CN112359096A - Kit for detecting key mutant gene of DNA base excision repair pathway - Google Patents

Abstract

The invention provides a kit for detecting key mutant genes of DNA base excision repair pathways, wherein a probe library consists of probes shown in SEQ ID NO. 1-213, and the probe library is prepared into the detection kit which can be used for detecting the key mutant genes related to the DNA base excision repair pathways. The probe library is used for enriching target DNA fragments to be analyzed, and then sequencing is carried out on the enriched DNA fragments through a sequencing means, so that the effectiveness of gene detection is realized. The key mutant gene related to the DNA base excision repair pathway can be used for predicting the occurrence tendency of tumors, evaluating the malignancy degree and clinical prognosis of the tumors and guiding the screening and research and development of new drugs, so the invention has important guiding significance on the research direction of gene functionality.

Description

Kit for detecting key mutant gene of DNA base excision repair pathway

Technical Field

The invention belongs to the technical field of gene detection, and particularly relates to a kit for detecting a key mutant gene of a DNA base excision repair pathway.

Background

The abnormal DNA damage repair pathway is closely related to the tumor generation tendency and the sensitivity of the DNA damage inducing anti-tumor drugs to cells. The DNA base excision repair pathway is mainly used for repairing DNA bases damaged by endogenous oxygen free radicals and DNA damage caused by alkylating agents such as TMZ and the like, and is one of the most important ways in a DNA repair mechanism. The repair process is specifically divided into two pathways: single-nucleotide excision repair (SN-BER) and Long-fragment excision repair (Long-patch BER). When a base is damaged, both pathways begin with the recognition of the damage by DNA glycosylase followed by the excision of the glycosidic bond between the damaged base and the deoxyribose-phosphate backbone, forming an abasic/Apyrimidic (AP) site. All abasic sites were cleaved from the phosphodiester bond by the APE1 endonuclease, leaving 3 'hydroxyl (3' -OH) and 5 'deoxyribose phosphate (5' -dRP) moieties. Thereafter, DNA polymerase β (Pol β) was recruited. Normally, the hydrolase activity of Pol β cleaves off the deoxyphosphate backbone, the polymerase activity subsequently synthesizes mononucleotides to fill the gap, and the resulting gap structure is stitched by DNA Ligase III (DNA Ligase III) and repaired, a pathway known as SN-BER. However, when the hydrolase activity of Pol β is inhibited or the deoxyribose phosphate backbone is oxidized, Pol β can only continue to synthesize nucleotides, so that the downstream sequence where the deoxyribose phosphate backbone is located is replaced and forms a branched structure, similar to the alternative synthesis of Pol δ at the time of okazaki fragment maturation. Thus, the LP-BER repair path is initiated. Pol beta is an ideal substrate for FEN1 instead of synthetically formed branched structure, and DNA is cut into a gap by FEN1 and then repaired by suturing DNA Lig I or XRCC1/DNA Lig III complex. In addition, Pol β and FEN1 may also be repaired by a relay cooperation mechanism (Hit-and-Run model) in which 2-11 nucleotides are synthesized by substitution.

Synthetic Lethality (Synthetic Lethality) strategy has been widely used in recent years in the study of tumors. The concept of synthetic lethality in tumor studies is: when the function of two important genes that are not in the same repair pathway is suppressed from one of them, the cell is not lethal, but after the function of both genes is suppressed, the cell is dead. In clinic, if one of the mutations exists in the tumor cells, and the gene is normal in normal cells, the other gene can be used as a target point, and the function of the other gene is inhibited, so that the aims of specifically killing the tumor cells and individually treating the tumor cells are fulfilled.

Earlier researches show that the gene involved in DNA base excision repair pathway has embryonic genetic mutation and somatic mutation in specific tissue, and is highly related to the generation and treatment effect of tumor. Wherein, the activities of variant enzymes such as OGG1 gene R46Q, S326C and the like are reduced, and the probability of suffering from kidney cancer and lung cancer is increased; the PARP1 gene V762A variant enzyme activity is reduced, resulting in an increased rate of suffering from various tumors; in addition, DNA polymerase Pol β is mutated in about 30% of tumors, including frame shift mutations, fragment deletions, point mutations, and the like. The mutation of the genes causes the change (enhancement, attenuation or deletion) of the effectiveness of DNA base excision repair paths, and has important significance for the generation, prognosis and treatment strategy formulation of tumors.

Disclosure of Invention

The invention aims to provide a kit for detecting a key mutant gene of a DNA base excision repair pathway.

In order to achieve the purpose, the invention provides the following technical scheme:

a kit for detecting a DNA base excision repair pathway key mutant gene comprises a probe library which is formed by probes shown as SEQ ID NO. 1-213 aiming at the DNA base excision repair pathway key mutant gene.

Further, the DNA base excision repair pathway key mutant genes comprise POLB, PARP1, APEX1 and OGG 1.

Furthermore, a probe sequence corresponding to the gene POLB is shown as SEQ ID NO. 1-29, a probe sequence corresponding to the gene PARP1 is shown as SEQ ID NO. 30-152, a probe sequence corresponding to the gene APEX1 is shown as SEQ ID NO. 153-184, and a probe sequence corresponding to the gene OGG1 is shown as SEQ ID number 185-213.

A method for detecting a DNA base excision repair pathway key mutant gene comprises the following steps:

(1) obtaining a DNA sample library of a subject;

(2) hybridizing all probes shown as SEQ ID NO. 1-213 in the kit with the DNA sample library;

(3) and (3) separating the hybridization product in the step (2), releasing the gene segment hybridized with the probe in the kit, and sequencing the separated gene segment by adopting a sequencing technology so as to detect the mutation condition of the gene.

Further, the DNA sample library in the step (1) is composed of double-stranded DNA fragments;

the step (1) comprises the following steps: extracting whole genome DNA, and then fragmenting the whole genome DNA; or mRNA is extracted, fragmented, and then double-stranded cDNA is synthesized using the fragmented mRNA as a template.

Further, the probe in the step (2) is selectively labeled.

Preferably, the probe in step (2) is labeled with biotin.

Has the advantages that: the invention develops a kit for detecting a key mutant gene of a DNA base excision repair pathway, which mainly has the following meanings:

1. by analyzing the variation condition of the key mutant gene of the DNA base excision repair pathway, the effectiveness (enhancement, weakening or deletion) of the DNA base excision repair pathway in human tissues or cells is evaluated, the tendency of tumorigenesis is predicted, and a patient is guided to take corresponding preventive measures.

2. Through the analysis of the variation condition of the key mutant gene of the DNA base excision repair pathway, the effectiveness (enhancement, attenuation or deletion) of the DNA base excision repair pathway in the cells of the tumor patients is evaluated, and the method has important guiding significance for the evaluation of the malignancy degree and clinical prognosis of the tumor.

3. Through the analysis of the variation condition of the key mutant gene of the DNA base excision repair pathway, the effectiveness (enhancement, attenuation or deletion) of the DNA base excision repair pathway in the cells of the tumor patients is evaluated, and the method has important guiding significance for the selection of radiation and chemotherapy drugs and the formulation of treatment strategies (such as synthetic lethal schemes and the like).

4. Through the analysis of the variation condition of the key mutant gene of the DNA base excision repair pathway, the effectiveness (enhancement, weakening or deletion) of the DNA base excision repair pathway in the cells of the tumor patients is evaluated, and the method has important guiding significance for the selection of new therapeutic targets, the research and development of targeted drugs and the like.

Drawings

FIG. 1 is a flow chart of the detection method of the present invention.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The invention provides a method for detecting a key mutant gene of a DNA base excision repair pathway, which mainly comprises the following steps:

(1) obtaining a DNA sample library of a subject; extracting whole genome DNA, and then fragmenting the whole genome DNA; or extracting mRNA, fragmenting the mRNA, and synthesizing double-stranded cDNA by using the fragmented mRNA as a template;

(2) designing a DNA probe hybridized with the gene aiming at the gene segment to be detected, and screening a plurality of probes from the DNA probe library as a DNA probe library;

(3) hybridizing the DNA probe library with the DNA sample library, and enriching the DNA sample library to obtain a DNA fragment of the gene to be detected;

(4) and (4) separating the hybridization product in the step (3), releasing the gene segment hybridized with the probe in the kit, and sequencing the separated gene segment by adopting a sequencing technology so as to detect the mutation condition of the gene.

In the implementation process, each probe in the DNA probe library may be labeled with biotin, and then the magnetic beads labeled with streptavidin are used to adsorb the hybridization product after hybridization, releasing the enriched gene fragment to be detected from the magnetic beads, and then sequencing the gene fragment.

