CN110684848A - Multiple PCR primer group and kit for genetic tumor germ line mutation detection - Google Patents
Multiple PCR primer group and kit for genetic tumor germ line mutation detection Download PDFInfo
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Abstract
The invention discloses a multiple PCR primer group and a kit for genetic tumor germ line mutation detection. The multiplex PCR primer set includes at least SEQ ID No.: 1 to 276. The multiplex PCR primers of the invention are free from interference, and multiple genes can be amplified by mixing the primers for multiplex PCR at one time, thereby greatly reducing the operation amount of PCR and further greatly reducing the overall sequencing cost. In some embodiments of the invention, all exon regions of the 152 core CPG oncogenes inherited by the germ line and frequently mutated intron regions, including point mutations, small fragment deletions and insertions, can be amplified at one time, greatly facilitating subsequent high-throughput sequencing operations. By further reading the high-throughput sequencing data, the tumor incidence risk of the person to be detected can be evaluated, so that the person to be detected can avoid the related unhealthy life style, reduce the tumor incidence risk and guide the personalized health management.
Description
Technical Field
The invention relates to a multiple PCR primer group and a kit for genetic tumor germ line mutation detection.
Background
It is estimated that about 5% to 10% of tumors in humans occur due to inheritance of germline variation (germline mutation) of oncogenes. Germ line mutations are also called germ cell mutations, and generally all cells in a body have mutations and are familial. In individuals with germline mutations, the risk of developing tumors is tens or hundreds of times higher than in the general population. In parents, if one of the parents carries a mutation of a disease-causing gene and transmits the mutation to the next generation, the offspring will have a greatly increased risk of cancer compared to the ordinary people.
In 1990, researchers discovered a Gene directly associated with hereditary breast Cancer, named breast Cancer number 1 Gene (Brest Cancer surgery Gene 1, BRCA 1). In 1994, another gene was found to be associated with breast cancer, designated BRCA 2. Research shows that BRCA1/2 is two genes with the function of inhibiting malignant tumor and has important functions in regulating human body cell replication, genetic material DNA damage repair, cell growth, etc. The obvious characteristic of the tumor cell is that the tumor cell can be replicated and proliferated without limit, and the cancer suppressor gene such as BRCA1 can inhibit the proliferation of the tumor cell, promote the apoptosis of the tumor cell and achieve the effect of suppressing cancer. In addition, most tumors exhibit chromosomal instability, such as chromosomal abnormalities, aneuploidy, and chromosomal additions or deletions. DNA damage repair can transform abnormal cells into normal cells or into an apoptotic process. The mutation of BRCA1 gene leads the protein coded by BRCA1 to enter cytoplasm from nucleus, and loses the ability of combining with DNA, thereby losing the normal functions of inhibiting proliferation, promoting apoptosis, repairing DNA damage and the like. It has been found that families possessing mutations in the BRCA1 gene tend to have a high incidence of breast cancer, often at a younger age, with a high incidence of cancer in both breasts, and possibly ovarian cancer at the same time.
With the vigorous development of molecular biotechnology, partial gene mutations have been identified as risk factors for specific cancers. Among them, the typical and well-known relationship between BRCA gene mutations and breast and ovarian cancers is well known. In past studies, mutations in the BRCA1 and BRCA2 genes have been shown to be associated with a higher risk of developing breast (breast cancer) and ovarian (ovarian cancer). Approximately 5% to 10% of breast cancer cases and 10% to 18% of ovarian cancer cases can be attributed to mutations in the germline BRCA1 and BRCA2 genes.
In nature < responsive the progress of Cancer predisposing genes. nature 505, pages 302-308 (16January 2014) > introduction of genes whose germline mutations lead to a high or moderate increase in the risk of Cancer is called Cancer susceptibility genes (CPG), over 100 oncogenic CPG's have been identified and provide important scientific explanations in many fields, particularly the mechanism of Cancer etiology.
