprises upstream and downstream primer sequences shown by SEQ ID NO. 249-252.

TABLE 1 primer set for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation

The initial amount of gDNA of the blood sample was 50 ng/reaction tube; initial concentrations of DNA were quantified using qubits (life technologies). The primer set I, II was prepared in a PCR tube according to Table 2, and PCR amplification was performed according to Table 4. The primer set III was prepared in the reaction system according to Table 3, and PCR amplification was carried out according to Table 5. Each sample was subjected to 3 reactions with primer sets I, II, III, respectively, each primer at a concentration of 100 uM.

TABLE 2 reaction System of primer set I, II

TABLE 3 reaction System of primer set III

TABLE 4 PCR reaction amplification procedure for primer set I, II

TABLE 5 PCR reaction amplification procedure for primer set III

2.2 purification of the multiplex PCR products

And taking out the AMPure XP magnetic beads stored at 4 ℃, carrying out vortex oscillation, then placing the AMPure XP magnetic beads at room temperature for 25-30 minutes to balance the magnetic beads, and carrying out vortex oscillation on the resuspended magnetic beads before use, so as to ensure that the magnetic beads are uniform and consistent in concentration.

Adding 27 mul of AMPure XP magnetic beads which are balanced at room temperature into 30 mul of PCR combined products, and gently sucking and beating the mixture by using a pipette and uniformly mixing the mixture for 20 times; after incubation for 5 minutes at room temperature, the PCR tubes were placed on a DynaMag-96Side magnetic frame for 3 minutes; thoroughly removing the supernatant, taking the PCR tube off the magnetic frame, adding 40 μ l YF buffer B into the tube, and gently sucking and stirring the mixture for 20 times by using a pipettor; after incubation for 5 minutes at room temperature, the PCR tubes were placed on a DynaMag-96Side magnetic frame for 3 minutes; removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu l of 80% ethanol solution into the PCR tube, and standing for 30 seconds; removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu l of 80% ethanol solution into the PCR tube, standing for 30 seconds, and completely removing the supernatant; standing for 4 minutes at room temperature to completely volatilize residual ethanol; taking down the PCR tube from the magnetic frame, adding 24 μ l of clean-free water, gently sucking and mixing by a pipette for 20 times, resuspending the magnetic beads to avoid generating bubbles, and standing for 3 minutes at room temperature; placing the PCR tube on the magnetic frame again, and standing for 3 minutes; pipette 17. mu.l of the supernatant, which is the purified multiplex PCR product, into a new 200. mu.l PCR tube.

2.3 linker sequence PCR reaction

The linker sequence PCR reaction system is shown in Table 6 below:

TABLE 6 linker sequence PCR reaction System

Running a PCR program, and setting the parameters of a PCR instrument as follows, namely a hot cover is 105 ℃; 3 minutes and 30 seconds at 95 ℃; 9 cycles of 98 ℃ for 20 seconds, 58 ℃ for 1 minute and 72 ℃ for 30 seconds; 5 minutes at 72 ℃.

2.4 magnetic bead purification of PCR reaction product obtained in step 2.3

Adding 23 mul of AMPure XP magnetic beads which are balanced at room temperature into 25 mul of PCR reaction products, and gently sucking and beating the mixture by using a pipettor and uniformly mixing the mixture for 20 times; after incubation for 5 minutes at room temperature, the PCR tubes were placed on a DynaMag-96Side magnetic frame for 3 minutes; thoroughly removing the supernatant, taking down the PCR tube from the magnetic frame, adding 40 μ l YF buffer B into the tube, and gently sucking and mixing the mixture for 20 times by using a pipettor; after incubation for 5 minutes at room temperature, the PCR tubes were placed on a DynaMag-96Side magnetic frame for 3 minutes; removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu l of 80% ethanol solution into the PCR tube, and standing for 30 seconds; removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu l of 80% ethanol solution into the PCR tube, standing for 30 seconds, and completely removing the supernatant; standing for 4 minutes at room temperature to completely volatilize residual ethanol; taking the centrifuge tube off the magnetic frame, adding 24 μ l of clean-free water or 1 × TE buffer (pH 8.0), gently pipetting for 20 times, re-suspending the magnetic beads to avoid generating bubbles, and standing at room temperature for 3 min; placing the PCR tube on the magnetic frame again, and standing for 3 minutes; and (3) sucking 20 mu l of supernatant by using a liquid transfer machine, and transferring the supernatant into a new PCR tube, wherein the supernatant in the tube is the prepared multiplex PCR library, namely the sequencing library.