The specific experimental process is as follows:

first, prepare cDNA/DNA sample library

1. Preparation of DNA sample library

1.1 DNA extraction

Human genomic DNA was extracted using a DNA extraction kit from QIAGEN, Germany, and the quality and concentration of the DNA template were determined using a spectrophotometer and a gel electrophoresis system for the extracted DNA. The absorbance of the DNA template at 260nm is more than 0.05, and the ratio of the absorbance A260/A280 is between 1.8 and 2, which is qualified.

1.2 DNA fragmentation

Mu.g of qualified genomic DNA was diluted to 100. mu.L with TE buffer. And (3) fragmenting the DNA by using a tissue homogenizer, wherein the fragment length is 150-200 bases.

The DNA was purified using the purification kit from Biyuntian.

1.3 quality testing of DNA sample libraries

And (5) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and determining that the length peak value of the DNA fragment is reasonable.

2. Preparation of cDNA sample library

2.1 mRNA extraction

Human mRNA is extracted by adopting an mRNA extraction kit of Tiangen biology company, and the quality and the concentration of the extracted mRNA are detected by using a spectrophotometer and a gel electrophoresis system. The absorbance A260/A280 ratio is between 1.8 and 2.

2.2 fragmentation of mRNA

Fragmenting mRNA by using a Biyuntian company kit; then, mRNA was purified by column chromatography using Tiangen Bio Inc.

2.3 Synthesis of first and second Strand cDNA of mRNA Using the reagent cDNA Synthesis kit of Thermo Scientific, USA

And then purifying the cDNA by passing through a column by using a Biyuntian kit.

3. cDNA/DNA end repair

The DNA fragment is end-repaired using Klenow fragment, T4 DNA polymerase and T4 polynucleotide kinase, since Klenow fragment has 5 '-3' polymerase activity and 3 '-5' polymerase activity, but lacks 5 '-3' exonuclease activity. Therefore, the DNA fragment can be conveniently and accurately subjected to end repair. And (4) carrying out column purification on the repaired DNA fragment.

The cDNA/DNA was filled in at the 5 'protruding sticky ends and blunt-ended at the 3' protruding sticky ends using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. The reaction was carried out in a PCR amplification apparatus at 20 ℃ for 30 min.

TABLE 1

4. Adding base A to the 3' end of cDNA/DNA sample

A DNA fragment having a cohesive end A was obtained by adding a base A to the 3' -end of the end-repaired DNA fragment using Klenow having 3' -5 ' exonuclease activity, and the reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 min. The treated DNA fragment was subjected to column purification.

TABLE 2

5. Adding linkers at both ends of cDNA/DNA

TABLE 3

Performing the operations of steps 6 and 7, if mRNA → cDNA is used; if genomic DNA is used, step 8 is performed directly.

6. Isolating cDNA fragments of appropriate length

The DNA fragments were separated using an electrophoresis gel, and 150-and 250-base cDNA fragments were excised on the gel in accordance with the DNA molecular weight.

The gel samples containing the cDNA library were column purified using the Biyuntian kit.

7. Quality detection of cDNA fragment sample library

And (3) carrying out qualitative and quantitative analysis on the cDNA by using a bioanalyzer, and confirming that the length peak value of the separated cDNA fragment is reasonable.

PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 95 deg.C for 10 s, denaturing at 95 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 1min, co-circulating 20 times (cDNA sample bank) or 5-8 times (DNA sample bank), and finally extending at 72 deg.C for 5 min.

And (3) purifying the PCR amplification product by a column by using a commercial kit.

8. Amplification of DNA templates

And (3) carrying out polymerase chain reaction on the treated DNA sample by using a PCR amplification instrument to increase the content of the DNA so as to meet the requirement of hybridization with the probe.

TABLE 4

PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 95 deg.C for 10 s, denaturing at 95 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 1min, co-circulating 20 times (cDNA sample bank) or 5-8 times (DNA sample bank), and finally extending at 72 deg.C for 5 min.

And (3) purifying the PCR amplification product by a column by using a Biyuntian kit.

9. Quality detection of amplified cDNA/DNA sample library

And (3) carrying out cDNA/DNA qualitative and quantitative analysis by using a bioanalyzer, and confirming that the length peak value of the purified fragment is reasonable and about 200 bp.

Second, design principle of probe

According to a human reference genome HG19, the gene related to the DNA base excision repair pathway and all coding sequences thereof are obtained by combining Ensembl, CCDS, Gencode, VEGA, SNP and CytoBand databases.

The design of the probe has several major considerations:

(1) specificity of

Stringent specificity parameters may be effective in reducing probe binding in non-target regions, but also limit regions of a part of exons that have a lower degree of reproducibility, resulting in reduced coverage of the probe design. Too loose specificity parameters will directly reduce the amount of data on the target area, resulting in reduced capture efficiency and increased sequencing costs. Therefore, a reasonable specificity parameter is key to balancing coverage and capture efficiency. We consider a probe with a sequence similarity across the genome of no more than 3 as the optimal threshold for specificity parameters.

(2) Stability of binding

The indicators of the binding ability of the probe to the template are the minimum free energy (. DELTA.G) and the melting temperature (Tm), and since these two parameters are somewhat complicated to calculate, the binding ability of the probe to the template is generally indirectly expressed by a more direct and simple GC content. In general, the greater the GC content, the greater the binding stability. In fact, the closer the GC content is to 50% of the probe, the better the capture effect. The higher the GC content, the better the binding ability of the probe to the target DNA fragment is, however, the better the binding ability to the target DNA fragment is to its original complementary fragment (consistent length on the one hand, no mutation mismatch on the other hand), and therefore, the higher the concentration of the probe is needed to promote it to take some advantage in competition for binding to the DNA complementary fragment.

We will prefer to design probes with GC contents close to 50%, and for unavoidable GC extremes we will add modified bases to the probes and thus alter the GC extremes.

(3) Concentration of Probe

As can be seen from the above binding stability, the capture effect of the probes with different binding capacities is greatly different. Too high and too low binding capacity are detrimental to capture, and therefore, different concentrations of probes for different GC contents need to be set in order to maximize uniformity of capture efficiency. Through summarizing historical capture data, different probe concentration parameters are set for probes with different GC contents, the concentration base number of the probe with the GC content of 40-60% is set to be 1, then the concentration base numbers of the probes with the GC contents of 30-40% and 60-70% are 1 x 22, and the concentration base numbers of the probes with the GC contents of 20-30% and 70-80% are 1 x 24.

(4) Position of the probe

1. The optimal probe placement position is 100% coincident with the target region, but due to the effects of randomly interrupted libraries, sequence repeatability, GC content, mutation types, etc., the actual probe design strategy is as follows:

2. since libraries are typically generated by random disruption, to maximize capture of target libraries, probes are typically designed in a tiling fashion;

3. if the target area has a repeated sequence area, the probe design can be extended to two sides to search areas with better specificity under the condition of ensuring the coverage of the target area during design;

4. if the target area is an extreme GC area, in the design process, under the condition of ensuring the coverage of the target area, a probe is designed by extending and searching areas with more proper GC content to two sides;

different design considerations are also required for different mutation types:

1. for insertions or deletions exceeding 60bp, probes need to be designed on both sides of a mutation breakpoint besides the probes need to be covered, and the closer to the breakpoint, the better;

2. for CNV regions exceeding 10 kbp, the optimal probe design strategy is to place sparse probes at intervals, so that on one hand, CNV can be detected, and on the other hand, the sequencing cost can be reduced;

3. for intron regions where fusion occurs, if the fusion site is not defined, then full coverage of the entire intron is required; if the fusion site is defined, probes need to be designed on both sides of the fusion site.

4. For short-chain repeat regions such as MSI and STR, probes need to be designed to extend 60bp on both sides in addition to the probes covering them.

Hybridization of DNA capture probes

1. Hybridization of DNA sample libraries with biotinylated DNA Probe libraries

The cDNA/DNA sample pool was mixed with hybridization buffer at 95 ℃ for 5 minutes, and then maintained at 65 ℃. The reaction was performed in a PCR amplificator.

The mixture was then mixed with a pool of probes at 65 ℃ for 5 minutes. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65 ℃ for 24 hours.

Fourthly, obtaining the hybridized gene segment

1. Preparation of streptavidin magnetic beads

The separation of the hybridization products was performed using Dynabeads streptavidin magnetic beads. The beads were placed on a homogenizer and mixed, requiring 50. mu.L of beads per sample.