"New England journal of medicine" < Germine details in Predisposition Genes in Pediatric cancer. NEJM.2015Dec 10; 2336-2346 DNA sequence analysis of healthy and tumor tissues of 1120 children and adolescent cancer patients under 20 years old showed that 8.5% of patients had germ line mutations of cancer-susceptible genes which were or may be pathogenic among children and adolescent cancer patients. For most patients, family history does not predict the presence of cancer susceptibility, and children may carry cancer susceptibility genes even if no cancer has occurred in the family history. This study provides the most comprehensive of children's cancer susceptibility gene mutations, and found that children with tumors have a higher cancer susceptibility gene mutation rate than thought, and these mutations increase the risk of cancer. It is helpful for people to further understand the mechanism of cancer.
(American journal of medical society), < Association Between associated geological details Cancer Predisposition Genes and Risk of functional Cancer. JAMA.2018; 319(23) 2401-2409 > the results of the gene tests were compared between 2000-2016, 3046 patients with pancreatic cancer with 21 oncogenes and 123000 patients without pancreatic cancer. The results show that 6 genes are clearly associated with increased risk of pancreatic cancer: BRCA1, BRCA2, CDKN2A, TP53, MLH1 and ATM. Mutations in these genes occur in 5.5% of patients, including 5.2% of patients with no family history of pancreatic cancer. Therefore, they recommend that all pancreatic cancer patients undergo genetic testing regardless of family history of pancreatic cancer, so that the molecular mechanism behind the genetic factors causing pancreatic cancer is more clear.
In the < Pathological Germine Variants in 10,389 adolt cancers. cell,173(2),355-370> study analyzed the patterns of Pathogenic variation in a total of 10389 samples of 33 cancer species in TCGA, found that the proportion of Pathogenic or suspected Germline variation was 8%, 33 variations were identified in oncogenes, and then the functional consequences of these variations were investigated by validation in other cancer species populations and testing for RET alleles. Finally, 18 copy number deletions and 47 VUSs based on multiple functional evidence were found. This study presented the results of maximal systematic analysis of rare germline susceptibility variations, and also provided a powerful fundamental support for the solution of cancer functional problems.
In the past, doctors have suggested that high-risk groups with family history of cancer be tested for related genes. However, according to the recent article published by American Journal of modern Medicine in the department of Texas university Medicine in America at the front, the proportion of women who do not have a history of related cancers (breast cancer or ovarian cancer) but still like to actively carry out BRCA gene mutation detection appears to be remarkably increased according to the statistical result. Before cancer develops, more carriers of the BRCA1/2 mutation are discovered early, and appropriate preventive treatment measures (such as mastectomy and oophorectomy) are taken, so that the incidence rate of breast cancer and ovarian cancer can be reduced. For patients who have been diagnosed with breast cancer and are positive for BRCA mutation, prophylactic treatment can reduce the risk of the onset of another cancer.
The 2020 standard of health promulgated by the U.S. department of health and human services indicates that diseases caused by genetic mutations will significantly affect the lives of patients and their families. In the 2020 target, genomic medicine was first placed on a priority list, emphasizing the importance of understanding family and genetic history.
Modern life science research teaches that "all diseases are related to genes except for injuries". There is a lot of data to show that cancer is a genetic disease, and congenital familial inheritance and acquired gene mutation can change genes, thereby causing cancer. With the continuous development of gene and genomics research, gene detection is leading the development of predictive medicine and is applied to various aspects such as disease prevention, personalized medicine and the like.
However, the genetic tumor germ line mutations are numerous, the conventional sequencing detection is high in cost, and the number of detected genes is required to be reduced for reducing the cost. The development of a low-cost genetic tumor germ line mutation detection technology has very important significance.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a multiplex PCR primer group and a kit for genetic tumor germ line mutation detection.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided: a multiplex PCR primer set for genetic tumor germline mutations, said multiplex PCR primer set comprising at least the following primer sequences:
in the table, SEQ ID No. is, from top to bottom, in sequence from left to right: 1 to 276.