2.5 library quantification

2 μ l of the sequencing library was used

3.0fluorometer (qubit dsDNA HS Assay kit) to perform sequencing library concentration determination, record library concentration, and finally obtain the concentration range of gDNA sequencing library of blood 34 ng/. mu.l-50 ng/. mu.l.

2.6 library quality testing

The length and purity of the library fragment were measured using Agilent 2100Bioanalyzer system (High Sensitivity DNAkit) or Qsep400 full-automatic nucleic acid protein analysis system (Ganzea) from 1. mu.l of the sequencing library, and the distribution interval of the target fragment of the obtained sequencing library was between 300bp and 450bp, with a main peak around 417 bp.

3. Data processing of sequencing libraries

Using Trimmomatic to perform quality control on original data (in a FASTQ file format) obtained by sequencing, and performing preliminary filtering on Raw reads to obtain Clean reads, wherein the content of data filtering is mainly as follows: (1) cutting off sequences with the average base quality value less than 20 in an 8bp sliding window mode; (2) removing the linker sequence at the tail of the sequence; (3) if the first base or the tail base of the sequence is less than 20, the base is directly sheared off; (4) typically, if the remaining sequence length after removal is less than 40 (double ends), the pair of sequences is discarded.

Clean reads were aligned to the reference genome using BWA software to obtain bam results file (the human hg19 reference genome is the human (Homo sapiens) standard reference genome (GRCh37/hg 19)). Based on a bam result file aligned with a genome reference sequence, mutation analysis is carried out by VarScan software to obtain the information of the gene variation and copy number variation of all exon and intron splicing regions of 5 genes (HJV, SLC40A1, SUGP2, DENND3 and ATP7B), and ANNOVAR software is used for annotating the gene variation and copy number variation sites to determine the gene information, functional information, harmfulness and the like corresponding to the mutation sites, so that the detection results of the hemochromatosis and hepatolenticular degeneration susceptibility gene mutation of a blood sample are determined.

4. Sequencing quality analysis of clinical samples

The assay was performed on 17 samples of whole blood collected clinically as described above, with the sequencing base mass profile for sample number H25 shown in FIG. 1 and the sequencing depth cumulative profile shown in FIG. 2. Performing sequencing quality analysis by using Trimmomatic after sequencing is completed, wherein a filtering ratio (QCrate) is the residual total base number (Mb)/the original data total base number (Mb) after the original data are filtered; the target area ratio with depth > 30X was 30X coverage, and the results are shown in table 7.

TABLE 7 sequencing Mass analysis results

5. HJV, SLC40A1, SUGP2, DENND3 and ATP7B mutation site analysis of clinical samples, and gene variation and/or copy number variation of the five genes are determined according to HGMD database reports, and are respectively shown in tables 8 to 12.

TABLE 8 genetic variation and/or copy number variation of HJV Gene

TABLE 9 genetic variation and/or copy number variation of SLC40A1 Gene

TABLE 10 genetic variation and/or copy number variation of SUJP2 Gene

Serial number	Location of gene	Base change	Protein changes
					1	Chr19:19121086-19121086	1916G>A	Arg639Gln

TABLE 11 genetic variation and/or copy number variation of DENND3 Gene

Serial number	Location of gene	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 genetic variation and/or copy number variation of ATP7B Gene

And (3) detecting a clinical sample: 14 patients with hepatolenticular disease and 3 patients with hemochromatosis were sequenced according to the above method, and the results of detecting mutations in HJV, SLC40A1, DENND3 and SUJP2 genes of hemochromatics are shown in Table 13, and the results of detecting mutations in human ATP7B gene of hepatolenticular degeneration are shown in Table 14.

Results of testing HJV, SLC40A1, DENND3 and SUJP2 gene mutation of patients with Table 13 hemochromatosis

TABLE 14 detection results of mutations in hepatolenticular degeneration human ATP7B gene

14 liver bean patients detected 19 mutation types in total, mean sequencing depth of 2584 ×, 3 hemochromatosis patients detected 7 mutation types of HJV gene, 5 mutations of DENND3 gene, 3 mutations of SUJP2 gene, 8 mutations of SLC40A1 gene, mean sequencing depth of 3558 ×, according to the requirement of sequencing depth of 100 × on the market,using NextSeq^TMThe 500/550Mid Output Kit v2.5(300Cycles) Kit can detect more than 600 samples at a time. The heterozygous mutation frequency is between 31.1% and 61.4%, and the homozygous mutation frequency is between 94.6% and 100%, which completely meets the result judgment requirement of the invention. Therefore, the detection method has the advantages of good accuracy, low cost, high flux and good feasibility of being applied to clinical research.