And (3) washing magnetic beads: mixing 50 μ L of magnetic beads and 200 μ L of binding buffer solution, separating and purifying the magnetic beads and the buffer solution by an external magnetic field, discarding the buffer solution, and repeating the steps for three times.

2. Isolation of the hybridization product

The hybridization product was mixed well with streptavidin magnetic beads and shaken for 30 minutes at room temperature. Separating and purifying the magnetic beads by an external magnetic field.

To the beads, 500. mu.L of washing buffer was added, incubated at 65 ℃ for 10 minutes, and the beads were isolated and purified by an external magnetic field. The above steps were repeated three times.

3. cDNA/DNA enrichment sample Release

Mixing the magnetic beads with 50 mu L of elution buffer solution, incubating for 10 minutes at room temperature, separating and discarding the magnetic beads through an external magnetic field, and collecting supernatant, wherein the supernatant is the enriched gene segments related to the DNA base excision repair pathway.

Fifth, PCR amplification and purification

The enriched gene fragments were further amplified in preparation for loading into a sequencing instrument.

TABLE 5

PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 95 deg.C for 10 s, denaturing at 95 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 1min, co-circulating 20 times (cDNA sample bank) or 5-8 times (DNA sample bank), and finally extending at 72 deg.C for 5 min.

And (3) purifying the PCR amplification product by a column by using a Biyuntian kit.

Sixthly, detecting gene mutation by adopting next generation sequencing technology

Sequencing was performed using the Illumina X10 sequencing instrument platform and the sequencing results were analyzed using the existing sequencing software analysis package.

Example 1 detection of mutations in the POLB, PARP1, APEX1, OGG1 genes

Firstly, constructing a sample library

1. Extraction of DNA

The DNA of the sample was extracted using a kit from QIAGEN, Germany, according to a conventional method for extracting DNA from a tissue sample.

2. DNA fragmentation

The DNA sample was fragmented using a DNA fragmenter to a fragment length of 150-200 bp.

3. DNA sample pool quality detection

And (5) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and determining that the length peak value of the DNA fragment is reasonable.

4. DNA end repair

The DNA was filled in at the 5 'protruding sticky ends and blunt-ended at the 3' protruding sticky ends using T4 polymerase and Klenow E.coli polymerase fragments to generate blunt ends for subsequent blunt end ligation. The reaction system is shown in Table 1, and the reaction is carried out in a PCR amplification instrument at 20 ℃ for 30 min.

5. Adding base A to the 3' end of the DNA sample

The reaction was carried out in a PCR amplification apparatus at 37 ℃ for 30 minutes, and the reaction system is shown in Table 2.

6. Adding linkers at both ends of DNA

The reaction system is shown in Table 3.

7. DNA fragment sample library obtained by amplification

The Polymerase Chain Reaction (PCR) was carried out in a PCR amplification apparatus, and the reaction system is shown in Table 4.

PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 95 deg.C for 10 s, denaturing at 95 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 1min, co-circulating 20 times (cDNA sample bank) or 5-8 times (DNA sample bank), and finally extending at 72 deg.C for 5 min.

8. Quality detection of amplified DNA sample libraries

And (3) carrying out qualitative and quantitative analysis on the DNA by using a biological analyzer, and confirming that the length peak value of the purified fragment is reasonable and about 200 bp.

If the concentration of the obtained DNA sample library is low, the sample is dried at low temperature (below 45 ℃) by a vacuum concentrator and then dissolved with nuclease-free water to reach the required concentration.

Secondly, preparing DNA probe library of the POLB, PARP1, APEX1 and OGG1 genes

According to the above-mentioned method and idea of designing a probe, a probe having Biotin (Biotin) at the 5' end was designed and synthesized for the test.

Thirdly, hybridizing the DNA sample library with a biotinylated DNA probe library

The DNA pool was mixed with a hybridization buffer (10 mM Tris-HCl, 2% bovine serum albumin, pH 8.0) (after mixing, the concentration of the DNA pool did not exceed 50 ng/. mu.L at most) under reaction conditions of 95 ℃ for 5 minutes, and then maintained at 65 ℃. The reaction was performed in a PCR amplificator.

Then, with a DNA sample library: the probe pool was added to the above mixture at a molar ratio of 1:100 under reaction conditions of 65 ℃ for 5 minutes. The hybridization reaction was placed in a PCR amplification apparatus and incubated at 65 ℃ for 24 hours.

Fourthly, obtaining the hybrid enriched gene fragments of the POLB, the PARP1, the APEX1 and the OGG1

1. Preparation of streptavidin magnetic beads

Dynabeads streptavidin magnetic beads are placed on a mixing instrument to be mixed uniformly, 50 mu L of magnetic beads and 200 mu L of binding buffer (10 mM Tris-HCl, 2% bovine serum albumin, pH8.0) are mixed uniformly on the mixing instrument, the magnetic beads and the buffer are separated and purified by using an external magnetic field, the buffer is discarded, and the steps are repeated three times, and 200 mu L of binding buffer is added each time.

2. Isolation of the hybridization product

The hybridization reaction mixture in step three was mixed with the treated streptavidin magnetic beads of step four and shaken for 30 minutes at room temperature. The magnetic beads were separated and purified from the buffer using an external magnetic field, and then 500. mu.l of a washing buffer (phosphate buffer, 0.1% Tween-20, 0.1% SDS, pH 7.4) was added to the magnetic beads, incubated at 65 ℃ for 10 minutes, and mixed uniformly every 5 minutes. And separating and purifying the magnetic beads and the buffer solution by using an external magnetic field, and repeating the steps for three times.

3. Release of enriched DNA samples

The beads were mixed with 50. mu.l of elution buffer (10 mM sodium hydroxide solution), incubated at room temperature for 10 minutes, and mixed uniformly every 5 minutes. Separating and purifying the magnetic beads and the buffer solution by using an external magnetic field, reserving supernatant, and discarding the magnetic beads, wherein the supernatant contains the enriched DNA sample libraries of the POLB, PARP1, APEX1 and OGG1 gene fragments.

Fifth, PCR amplification and purification

The enriched DNA sample library was further amplified in preparation for loading into a sequencing instrument, and the reaction system is shown in table 5.

PCR conditions were as follows: placing in a PCR amplification apparatus, pre-denaturing at 95 deg.C for 10 s, denaturing at 95 deg.C for 30 s, annealing at 65 deg.C for 30 s, extending at 72 deg.C for 1min, co-circulating 20 times (cDNA sample bank) or 5-8 times (DNA sample bank), and finally extending at 72 deg.C for 5 min.

Sixthly, verification of detection sensitivity and specificity

Mutant and wild type plasmids were constructed for the disease-associated pathway typical representative mutation sites, POLB (c.725c > G), PARP1 (c.2285t > C), APEX1 (c.444t > a), and OGG1 (c.461g > a), respectively, and samples of different abundances were mixed according to the copy number ratio of the mutant in the wild type, and simultaneously, human genomic DNA samples extracted from 293T cells of the same mass were added and mixed uniformly. The probe library is adopted to carry out capture, sequencing and investigation on sensitivity and specificity, each sample is tested repeatedly for 3 times, and the results are as follows:

TABLE 6

As can be seen from the table, the detection probe library and the detection method provided by the invention have better detection sensitivity and specificity to low-abundance samples, and can basically and accurately detect 0.5% of mutation conditions.

The optimized probe library is adopted to detect 4 pathway key genes of tumor samples of 3 patients with lung cancer, and the detection results are shown in table 7. Selecting a part of east Asia population, wherein the frequencies in a 1000g2015aug _ EAS database, an ExAC _ EAS database and a gnomaD _ exome _ EAS database are all less than 0.01, so that amino acid changes are caused, and at least one prediction software is used for predicting the gene mutation results of harmful mutations in the harmfulness prediction software such as SIFT, Polyphen, CADD and the like for displaying.

Note:

1000g2015aug _ eas: the allele frequencies of this mutation in the thousand human genome project database 2015, month 8, contain only those in east asia.

ExAC _ EAS: the Exome Aggregation Consortium database contains data of 60000 unrelated individuals and only contains east Asian population.

gnomAD _ isomer _ EAS: the Genome Aggregation Database is a Genome mutation Database jointly developed by researchers of various countries, wherein a data set comprises 123136 whole exon data and 15496 whole Genome data, and is derived from various disease research projects and large-scale population sequencing projects. The database contains all exon data of the east asian population.