In some examples of primers, the multiplex PCR primer set further includes a primer set for detecting genetic disease-associated conditions as follows:
ADH1B、AIP、ALDH2、ALK、APC、ARID1A、ATM、AXIN2、BAP1、BARD1、BLM、BMPR1A、BRCA1、BRCA2、BRIP1、BUB1、BUB1B、BUB3、CDC73、CDH1、CDK4、CDKN1C、CDKN2A、CEBPA、CEP57、CHEK2、CTNNB1、CYLD、DDB2、DICER1、DIS3L2、EGFR、EPCAM、EPHB2、ERBB4、ERCC1、ERCC2、ERCC3、ERCC4、ERCC5、EXT1、EXT2、EZH2、FANCA、FANCB、FANCC、FANCD2、FANCE、FANCF、FANCG、FANCI、FANCL、FANCM、FH、FLCN、FOXP1、GATA2、GPC3、HDAC9、HNF1A、HOXB13、HRAS、KIT、KLLN、KRAS、LZTR1、MAX、MEN1、MET、MITF、MLH1、MLH3、MPL、MRE11、MRE11A、MSH2、MSH3、MSH6、MUTYH、NAT1、NBN、NF1、NF2、NSD1、PALB2、PBRM1、PDE11A、PHOX2B、PIK3CA、PMS1、PMS2、POLE、POLH、PRF1、PRKAR1A、PRSS1、PTCH1、PTEN、PTPN11、RAD50、RAD51、RAD51C、RAD51D、RB1、RECQL4、RET、RHBDF2、RUNX1、SBDS、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、SLX4、SMAD4、SMARCB1、SPINK1、STK11、SUFU、TGFBR2、TMEM127、TP53、TSC1、TSC2、TSHR、VHL、WRN、WT1、XPA、XPC。
in a second aspect of the present invention, there is provided: a multiplex PCR kit for genetic tumor germline mutation using a multiplex PCR primer set as described in the first aspect of the invention.
In a third aspect of the present invention, there is provided: a multiplex PCR method using a multiplex PCR primer set as described in the first aspect of the present invention.
In some multiplex PCR examples, the initial concentration of dNTPs in the PCR reaction system is 0.3mM, Mg2+Was 2.5mM, and 5U of polymerase was added per 50. mu.L of the reaction system.
In some multiplex PCR examples, the concentration of primers in the PCR reaction system is 0.15. mu.M.
In some examples of multiplex PCR, the PCR amplification system is as follows:
| name of reagent | Volume (μ L) |
| Sample DNA | 4.5 |
| Capture primer mixture | 8 |
| Library amplification reaction solution | 12.5 |
| Total amount of | 25 |
。
In some multiplex PCR examples, the reaction conditions for PCR amplification are:
in some examples of multiplex PCR, further comprising purification of PCR products, the purification comprising:
1) adding 9 mu L of AMPure XP magnetic beads into each tube in a system after the PCR reaction is finished, uniformly mixing, and standing;
2) adsorbing the magnetic beads until the solution is clarified, and sucking the supernatant into a new 0.2ml PCR tube;
3) adding 15 mu L of magnetic beads into the new PCR tube, uniformly mixing, standing, adsorbing the magnetic beads until the solution is clarified, discarding the supernatant, and leaving the magnetic beads;
4) adding 100 mu L of 70% ethanol into a PCR tube, fully suspending magnetic beads, adsorbing the magnetic beads until the solution is clarified, discarding supernatant, retaining the magnetic beads, and volatilizing the ethanol;
5) adding 15 mu L of eluent, fully suspending the magnetic beads, and eluting the DNA, wherein the eluent is 10mM Tris-HCl, the pH value is 7.5-8.0, and the temperature is 55-65 ℃.
In some multiplex PCR examples, the temperature of the eluate is 60 ℃.
The invention has the beneficial effects that:
the multiplex PCR primers of the invention are free from interference, and multiple genes can be amplified by mixing the primers for multiplex PCR at one time, thereby greatly reducing the operation amount of PCR and further greatly reducing the overall sequencing cost.
In some embodiments of the invention, all exon regions of the 152 core CPG oncogenes inherited by the germ line (reference includes 468 genes in MSK-IMPACT) and frequently mutated intron regions, including point mutations, small fragment deletions and insertions (indels) of the mutation types, may be amplified at once. Greatly facilitates the subsequent high-throughput sequencing operation. By further reading the high-throughput sequencing data, the tumor incidence risk of the person to be detected can be evaluated, so that the person to be detected can avoid the related unhealthy life style, reduce the tumor incidence risk and guide the personalized health management.
Detailed Description
The technical scheme of the invention is further explained by combining the embodiment.