It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.

SEQUENCE LISTING

<110> affiliated Beijing friendship hospital of capital medical university

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<213> Artificial Sequence (Artificial Sequence)

<400>43

gagccagggg aatgagaact g 21

<210>44

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>44

gcattgtaag tcttgcgtct tgaata 26

<210>45

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>45

aaggtctctt tgggttagtg cttt 24

<210>46

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>46

gccagtcctg tgtcagctc 19

<210>47

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>47

ctgcaatgct ggcctcga 18

<210>48

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>48

ctatgaaggt ggtctggatg gc 22

<210>49

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>49

aacgcgggga ggaaaatcc 19

<210>50

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>50

tctgcgcctc ctctccc 17

<210>51

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>51

taatcacaag ctgcagcatc ct 22

<210>52

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>52

gagaactgtt agccaaaata gactgc 26

<210>53

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>53

tgctccagcc aggtcgt 17

<210>54

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>54

gtgacaccac gggttctcag 20

<210>55

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>55

tctctctgag aaagaaaaag gtgtgat 27

<210>56

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>56

ttataatcag cctccacaac cctg 24

<210>57

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>57

ctggcgggca gtccttg 17

<210>58

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>58

gggctgtccg caacctc 17

<210>59

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>59

tttctgccaa cttcagcttg taatatttat a 31

<210>60

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>60

gtggtcactc ttagcagggc 20

<210>61

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>61

taccaaagaa acacgaaaaa caacaaatag 30

<210>62

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>62

ttccaaactt cgaagactcc actt 24

<210>63

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>63

gacttccggc agagagagtt g 21

<210>64

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>64

agtttttaaa catgctttta gacaaaggtg 30

<210>65

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>65

gaggcaagca ttttagcaca gg 22

<210>66

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>66

cctctggggc tggatctga 19

<210>67

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>67

tttctcaatg tgacaagttt cccaac 26

<210>68

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>68

caggctgatt gagaaagagt gtttg 25

<210>69

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>69

cacggagcgc aaatttccag 20

<210>70

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>70

actctagaga tgccggaaga gaa 23

<210>71

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>71

acccctacca aaacaacaac taca 24

<210>72

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>72

cgcccagctc caagttgt 18

<210>73

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>73

acatgtgctc tatataatct agtaacagga tag 33

<210>74

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>74

atggtcatcc tggctccaaa tc 22

<210>75

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>75

acctacatta caaaaagaca ctttagttca tta 33

<210>76

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>76

tgaagatatc cgatcaaggt tcattca 27

<210>77

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>77

cctgtccgaa ccaaaccaca 20

<210>78

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>78

gagatggatg ggtctcctac taca 24

<210>79

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>79

aggtctgaac atgagaacaa aagga 25

<210>80

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>80

taaccaacat cttagccccc atg 23

<210>81

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>81

agcctcattt atcaccaccg attta 25

<210>82

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>82

gtacagtgtg gtaaactgac attataactc 30

<210>83

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>83

tgttgtttcc tttgctacag aggta 25

<210>84

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>84

attttctctt ttcatttaag ggagatcgg 29

<210>85

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>85

aaattgcgca actgtgttgt aaga 24

<210>86

<211>35

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>86

tgcaaagtag tttttaataa tcatgttcta atgag 35

<210>87

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>87

tccccagggg tctgcg 16

<210>88

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>88

gtggcccacg tccgc 15

<210>89

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>89

tccttttatc agtaaagagg acagtcaa 28

<210>90

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>90

gtggccggca gcagg 15

<210>91

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>91

ggaggtgacc atgtcgctg 19

<210>92

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>92

aaagagctgc cccctacca 19

<210>93

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>93

gctaacgatg acaactgcgt tc 22

<210>94

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>94

tgtgagcatt tacattatct tcaacatctt 30

<210>95

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>95

agcatgtact cagtgcggc 19

<210>96

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>96

gcccatcagg aaggacgtg 19

<210>97

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>97

tccgaaattc ttttatcaat ctttacagat ga 32

<210>98

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>98

tactcgaagt ggcaaataca gaatct 26

<210>99

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>99

ccaccctgca gctgctc 17

<210>100

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>100

aagaacaaac tccctgtaac tgct 24

<210>101

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>101