TABLE 7

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<400> 15

tttgattaga attgagagtg tcacttggtg aaaagccatt ttgggaatac tgacttaatt 60

tttcttctat taggatattg tactaaatga agttaaaaaa 100

<210> 16

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 16

ctattaggat attgtactaa atgaagttaa aaaagtggat tctgaataca ttgctacagt 60

ctgtggcagt ttcagaagag gtaacatact tcctaatctt 100

<210> 17

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 17

gcaatgtata tctaatgcca attactgttg tcatcacaga ttctgctgtc tacatcaata 60

cacctgaata gttggacaga aaattgaaat cttttaacta 100

<210> 18

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 18

tctaatgcca attactgttg tcatcacaga ttctgctgtc tacatcaata cacctgaata 60

gttggacaga aaattgaaat cttttaacta attctaacta 100

<210> 19

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 19

ttctgtcatc acaggtgcag agtccagtgg tgacatggat gttctcctga cccatcccag 60

cttcacttca gaatcaacca aacaggtgcc tcagagttta 100

<210> 20

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 20

tcttgccatt acaaagtaat tactcttttt cttattccct aattatgatt ctacagccaa 60

aactgttaca tcaggttgtg gagcagttac aaaaggttca 100

<210> 21

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 21

tacatcaggt tgtggagcag ttacaaaagg ttcattttat cacagatacc ctgtcaaagg 60

gtgagacaaa gttcatggta agtacttgtt agagttagca 100

<210> 22

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 22

tggtttaaaa tgttcatttt agggtgtttg ccagcttccc agtaaaaatg atgaaaaaga 60

atatccacac agaagaattg atatcaggta ttgttcagac 100

<210> 23

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 23

tgttcatttt agggtgtttg ccagcttccc agtaaaaatg atgaaaaaga atatccacac 60

agaagaattg atatcaggta ttgttcagac tttgttgctg 100

<210> 24

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 24

ccattttttt taggttgata cccaaagatc agtattactg tggtgttctc tatttcactg 60

ggagtgatat tttcaataag aatatgaggg ctcatgccct 100

<210> 25

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 25

atattttcaa taagaatatg agggctcatg ccctagaaaa gggtttcaca atcaatgagt 60

acaccatccg tcccttggga gtcactggtg agtgtccatg 100

<210> 26

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 26

gtagtttctt tgtattttta gtccttcagg caacgagaga ggatttaatg cataacatgc 60

tcctaggccc tcagttgaag gccatcaagg caagcgttag 100

<210> 27

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 27

tgtattttta gtccttcagg caacgagaga ggatttaatg cataacatgc tcctaggccc 60

tcagttgaag gccatcaagg caagcgttag ttccagcttt 100

<210> 28

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 28

cctgccagtg gatagtgaaa aagacatctt tgattacatc cagtggaaat accgggaacc 60

caaggaccgg agcgaatgag gcctgtatcc tccctggcag 100

<210> 29

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 29

ccggagcgaa tgaggcctgt atcctccctg gcagacacaa cccaatagga gtcttaattt 60

atttcttaac ctttgctatg taagggtctt tggtgttttt 100

<210> 30

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 30

tggtgcagaa gcgcttcggg tgaattcata ccagagccac cgggtgtgac tcggctacct 60

ctcccaatta ccacagggag gtcttaaaat tgaatttcag 100

<210> 31

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 31

cagaagcgct tcgggtgaat tcataccaga gccaccgggt gtgactcggc tacctctccc 60

aattaccaca gggaggtctt aaaattgaat ttcagtttca 100

<210> 32

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 32

aatatcatag acaatgtacc tgaggggaag cttgttaagg agccaacagc catacaacag 60

agggtatgta catgtgcctc atctccccca ggctggcagg 100

<210> 33

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 33

cacataagtg actctgtagc tcgagaacat ccctttgtgc tcacacagca tactcaagaa 60

aggatactcg ttatatagta gagaggtgtc attcacacca 100

<210> 34

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 34

aagtgactct gtagctcgag aacatccctt tgtgctcaca cagcatactc aagaaaggat 60

actcgttata tagtagagag gtgtcattca caccagatga 100

<210> 35

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 35

tcattcacac cagatgaaat cccggtccca agaggaacgt ctacaccatc cagactaatg 60

ttagctgaag gatcaggggt agttttgccc aaacctgaaa 100

<210> 36

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 36

tcccggtccc aagaggaacg tctacaccat ccagactaat gttagctgaa ggatcagggg 60

tagttttgcc caaacctgaa aaacagaagt cacaagtgac 100

<210> 37

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 37

gactaatgtt agctgaagga tcaggggtag ttttgcccaa acctgaaaaa cagaagtcac 60

aagtgacatg aactgtgagt acagatgtgg caaagcttcc 100

<210> 38

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 38

tgcccaaacc tgaaaaacag aagtcacaag tgacatgaac tgtgagtaca gatgtggcaa 60

agcttccagg gagatgagct ctatttgatg ttgggtccat 100

<210> 39

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 39

acttgctgat atgtgaagcg tgcttcagtt catacctatt caaaagagga cagtctcaga 60

agaggccttc acactgggct ctcgaaacca acagtttgat 100

<210> 40

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 40

ctgatatgtg aagcgtgctt cagttcatac ctattcaaaa gaggacagtc tcagaagagg 60

ccttcacact gggctctcga aaccaacagt ttgattagga 100

<210> 41

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 41

atgtgaagcg tgcttcagtt catacctatt caaaagagga cagtctcaga agaggccttc 60

acactgggct ctcgaaacca acagtttgat taggagaata 100

<210> 42

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 42

attccacctg tccccatgaa atgccatcag tcccagagcc aggtttactc acatgtttcc 60

aagggcaact tctcccaaca ggattaagcc tattgggtct 100

<210> 43

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 43

cagagccagg tttactcaca tgtttccaag ggcaacttct cccaacagga ttaagcctat 60

tgggtctccc tgagacgtat ggcagtagtt ggcactcttg 100

<210> 44

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 44

aacttctccc aacaggatta agcctattgg gtctccctga gacgtatggc agtagttggc 60

actcttggag accatgtcag cgaaatagat ccctttacca 100

<210> 45

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 45

tccctgagac gtatggcagt agttggcact cttggagacc atgtcagcga aatagatccc 60

tttaccaaac atgtagcctg tctggaaggt cggaaaaggg 100

<210> 46

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 46

ggagaccatg tcagcgaaat agatcccttt accaaacatg tagcctgtct ggaaggtcgg 60

aaaagggaga gctgagaatc ttctgatggg aaattaggag 100

<210> 47

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 47

ccctcgccag gccaggcaca taccacgggc gcttcaggcg gggctatccg aagaccctgg 60

gacaggatcc cagcaaagtt ggtggtcctg gacccgtgcc 100

<210> 48

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 48

tcaggcgggg ctatccgaag accctgggac aggatcccag caaagttggt ggtcctggac 60

ccgtgccaca gcaatcttcg gttatgaagc tgcttaaagg 100

<210> 49

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 49

atcccagcaa agttggtggt cctggacccg tgccacagca atcttcggtt atgaagctgc 60

ttaaagggct tgtaacgctg gcattcgcct tcacgctcta 100

<210> 50

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 50

cacagcaatc ttcggttatg aagctgctta aagggcttgt aacgctggca ttcgccttca 60

cgctctatct taaagatctg gttggagtag taaacaaagg 100

<210> 51

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 51

ggaagagctg gcaggacaca gaaggaagtg ggggaagaag ggattcttac atcgatgact 60

tccaagtcat acgcattgtg tgtggttgca tgagtgttct 100

<210> 52

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 52

gaagaaggga ttcttacatc gatgacttcc aagtcatacg cattgtgtgt ggttgcatga 60

gtgttcttaa catacttcct gatgatctcg gcttcttcag 100

<210> 53

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 53

tcatacgcat tgtgtgtggt tgcatgagtg ttcttaacat acttcctgat gatctcggct 60

tcttcagaat ctctgtcaac cacctggata aacagaatct 100

<210> 54

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 54

cttaacatac ttcctgatga tctcggcttc ttcagaatct ctgtcaacca cctggataaa 60

cagaatcttg ggattagcac aaaagagacc caggagagct 100

<210> 55

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 55

gaatctctgt caaccacctg gataaacaga atcttgggat tagcacaaaa gagacccagg 60

agagctacag aggaggaatg ctcaggctca gtcaatgcat 100

<210> 56

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 56

ctgctctcag gatgagaggt taagatgctt gaggaaggcc tgaccctgtt accttaatgt 60

cagttttgag cttctcatag ttgacatcga tgggatcctt 100

<210> 57

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 57

gaaggcctga ccctgttacc ttaatgtcag ttttgagctt ctcatagttg acatcgatgg 60

gatccttgct gctatcatca gaccctcccc tgagcagact 100

<210> 58

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 58

tgagcttctc atagttgaca tcgatgggat ccttgctgct atcatcagac cctcccctga 60

gcagactgta ggccacctcg atgtccagca ggttgtcaag 100

<210> 59

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 59

tgctgctatc atcagaccct cccctgagca gactgtaggc cacctcgatg tccagcaggt 60

tgtcaagcat ttccaccttg gcctggagga gcaaaagaaa 100

<210> 60

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 60

tgtaggccac ctcgatgtcc agcaggttgt caagcatttc caccttggcc tggaggagca 60

aaagaaagcc cccgacttag gtatcatggt gaatgagaca 100

<210> 61

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 61

gcatttccac cttggcctgg aggagcaaaa gaaagccccc gacttaggta tcatggtgaa 60

tgagacagac tcacccaggt ggcagagaaa acctacatgc 100

<210> 62

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 62

ggtagtcaca ccccagccca gcagacctgc agtcccttct gaacccttgc gctacctgca 60

cactgtctgc attgttcagg agcggaggct tcttcatccc 100

<210> 63

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 63

cccttctgaa cccttgcgct acctgcacac tgtctgcatt gttcaggagc ggaggcttct 60

tcatcccaaa gtcgtggggg atcagggtgt aaaagcgatt 100

<210> 64

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 64

ctgcattgtt caggagcgga ggcttcttca tcccaaagtc gtgggggatc agggtgtaaa 60

agcgatttga gagatccagg atctgagagt cgctgctgcc 100

<210> 65

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 65

caaagtcgtg ggggatcagg gtgtaaaagc gatttgagag atccaggatc tgagagtcgc 60

tgctgccctg agacaccgcc tggagaggag gggacagaag 100

<210> 66

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 66

ttgagagatc caggatctga gagtcgctgc tgccctgaga caccgcctgg agaggagggg 60

acagaaggaa tgcaagactg agggagcagc tccaagcccc 100

<210> 67

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 67

cctgagacac cgcctggaga ggaggggaca gaaggaatgc aagactgagg gagcagctcc 60

aagcccccgg ccaggctttc tcttttagca ggtgggtgct 100

<210> 68

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 68

caccccctaa gtcggccaga ggagggttcc aggaggcccc tggtagccct gtgcttacct 60

gctggacctc actgaggatg gagtatgcgg cctggatctg 100

<210> 69

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 69

aggcccctgg tagccctgtg cttacctgct ggacctcact gaggatggag tatgcggcct 60

ggatctgcct tttgctcagc ttccccaagg gcatcttctg 100

<210> 70

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 70

cctcactgag gatggagtat gcggcctgga tctgcctttt gctcagcttc cccaagggca 60

tcttctgaag gtcgatctga ggagacaggg gatttcgctc 100

<210> 71

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 71

gccttttgct cagcttcccc aagggcatct tctgaaggtc gatctgagga gacaggggat 60

ttcgctctga aagctgggca ctgtgcagtg tgatcgcagg 100

<210> 72

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 72

gaaggtcgat ctgaggagac aggggatttc gctctgaaag ctgggcactg tgcagtgtga 60

tcgcaggaga gctggaccgg gcagcccacc ctcaggcccc 100

<210> 73

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 73

aaataatcat ccacagcaaa tgctcacaga taaaatgata aagcgcaata acctcatact 60

ccaccatggc tttcttcata ctttccacat caaagatcat 100

<210> 74

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 74

aatgataaag cgcaataacc tcatactcca ccatggcttt cttcatactt tccacatcaa 60

agatcatctt gatgaggtcc tgaactggct tggggagctt 100

<210> 75

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 75

tggctttctt catactttcc acatcaaaga tcatcttgat gaggtcctga actggcttgg 60

ggagcttgga cttggtgcca ggatttactg tcagcttctt 100

<210> 76

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 76

tcttgatgag gtcctgaact ggcttgggga gcttggactt ggtgccagga tttactgtca 60

gcttcttcac tgcctcttca tccttcagga aaaaagcaca 100

<210> 77

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 77

tggacttggt gccaggattt actgtcagct tcttcactgc ctcttcatcc ttcaggaaaa 60

aagcacattg ctaagagacc caaatcaaca actcaagcaa 100

<210> 78

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 78

tcactgcctc ttcatccttc aggaaaaaag cacattgcta agagacccaa atcaacaact 60

caagcaaggt atctgcgtct gtggggctgg actgcagaca 100

<210> 79

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 79

agggcattat gtggttacct ggccatagtc aatctccagg gggtagaact ttttgggata 60

cttcgtgaaa tttttggagt gccaagcgtt cccggttttt 100

<210> 80

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 80

ctccaggggg tagaactttt tgggatactt cgtgaaattt ttggagtgcc aagcgttccc 60

ggttttttct tcatataatt tcatgaagtg ctcaatggca 100

<210> 81

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 81

gaaatttttg gagtgccaag cgttcccggt tttttcttca tataatttca tgaagtgctc 60

aatggcatcc tccttggacg gcatctgttc cagtttgttg 100

<210> 82

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 82

ttcttcatat aatttcatga agtgctcaat ggcatcctcc ttggacggca tctgttccag 60

tttgttgcta ccgatcaccg tacccacacg gccccaggac 100

<210> 83

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 83

atcctccttg gacggcatct gttccagttt gttgctaccg atcaccgtac ccacacggcc 60

ccaggacctg aatatccaat acctgcagtg ggaaggaggg 100

<210> 84

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 84

caggacgggc ccatgtctct gcacaaccag gctacacctg cagaactcac ctgttttcct 60

tgtcgtcctc cagaagctgc agcttgtagt aggagttggt 100

<210> 85

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 85

acacctgcag aactcacctg ttttccttgt cgtcctccag aagctgcagc ttgtagtagg 60

agttggttcc tttaacgatg tccaccaggc caagggtggc 100

<210> 86

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 86

cctccagaag ctgcagcttg tagtaggagt tggttccttt aacgatgtcc accaggccaa 60

gggtggcact gaagaccttc ccacctttct ccaggacatg 100

<210> 87

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 87

ttcctttaac gatgtccacc aggccaaggg tggcactgaa gaccttccca cctttctcca 60

ggacatgcgc agagtgttcc agtcctgtcc cagaggaaaa 100

<210> 88

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 88

cactgaagac cttcccacct ttctccagga catgcgcaga gtgttccagt cctgtcccag 60

aggaaaagca cctacagttt tctctcccct tgaggtgcta 100

<210> 89

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 89

gcgcagagtg ttccagtcct gtcccagagg aaaagcacct acagttttct ctccccttga 60

ggtgctagca ctctcacgga gaagggatct gcaggcctga 100

<210> 90

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 90

tggcagtcct gtttgcttac cagaatcagg atccacagct gctcctcctt taagagttaa 60

tttcattctc ttttcagatt tgttgatacc ttggaggaga 100

<210> 91

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 91

gtcctgtttg cttaccagaa tcaggatcca cagctgctcc tcctttaaga gttaatttca 60

ttctcttttc agatttgttg ataccttgga ggagaacaga 100

<210> 92

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 92

gtttgcttac cagaatcagg atccacagct gctcctcctt taagagttaa tttcattctc 60

ttttcagatt tgttgatacc ttggaggaga acagaaaaat 100

<210> 93

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 93

ggggccctca ccttcctcct tgacctggcc cttgcttttt ttggagagcg cagcccctga 60

cttccctctt ggggccacaa cttcaacagg ctctgccttc 100

<210> 94

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 94

gctttttttg gagagcgcag cccctgactt ccctcttggg gccacaactt caacaggctc 60

tgccttcacc tctgcccccc aaggggacaa gatgtgcgct 100

<210> 95

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 95

tcttggggcc acaacttcaa caggctctgc cttcacctct gccccccaag gggacaagat 60

gtgcgctaag aacaactcct gaaggctctt ggtggaggcg 100

<210> 97

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 97

cacctctgcc ccccaagggg acaagatgtg cgctaagaac aactcctgaa ggctcttggt 60

ggaggcggag acgtcctgga ggaagtcctc agacacaact 100

<210> 98

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 98

taagaacaac tcctgaaggc tcttggtgga ggcggagacg tcctggagga agtcctcaga 60

cacaactcgg atgttggctt cctttacttc ctccatcttc 100

<210> 99

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 99

ggagacgtcc tggaggaagt cctcagacac aactcggatg ttggcttcct ttacttcctc 60

catcttctta ttcatctttt ccacctcctc tgttcaaatt 100

<210> 100

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 100

gatacagaag cattgtccct gttgcacaaa ttcagattca actcactttt ggtgctgatg 60