ggcacagccc agtgacc 17

<210>102

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>102

cttgaacact gtgattccta aagagc 26

<210>103

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>103

ttgccatgcc tgagctgg 18

<210>104

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>104

tattctaaac atttcaaata actcagctgc ta 32

<210>105

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>105

ctttctggcc cccgataact c 21

<210>106

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>106

aggatctcgc tgatgagcct 20

<210>107

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>107

gaaggacgcc agcatcatac a 21

<210>108

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>108

aaaatactaa aattagccgg gcacg 25

<210>109

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>109

tgtgtgtaag ttgtccagct cc 22

<210>110

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>110

gctggtactt ggcatggct 19

<210>111

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>111

ccggtgaact gatcttagtg attct 25

<210>112

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>112

cagataaaat cacttaaggc gtgtaatca 29

<210>113

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>113

ccagtcacct gctgctacag 20

<210>114

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>114

cacgtgaatt cagaggcttc tttc 24

<210>115

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>115

gcaacaagca gctcacagc 19

<210>116

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>116

gttctccgca cccgtgag 18

<210>117

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>117

ctggcgtctt atgttgccaa tg 22

<210>118

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>118

gggcacggtg tgggc 15

<210>119

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>119

gtgaagaagc aggtagggtg g 21

<210>120

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>120

gccatccagt ttgggggaat 20

<210>121

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>121

ctcttgggag gggcttcttt c 21

<210>122

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>122

ctttccaaat ggcgactttc cc 22

<210>123

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>123

gtctgaggac cgtctcacaa tc 22

<210>124

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>124

caggtgcggg cggtg 15

<210>125

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>125

tccctgcccc ggacc 15

<210>126

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>126

cggtagcgtt ggcccc 16

<210>127

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>127

atgcactgaa cccaaaatga actg 24

<210>128

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>128

tccacatggt tcccagggt 19

<210>129

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>129

atggccttct cagctgaaca g 21

<210>130

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>130

catcctccag tgctgcctg 19

<210>131

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>131

gctccactcc tttctgggc 19

<210>132

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>132

cacagagatc cggaatgcag taa 23

<210>133

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>133

gcttgtcgga cgtcaggg 18

<210>134

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>134

aagccttgtt tctagaatgg ctca 24

<210>135

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>135

cactaacccc agcaggaacc 20

<210>136

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>136

caggctgtgg gtgctgg 17

<210>137

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>137

tattctggag ctcctggacc tt 22

<210>138

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>138

ggtgttgggg caggagc 17

<210>139

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>139

ttgcgatcat cccacagagc 20

<210>140

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>140

tgcgaagatt tcaattatat tgcttcca 28

<210>141

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>141

accgttgcgc ctcagc 16

<210>142

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>142

attcataccc acatgtccta cacatt 26

<210>143

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>143

gtccgtgcag tatcccaagg 20

<210>144

<211>32

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>144

caaaggaggg agaaataatt acaagttact ag 32

<210>145

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>145

cagcaggagc acccgc 16

<210>146

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>146

agattgaacg acagaggatc acg 23

<210>147

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>147

gcaatgcaca gcaccgtg 18

<210>148

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>148

atgatggcag agcagtgtgg 20

<210>149

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>149

gatttcccag aactcttcac ataatttcta a 31

<210>150

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>150

cagttgtctc tttcctacgt ctagg 25

<210>151

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>151

gacagctgct atgatatcct cctg 24

<210>152

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>152

tattgtaaca gctggcctag aacc 24

<210>153

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>153

tgtttactga aggagcagct cttt 24

<210>154

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>154

tacgttcagg cctacaaatc tctg 24

<210>155

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>155

agggccacac acagcatg 18

<210>156

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>156

ctgcatttgc tttccaggtg g 21

<210>157

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>157

gaggaaggca ccatgggaag 20

<210>158

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>158

aatgcatatt ttaaccaagt accttcctc 29

<210>159

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>159

gccatttgtc ctcgtgagtt tg 22

<210>160

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>160

gctggaggaa cgttgagaat ct 22

<210>161

<211>21

<212>DNA

<213> Artificial sequence (artificacial sequence)