cacagggaag ccttgttggc cgtccccgtc aacttccccc 100

<210> 101

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 101

agattcaact cacttttggt gctgatgcac agggaagcct tgttggccgt ccccgtcaac 60

ttccccccga gtttctcaat catggccttc acttcatcct 100

<210> 102

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 102

gaagccttgt tggccgtccc cgtcaacttc cccccgagtt tctcaatcat ggccttcact 60

tcatccttgt tccgggacag cttcccgaga gtcaggatct 100

<210> 103

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 103

ccgagtttct caatcatggc cttcacttca tccttgttcc gggacagctt cccgagagtc 60

aggatcttca tgttggataa tggcttatct gggatgaaag 100

<210> 104

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 104

ttgttccggg acagcttccc gagagtcagg atcttcatgt tggataatgg cttatctggg 60

atgaaaggag agaatcattc agcaaggcca gctctggtgc 100

<210> 105

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 105

ttcatgttgg ataatggctt atctgggatg aaaggagaga atcattcagc aaggccagct 60

ctggtgctgc tccccagtaa ggggaggtgg aggagcaggt 100

<210> 106

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 106

taagaccggg gtcccaaatg ctgtacctgc tgaagcagag gagttcacag cagcaggagc 60

cgaggctgtg gagggcggag gcgtggccgc cacggaggcg 100

<210> 107

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 107

agcagaggag ttcacagcag caggagccga ggctgtggag ggcggaggcg tggccgccac 60

ggaggcgctg gtttctgggg ggaatatacg gtcctgtttt 100

<210> 108

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 108

tgtggagggc ggaggcgtgg ccgccacgga ggcgctggtt tctgggggga atatacggtc 60

ctgtttttta accttcaatt tcttgaggta agagatttct 100

<210> 109

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 109

gctggtttct ggggggaata tacggtcctg ttttttaacc ttcaatttct tgaggtaaga 60

gatttctcgg aattcctaaa aaatattaag ttttagttaa 100

<210> 110

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 110

tttaaccttc aatttcttga ggtaagagat ttctcggaat tcctaaaaaa tattaagttt 60

tagttaagaa gccagctctc ccttgaggta accaccccat 100

<210> 111

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 111

ctacccctta cctttggggt tacccactcc ttccggttgg gtgtctgtgt cttgaccata 60

cacttggtcc aggcagtgac gtccccagtg cagtaatagg 100

<210> 112

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 112

cggttgggtg tctgtgtctt gaccatacac ttggtccagg cagtgacgtc cccagtgcag 60

taataggcat cgctcttgaa gaccagctga cccgagcatt 100

<210> 112

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 112

gtccaggcag tgacgtcccc agtgcagtaa taggcatcgc tcttgaagac cagctgaccc 60

gagcattcct cgcagggaag gagggcaccg aacaccatgc 100

<210> 113

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 113

gcatcgctct tgaagaccag ctgacccgag cattcctcgc agggaaggag ggcaccgaac 60

accatgccat cagctactcg gtccaagatc tgcagccagt 100

<210> 114

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 114

tccacaacga cggggtcggc ctcacatgcg tgtcccactt aacacaaagg cagctcaccg 60

ccgactcccc agaaggcact tgctgcttgt tgaagatgag 100

<210> 115

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 115

cccacttaac acaaaggcag ctcaccgccg actccccaga aggcacttgc tgcttgttga 60

agatgagtag ctccttcagg tcattagttg aacacacttt 100

<210> 116

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 116

ccccagaagg cacttgctgc ttgttgaaga tgagtagctc cttcaggtca ttagttgaac 60

acactttctt tagctcgtcc ttgatgttcc agatcaggtc 100

<210> 117

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 117

gtagctcctt caggtcatta gttgaacaca ctttctttag ctcgtccttg atgttccaga 60

tcaggtcgtt ctgagcctat ggacaagacc cagtggctga 100

<210> 118

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 118

tctttagctc gtccttgatg ttccagatca ggtcgttctg agcctatgga caagacccag 60

tggctgagag gctagctctt ttcaaaggaa gaatccaact 100

<210> 119

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 119

cgttctgagc ctatggacaa gacccagtgg ctgagaggct agctcttttc aaaggaagaa 60

tccaactgga ggaggagccc tggtcatgct gagcctccag 100

<210> 120

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 120

agaatccaga cagcagaatg tcgaaaggag acacagagct gagaactcac ctttagggct 60

ttttcaagct tactatcctt gtctttttct tttttagatt 100

<210> 121

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 121

ggagacacag agctgagaac tcacctttag ggctttttca agcttactat ccttgtcttt 60

ttctttttta gatttcttct tcgccacttc atccactcca 100

<210> 122

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 122

cacagagctg agaactcacc tttagggctt tttcaagctt actatccttg tctttttctt 60

ttttagattt cttcttcgcc acttcatcca ctccatccac 100

<210> 123

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 123

ttcatccact ccatccacct catcgccttt tctctttctg aaggagacac aggatatgag 60

agacagccag agccattaaa agtctgacac ccaatgactt 100

<210> 124

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 124

ggcaaccccg cagtgctcca cccacccttc actcttgact cctgggagct gcttcttcag 60

ggcttcttta tcctctgtag caaggaggct gaagcccttg 100

<210> 125

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 125

cttgactcct gggagctgct tcttcagggc ttctttatcc tctgtagcaa ggaggctgaa 60

gcccttgagc tgactcgcac tgtactcggg ccggaaaccc 100

<210> 126

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 126

tttatcctct gtagcaagga ggctgaagcc cttgagctga ctcgcactgt actcgggccg 60

gaaacccagc tcctccctgt tcttgacaaa gcagcctgga 100

<210> 127

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 127

gagctgactc gcactgtact cgggccggaa acccagctcc tccctgttct tgacaaagca 60

gcctggatgg taccagcggt caatcatgcc tagctgtggc 100

<210> 128

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 128

cagctcctcc ctgttcttga caaagcagcc tggatggtac cagcggtcaa tcatgcctag 60

ctgtggcttc tccgggtcca ccatcttctt ggacaggcgc 100

<210> 129

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 129

atggtaccag cggtcaatca tgcctagctg tggcttctcc gggtccacca tcttcttgga 60

caggcgcacc tggccctgca ggaaaaacca tatgtggtac 100

<210> 130

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 130

aataacatca ttgcaagcaa tccataaagt tcagggatct gggcccccaa gatcttacct 60

tttctatctt ctccatacac cccttgcacg tacttctgtt 100

<210> 131

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 131

gggatctggg cccccaagat cttacctttt ctatcttctc catacacccc ttgcacgtac 60

ttctgttgga cttggcatac tctgctgcaa agtcacccag 100

<210> 132

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 132

tcttctccat acaccccttg cacgtacttc tgttggactt ggcatactct gctgcaaagt 60

cacccagagt cttctctgcc ttgctaccaa ttccatcctg 100

<210> 133

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 133

tggacttggc atactctgct gcaaagtcac ccagagtctt ctctgccttg ctaccaattc 60

catcctggcc tttgcctgga gaatcaaaca gacagcaatg 100

<210> 134

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 134

gagtcttctc tgccttgcta ccaattccat cctggccttt gcctggagaa tcaaacagac 60

agcaatgctc atctcaacag ccccaaaatg cagctcagag 100

<210> 135

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 135

ctggggatca ctggctagcc atttgggaca aaaactggat tcttctctta ttataaaaat 60

aaattcccag gttgaatgtt tcttgcctaa gattaggaat 100

<210> 136

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 136

gatcactggc tagccatttg ggacaaaaac tggattcttc tcttattata aaaataaatt 60

cccaggttga atgtttcttg cctaagatta ggaataagac 100

<210> 137

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 137

aaaacaaaat accaaatatc atgcaacaga tgatgctgag tccaggaggt gttgctgaaa 60

taacatgggc accatacgct tgatctgcac atgtgggaga 100

<210> 138

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 138

acagatgatg ctgagtccag gaggtgttgc tgaaataaca tgggcaccat acgcttgatc 60

tgcacatgtg ggagagggca agctggggga ggtttgcttt 100

<210> 139

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 139

aataacatgg gcaccatacg cttgatctgc acatgtggga gagggcaagc tgggggaggt 60

ttgctttgct ctctgagacg aggccctcct gggagcttca 100

<210> 140

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 140

tgtgggagag ggcaagctgg gggaggtttg ctttgctctc tgagacgagg ccctcctggg 60

agcttcaggg tgggggctga atgcccatgc tgccccagta 100

<210> 141

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 141

tgctctctga gacgaggccc tcctgggagc ttcagggtgg gggctgaatg cccatgctgc 60

cccagtatgt acacacctgt cactcctcca gcttccgctg 100

<210> 142

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 142

agggtggggg ctgaatgccc atgctgcccc agtatgtaca cacctgtcac tcctccagct 60

tccgctgtct tcttgacttt ctgctggtca tcccaccgaa 100

<210> 143

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 143

atgtacacac ctgtcactcc tccagcttcc gctgtcttct tgactttctg ctggtcatcc 60

caccgaagct cagagaaccc atccacctca acgtcagggt 100

<210> 144

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 144

gtcttcttga ctttctgctg gtcatcccac cgaagctcag agaacccatc cacctcaacg 60

tcagggtgcc ggatggagtg gcccaccttc cagaagcagg 100

<210> 145

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 145

agctcagaga acccatccac ctcaacgtca gggtgccgga tggagtggcc caccttccag 60

aagcaggaga agtggtacca gtgtgggact tttccatcaa 100

<210> 146

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 146

tgccggatgg agtggcccac cttccagaag caggagaagt ggtaccagtg tgggactttt 60

ccatcaaaca tgggcgacta gaaggaagag aaacagaggg 100

<210> 147

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 147

gagaagtggt accagtgtgg gacttttcca tcaaacatgg gcgactagaa ggaagagaaa 60

cagagggaag taagtaagca gttaactttt gacttagacc 100

<210> 148

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 148

cacagttaac ccggggcgcc gcgtccccgc cccgccgccc gcacagcggc ccgcacctgc 60

accatgatgg ccatccggag cgagtccttg gggatgctct 100

<210> 149

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 149

gccgcccgca cagcggcccg cacctgcacc atgatggcca tccggagcga gtccttgggg 60

atgctctcgc tgcatttctt gcaagaggcg cgcccgctct 100

<210> 150

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 150

atggccatcc ggagcgagtc cttggggatg ctctcgctgc atttcttgca agaggcgcgc 60

ccgctcttgg cgtactcgac tcgatagagc ttatccgaag 100

<210> 151

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 151

tcgctgcatt tcttgcaaga ggcgcgcccg ctcttggcgt actcgactcg atagagctta 60

tccgaagact ccgccatcct cccctagctg ccgccaaagc 100

<210> 152

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 152

ttggcgtact cgactcgata gagcttatcc gaagactccg ccatcctccc ctagctgccg 60

ccaaagctcc ggaagcccga cgccacgacc tagaaacacg 100

<210> 153

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 153

attgcttcgg tgggtgacgc ggtacagctg cccaagggcg ttcgtaacgg gaatgccgaa 60

gcgtgggaaa aagggagcgg tggcggaaga cggggatgag 100

<210> 154

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 154

aagggcgttc gtaacgggaa tgccgaagcg tgggaaaaag ggagcggtgg cggaagacgg 60

ggatgagctc aggacaggta agggaatgaa atcagccctt 100

<210> 155

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 155

gaaaaaggga gcggtggcgg aagacgggga tgagctcagg acaggtaagg gaatgaaatc 60

agcccttctt cctagaagct gcggcggggg tgtttgtcat 100

<210> 156

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 156

gctcaggaca ggtaagggaa tgaaatcagc ccttcttcct agaagctgcg gcgggggtgt 60

ttgtcattcc cttgatgtac ggtaagtacg ggccgactca 100

<210> 157

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 157

tcttcctaga agctgcggcg ggggtgtttg tcattccctt gatgtacggt aagtacgggc 60

cgactcattt ttgcaggggt ttgtgaagaa gtcgcaggaa 100

<210> 158

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 158

gtatatatta ctcattttat agagccagag gccaagaaga gtaagacggc cgcaaagaaa 60

aatgacaaag aggcagcagg agagggccca gccctgtatg 100

<210> 159

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 159

aagaagagta agacggccgc aaagaaaaat gacaaagagg cagcaggaga gggcccagcc 60

ctgtatgagg accccccaga tcagaaaacc tcacccagtg 100

<210> 160

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 160

caaagaaaaa tgacaaagag gcagcaggag agggcccagc cctgtatgag gaccccccag 60

atcagaaaac ctcacccagt ggcaaacctg ccacactcaa 100

<210> 161

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 161

gaggaccccc cagatcagaa aacctcaccc agtggcaaac ctgccacact caagatctgc 60

tcttggaatg tggatgggct tcgagcctgg attaagaaga 100

<210> 162

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 162

gcaaacctgc cacactcaag atctgctctt ggaatgtgga tgggcttcga gcctggatta 60

agaagaaagg attagatgtg agtggaattt gagggaaaga 100

<210> 163

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 163

acctttcttt tcacttacag tgggtaaagg aagaagcccc agatatactg tgccttcaag 60

agaccaaatg ttcagagaac aaactaccag ctgaacttca 100

<210> 164

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 164

aagccccaga tatactgtgc cttcaagaga ccaaatgttc agagaacaaa ctaccagctg 60

aacttcagga gctgcctgga ctctctcatc aatactggtc 100

<210> 165

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 165

aatgttcaga gaacaaacta ccagctgaac ttcaggagct gcctggactc tctcatcaat 60

actggtcagc tccttcggac aaggaagggt acagtggcgt 100

<210> 166

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 166

aggagctgcc tggactctct catcaatact ggtcagctcc ttcggacaag gaagggtaca 60

gtggcgtggg cctgctttcc cgccagtgcc cactcaaagt 100

<210> 167

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 167

cagctccttc ggacaaggaa gggtacagtg gcgtgggcct gctttcccgc cagtgcccac 60

tcaaagtttc ttacggcata ggtgagaccc tattgatgcc 100

<210> 168

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 168

tgggcctgct ttcccgccag tgcccactca aagtttctta cggcataggt gagaccctat 60

tgatgcctaa tgcctgaact cttcaaaacc aattgctaat 100

<210> 169

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 169

gctaattctg tttcatttct ataggcgatg aggagcatga tcaggaaggc cgggtgattg 60

tggctgaatt tgactcgttt gtgctggtaa cagcatatgt 100

<210> 170

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 170

agcatgatca ggaaggccgg gtgattgtgg ctgaatttga ctcgtttgtg ctggtaacag 60

catatgtacc taatgcaggc cgaggtctgg tacgactgga 100

<210> 171

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 171

aatttgactc gtttgtgctg gtaacagcat atgtacctaa tgcaggccga ggtctggtac 60

gactggagta ccggcagcgc tgggatgaag cctttcgcaa 100

<210> 172

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 172

tacctaatgc aggccgaggt ctggtacgac tggagtaccg gcagcgctgg gatgaagcct 60

ttcgcaagtt cctgaagggc ctggcttccc gaaagcccct 100

<210> 173

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 173

agtaccggca gcgctgggat gaagcctttc gcaagttcct gaagggcctg gcttcccgaa 60

agccccttgt gctgtgtgga gacctcaatg tggcacatga 100

<210> 174

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 174

agttcctgaa gggcctggct tcccgaaagc cccttgtgct gtgtggagac ctcaatgtgg 60

cacatgaaga aattgacctt cgcaacccca aggggaacaa 100

<210> 175

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 175

ttgtgctgtg tggagacctc aatgtggcac atgaagaaat tgaccttcgc aaccccaagg 60

ggaacaaaaa gaatgctggc ttcacgccac aagagcgcca 100

<210> 176

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 176

aagaaattga ccttcgcaac cccaagggga acaaaaagaa tgctggcttc acgccacaag 60

agcgccaagg cttcggggaa ttactgcagg ctgtgccact 100

<210> 177

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 177

aaaagaatgc tggcttcacg ccacaagagc gccaaggctt cggggaatta ctgcaggctg 60

tgccactggc tgacagcttt aggcacctct accccaacac 100

<210> 178

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 178

aaggcttcgg ggaattactg caggctgtgc cactggctga cagctttagg cacctctacc 60

ccaacacacc ctatgcctac accttttgga cttatatgat 100

<210> 179

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 179

tggctgacag ctttaggcac ctctacccca acacacccta tgcctacacc ttttggactt 60

atatgatgaa tgctcgatcc aagaatgttg gttggcgcct 100

<210> 180

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 180

caccctatgc ctacaccttt tggacttata tgatgaatgc tcgatccaag aatgttggtt 60

ggcgccttga ttactttttg ttgtcccact ctctgttacc 100

<210> 181

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 181

tgaatgctcg atccaagaat gttggttggc gccttgatta ctttttgttg tcccactctc 60

tgttacctgc attgtgtgac agcaagatcc gttccaaggc 100

<210> 182

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 182

ttgattactt tttgttgtcc cactctctgt tacctgcatt gtgtgacagc aagatccgtt 60

ccaaggccct cggcagtgat cactgtccta tcaccctata 100

<210> 183

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 183