<400>161

aaccaacacg gagagaacac c 21

<210>162

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>162

cacctaccct gggatactct gat 23

<210>163

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>163

tgattgtggg gactttgcca a 21

<210>164

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>164

aaccctcacc aagagccct 19

<210>165

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>165

tcggccaaag acaccgatat tt 22

<210>166

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>166

ccacctggga attttaaagt ttctctt 27

<210>167

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>167

ccctaggagc tggccaatat ttt 23

<210>168

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>168

gaagctgcca tcaagagcaa ag 22

<210>169

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>169

ttggttgctg agtgagactt tga 23

<210>170

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>170

gtgccactgt gaaatatgtg cc 22

<210>171

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>171

aagtcatgcc caagatcctg ac 22

<210>172

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>172

gcattgtttt ccattttctc agtgc 25

<210>173

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>173

cagggaacat caactttgtt ggg 23

<210>174

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>174

tgggctcagg tgcacaac 18

<210>175

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>175

cccaggttct tatcggtcag c 21

<210>176

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>176

gacctctgac ttagaagttc catcc 25

<210>177

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>177

agcccaccaa ccaagaactg 20

<210>178

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>178

aggggcgggc aagtct 16

<210>179

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>179

tcttcaaact ctgcttcccc ttc 23

<210>180

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>180

cagcaaataa aaatgttgag agattccatc 30

<210>181

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>181

cccccaattc taaagagacc c 21

<210>182

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>182

caaaaccatc cctgccagac 20

<210>183

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>183

cagccccgga gccct 15

<210>184

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>184

gtcccgttat gcttgttttg ttatct 26

<210>185

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>185

aataccatca gagtgaccca aattttg 27

<210>186

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>186

gcccacattc agcatatttc tcag 24

<210>187

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>187

acctggaggg gacatccg 18

<210>188

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>188

cctcagtaac cccctggact 20

<210>189

<211>31

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>189

tcattatgta cttgtcaaaa ggctttatga t 31

<210>190

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>190

cttttccaga ctctctttga acttgaaa 28

<210>191

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>191

ctcatctttc ttctggggag cc 22

<210>192

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>192

cagggcctgc tcacagc 17

<210>193

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>193

gccctggaaa tacgtcactt ct 22

<210>194

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>194

ctgtttgcat tctctggtct aaagtatc 28

<210>195

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>195

cacccaccga cgacgc 16

<210>196

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>196

ctgcgcacca ggactcg 17

<210>197

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>197

ccacaaagga gactgaaatc aatacg 26

<210>198

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>198

tgaaatgtat gcctgtaaac taaaatctaa tct 33

<210>199

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>199

acccattaga catgtatatt tcagttgtaa ttt 33

<210>200

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>200

tataactgga ataatgggaa ctgtagctt 29

<210>201

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>201

gtcaaagccc aggacagtca tat 23

<210>202

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>202

catgtcctcc ccaacaaaat aatgg 25

<210>203

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>203

gactggggag ccaaatgtca t 21

<210>204

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>204

aacaacaaat atttttccaa caaaatgtct ttc 33

<210>205

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>205

ttcatccttt accactacca gatattcaa 29

<210>206

<211>28

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>206

attatgtgtg gataagaaca gtctcact 28

<210>207

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>207

ctgtttccat agagctctac cagaaa 26

<210>208

<211>26

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>208

caagaatatt ttccattgtg ctggga 26

<210>209

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>209

acctgggttt ccaccatatg c 21

<210>210

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>210

ctgttgtgtt tttagaggtg ctatctc 27

<210>211

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>211

aaaggatacc ctcgaacaag agc 23

<210>212

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>212

ctgggcttct ctctcttctt tctc 24

<210>213

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>213

ggtaacccag gccacttgg 19

<210>214

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>214

gaatgttgtg ttatgtgtat ttcaccataa taaa 34

<210>215

<211>30

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>215

tgaaaaatca gaaagacatc gtatagaacc 30

<210>216

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>216

gtggctcatt ctccagcatg a 21

<210>217

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>217

agcagctctc tgaactaatg acg 23

<210>218

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>218

aaactcgcat cttcatcatc tctga 25

<210>219

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>219