ctgcattgtg tgacagcaag atccgttcca aggccctcgg cagtgatcac tgtcctatca 60

ccctatacct agcactgtga caccacccct aaatcacttt 100

<210> 184

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 184

ccctcggcag tgatcactgt cctatcaccc tatacctagc actgtgacac cacccctaaa 60

tcactttgag cctgggaaat aagccccctc aactaccatt 100

<210> 185

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 185

ctggttctgg gtaggcgggg ctactacggg gcggtgcctg ctgtggaaat gcctgcccgc 60

gcgcttctgc ccaggcgcat ggggcatcgt actctagcct 100

<210> 186

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 186

ctgcccaggc gcatggggca tcgtactcta gcctccactc ctgccctgtg ggcctccatc 60

ccgtgccctc gctctgagct gcgcctggac ctggttctgc 100

<210> 187

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 187

cctcgctctg agctgcgcct ggacctggtt ctgccttctg gacaatcttt ccggtgagtg 60

actgagcctg agaagcctgt cccctcggac tggctcctgc 100

<210> 188

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 188

gggtagccaa cctgttacct ttaatatgtc tgtacagtga taattgtaag gatccagcgg 60

tataaccgat gcagaagata atagcacttg tttttctttt 100

<210> 189

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 189

agggttgtca tgtgccttgg gctcaggtgg agggagcaaa gtcctgcaca ctggagtggt 60

gtactagcgg atcaagtatg gacactgact cagactgagg 100

<210> 190

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 190

gcggatcaag tatggacact gactcagact gaggagcagc tccactgcac tgtgtaccga 60

ggagacaaga gccaggctag caggcccaca ccagacgagc 100

<210> 191

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 191

aagagccagg ctagcaggcc cacaccagac gagctggagg ccgtgcgcaa gtacttccag 60

ctagatgtta ccctggctca actgtatcac cactggggtt 100

<210> 192

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 192

gttaccctgg ctcaactgta tcaccactgg ggttccgtgg actcccactt ccaagaggtg 60

gctcagaaat tccaaggtga gtacaggacc tgggctgggg 100

<210> 193

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 193

tggtctccag gtgtgcgact gctgcgacaa gaccccatcg aatgcctttt ctcttttatc 60

tgttcctcca acaacaacat cgcccgcatc actggcatgg 100

<210> 194

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 194

tccaacaaca acatcgcccg catcactggc atggtggagc ggctgtgcca ggcttttgga 60

cctcggctca tccagcttga tgatgtcacc taccatggct 100

<210> 195

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 195

gacctcggct catccagctt gatgatgtca cctaccatgg cttccccagc ctgcaggccc 60

tggctggtga gtaggtgggt cccctgcccc caggccttcc 100

<210> 196

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 196

gtggaggctc atctcaggaa gctgggcctg ggctatcgtg cccgttacgt gagtgccagt 60

gcccgagcca tcctggaaga acagggcggg ctagcctggc 100

<210> 197

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 197

gccatcctgg aagaacaggg cgggctagcc tggctgcagc agctacgaga gtcctcatat 60

gaggaggccc acaaggccct ctgcatcctg cctggagtgg 100

<210> 198

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 198

gggctcattc tgtgtctgtc aaaggtggct gactgcatct gcctgatggc cctagacaag 60

ccccaggctg tgcccgtgga tgtccatatg tggcacattg 100

<210> 199

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 199

gctgtgcccg tggatgtcca tatgtggcac attgcccaac gtgactacag ctggcaccct 60

accacgtccc aggcgaaggg accgagcccc cagaccaaca 100

<210> 200

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 200

atcacttctg atttaggaaa ctttttccgg agcctgtggg gaccttatgc tggctgggcc 60

caagcggtga gtgtacctag gtgtcctccc taggtttcct 100

<210> 201

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 201

atttaggaaa ctttttccgg agcctgtggg gaccttatgc tggctgggcc caagcggtga 60

gtgtacctag gtgtcctccc taggtttcct ctcctccagc 100

<210> 202

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 202

tcctacaggt gctgttcagt gccgacctgc gccaatcccg ccatgctcag gagccaccag 60

caaagcgcag aaagggttcc aaagggccgg aaggctagat 100

<210> 203

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 203

gcagaaaggg ttccaaaggg ccggaaggct agatggggca ccctggacaa agaaattccc 60

caagcacctt cccctccatt ccccacttct ctctccccat 100

<210> 204

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 204

ccttcccctc cattccccac ttctctctcc ccatccccac ccagtctcat gttggggagg 60

ggcctccctg tgactacctc aaaggccagg cacccccaaa 100

<210> 206

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 206

cctgtgacta cctcaaaggc caggcacccc caaatcaagc agtcagtttg cacaacaaga 60

tggggtgggg gatattgagg gagacagcgc taaggatggt 100

<210> 206

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 206

gctatagaca ttattccgct atgcctcact aattcctact taactgacag ctctgtcagg 60

tcatcaccac ttttatgacc tttctcggac cccataggct 100

<210> 207

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 207

ccacttttat gacctttctc ggaccccata ggctggatca gatgcctcct gaagaattac 60

agacttcttc ctctagactt ggaggtgagg gaccctctct 100

<210> 208

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 208

tggatcagat gcctcctgaa gaattacaga cttcttcctc tagacttgga ggtgagggac 60

cctctctgga atagagaagg tgttgggagt gtttgttgaa 100

<210> 209

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 209

tgcatttgat ggccaccagc ttctgcgtcc tcttatcttc tgccaggatc acctccgaga 60

aggcccccct atcgggagag gggattcaca aggtgaagaa 100

<210> 210

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 210

ccctatcggg agaggggatt cacaaggtga agaactggaa ccccagcttc cctccagcct 60

ctcctccatt ccctatgggt tctgtgacca ctgctggacc 100

<210> 211

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 211

cattccctat gggttctgtg accactgctg gaccaaggac gtggatgacc ctcccctagt 60

cactcatcca tcccctggct ccagagatgg tcacatgacc 100

<210> 212

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 212

catgacccag gcctggccag tcaaagtagt ctctcccctg gccacagtaa ttggtcatgt 60

gatgcaagcc agcttactag cactttgaga atgagtctcc 100

<210> 213

<211> 100

<212> DNA

<213> Artificial sequence (chemical Synthesis)

<400> 213

gtagtctctc ccctggccac agtaattggt catgtgatgc aagccagctt actagcactt 60

tgagaatgag tctcctgttg agctggtagg atgtaagcct 100

Claims (7)

1. A kit for detecting a key mutant gene of a DNA base excision repair pathway is characterized by comprising a probe library which is formed by probes shown as SEQ ID NO. 1-213 aiming at the key mutant gene of the DNA base excision repair pathway.

2. The kit for detecting the key mutant genes of the DNA base excision repair pathway as claimed in claim 1, wherein the key mutant genes of the DNA base excision repair pathway comprise POLB, PARP1, APEX1 and OGG 1.

3. The kit for detecting the key mutant gene of the DNA base excision repair pathway as claimed in claim 1, characterized in that the probe sequence corresponding to the gene POLB is shown as SEQ ID NO. 1-29, the probe sequence corresponding to the gene PARP1 is shown as SEQ ID NO. 30-152, the probe sequence corresponding to the gene APEX1 is shown as SEQ ID NO. 153-184, and the probe sequence corresponding to the gene OGG1 is shown as SEQ ID number 185-213.

4. A method for detecting a DNA base excision repair pathway key mutant gene is characterized by comprising the following steps:

(1) obtaining a DNA sample library of a subject;

(2) hybridizing all probes shown as SEQ ID NO. 1-213 in the kit of claim 1 with the DNA sample library;

(3) and (3) separating the hybridization product in the step (2), releasing the gene segment hybridized with the probe in the kit, and sequencing the separated gene segment by adopting a sequencing technology so as to detect the mutation condition of the gene.

5. The method according to claim 4, wherein the DNA sample library in the step (1) is composed of double-stranded DNA fragments;

the step (1) comprises the following steps: extracting whole genome DNA, and then fragmenting the whole genome DNA; or mRNA is extracted, fragmented, and then double-stranded cDNA is synthesized using the fragmented mRNA as a template.

6. The method according to claim 4, wherein the probe in the step (2) is selectively labeled.

7. The method according to claim 6, wherein the probe in the step (2) is labeled with biotin.

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