caccccttcg tgcccatc 18

<210>220

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>220

cctctctgcg gcacacag 18

<210>221

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>221

gggctgtgtg ctggtgag 18

<210>222

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>222

gggaatgttc tctcacgtac ctg 23

<210>223

<211>33

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>223

tttctctgct actaaaagtg gtatattttt cat 33

<210>224

<211>29

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>224

tacagaaaag gataatggat tgaacacac 29

<210>225

<211>25

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>225

ttgtgtctat aaactaacgc gttgc 25

<210>226

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>226

agccggagcg aggagtt 17

<210>227

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>227

tgttcatttg gaaggtctgt gcta 24

<210>228

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>228

tgtccctgca tcagataaac gag 23

<210>229

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>229

gtgaagagga cggtggaatc c 21

<210>230

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>230

cctacagtca aggcctcgaa c 21

<210>231

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>231

gtatgtggct gtaagatacc tctcttt 27

<210>232

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>232

ttctgggtca tggcgatctt g 21

<210>233

<211>27

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>233

tctctgagtg tgacattcct aactttt 27

<210>234

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>234

aggcagccac agcaaagg 18

<210>235

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>235

ttgaggctgc aacagtaaca ct 22

<210>236

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>236

caaacctaag cgcatcagat tgg 23

<210>237

<211>34

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>237

cagtaaatgt atagaatatg acttcaccag tatc 34

<210>238

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>238

cagagcttac tgcacctggg 20

<210>239

<211>22

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>239

tgttttcagg ggcccaagtt aa 22

<210>240

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>240

ctgctctcgt tcagcatcct t 21

<210>241

<211>15

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>241

gcaggggctg tgggc 15

<210>242

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>242

cgagacctgc cgggga 16

<210>243

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>243

tttcatggtg tggtccaagc a 21

<210>244

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>244

caggttagag tgccacgtcc 20

<210>245

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>245

tcggctgagg acagatacgt 20

<210>246

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>246

gaaggcctga gagaaggtgc 20

<210>247

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>247

gccgccccta agcacag 17

<210>248

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>248

gtcattggcc accggaaatt g 21

<210>249

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>249

ggctgcaggt ggaaggac 18

<210>250

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>250

atgcctggac tctcctctct c 21

<210>251

<211>16

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>251

ctggcggcct ctgtcg 16

<210>252

<211>17

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>252

gggggacgac catgcag 17

Claims

1. The primer composition is used for detecting the mutation of the hemochromatosis and hepatolenticular degeneration susceptibility gene, and is characterized by comprising 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, wherein 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 gene which is related to the hepatolenticular degeneration.

2. The primer composition as claimed in claim 1, wherein the primer set comprises a primer set I, a primer set II and a primer set III, the primer set I comprises the upstream and downstream primer sequences shown in SEQ ID NO.1-122, the primer set II comprises the upstream and downstream primer sequences shown in SEQ ID NO.123-248, and the primer set III comprises the upstream and downstream primer sequences shown in SEQ ID NO. 249-252.

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

4. Use of the primer composition of claim 1 or 2 or the kit of claim 3 for the preparation of a product for detecting hemochromatosis and hepatolenticular degeneration susceptibility gene mutation.

5. The method for detecting the hemochromatosis and hepatolenticular degeneration susceptibility gene mutation is characterized by comprising the following steps of:

obtaining a DNA sample of a sample to be detected;

and processing the data of the sequencing library to obtain the information of the gene variation and copy number variation of all exons and intron shearing regions of the susceptibility genes of the hemochromatosis and the hepatolenticular degeneration of the sample to be detected, and determining the detection result of the hemochromatosis and the hepatolenticular degeneration susceptibility gene mutation of the sample to be detected.

6. The method as claimed in claim 5, wherein the primer set comprises a primer set I, a primer set II and a primer set III, the primer set I comprises the upstream and downstream primer sequences shown in SEQ ID NO.1-122, the primer set II comprises the upstream and downstream primer sequences shown in SEQ ID NO.123-248, and the primer set III comprises the upstream and downstream primer sequences shown in SEQ ID NO. 249-252.

7. The method according to claim 5 or 6, wherein the DNA sample of the test sample is human genomic DNA, preferably blood genomic DNA, peripheral blood cell genomic DNA, tumor tissue genomic DNA or body fluid DNA.

8. The method according to claim 5 or 6, wherein the sample to be tested is a Chinese population.