Primer design
As the amplification of 152 gene sequences needs to be completed in one system, proper specific primers need to be designed to ensure the effective amplification of the whole amplification primer pool. In order to solve the problem of designing a very multiple PCR primer, on the basis of the design principle, the following conditions are particularly applied:
1) because the genetic tumor embryonic system mutant gene detection sample is a blood card sample generally, the DNA has degradation conditions of different degrees. We require that the length kurtosis value of the amplification product be around 200 bp. It is ensured as far as possible that the maximum amount of information is available in the degraded DNA.
2) The Primer design process firstly uses authoritative software Primer3(MIT, Cambridge, MA) to design a preselected Primer combination m1, then carries out target amplification region prediction of the population on m1 combination through UCSC Genome Browser, confirms that no off-target of the target region occurs, and optimizes the m1 combination into the m2 combination. The unsuitable primers within the m2 combination were then knocked out using the inventor's MendelGene-PdesignV1.2 software by avoiding repetitive sequences by whole genome BLAST searches, avoiding highly folded regions by structural prediction, and then knocking out potential primer dimers by pairing tests.
3) The site-specific sequences of the primers were designed to be slightly longer than the typical PCR primers (24-35 bases), slightly higher in melting temperature (above 65 ℃ on average), and controlled to have a GC content of 50-60%.
4) Ensuring that each single primer concentration is lower than in the singleplex PCR would otherwise cause the total primer concentration in the reaction to be too high. For example, if the single primer is used in a conventional single PCR reaction at a primer concentration of 0.2. mu.M, then we will attempt to use a 0.15. mu.M concentration (the primer concentration typically ranges between 0.05. mu.M and 0.4. mu.M).
5) Typically multiplex PCR requires more dNTPs, magnesium ions and polymerase. We found that one of the better combinations is 0.3mM dNTP (instead of the standard 0.2mM), 2.5mM Mg2+(instead of 1.2 to 2mM) and 5units of polymerase per 50. mu.L reaction (instead of 1.25 units).
Based on the design steps, the scheme successfully designs a total of 3000 pairs of primer pairs, ensures that the primer pairs are not interfered with each other, and can complete the capture and library establishment of exons and intron regions including pathogenicity of 152 genes at one time.
First round PCR amplification system configuration
First round PCR amplification systems were prepared in 0.2ml PCR tubes according to Table 1. The PCR amplification system to which the DNA template has been added is vortexed, mixed and centrifuged briefly.
TABLE 1 first round PCR amplification System
First round PCR amplification
Setting PCR reaction conditions according to parameters in Table 2, placing the system prepared in the previous step into a PCR instrument, and starting the program.
TABLE 2 first round PCR amplification conditions
PCR product purification
Preheating the eluent to 60 c prior to performing this step of the experiment further improves the yield. The eluent is 10mM Tris-HCl, pH 7.5-8.0.
And 1, adding 9ul AMPure XP magnetic beads into each tube in the reaction system, and blowing and beating the mixture up and down for 10 times by using a50 ul pipette to fully mix the mixture. The mixture was allowed to stand at room temperature for 2 minutes.
2> magnetic shelf adsorb beads for 5 min until the solution is clear.
3> carefully pipette the supernatant into a new 0.2ml PCR tube to avoid pipetting into magnetic beads; discarding the magnetic beads. (for removal of large fragments exceeding 1Kb in PCR tubes).
4> 15ul of magnetic beads were added to the new PCR tube in 3>, and the mixture was thoroughly mixed by pipetting up and down 10 times with a50 ul pipette. The mixture was allowed to stand at room temperature for 2 minutes.
5> magnetic shelf adsorb beads, 5 min, until the solution is clear. Carefully pipette the supernatant, discard the supernatant, and retain the beads.
6> 100ul of 70% ethanol was added and the beads were repeatedly adsorbed back and forth on different sides with a magnetic rack to fully suspend the beads. The magnetic beads were adsorbed by the magnetic frame for 5 minutes until the solution was clear. The supernatant was carefully removed with a pipette to avoid attracting to the beads.
7, standing at room temperature for about 5 minutes until the ethanol is completely volatilized. This step can never be omitted, otherwise the residual ethanol would seriously affect the yield and subsequent experiments.
8> 15ul of eluent was added, the beads were suspended thoroughly, and the mixture was allowed to stand at room temperature for 2 minutes to elute the DNA.
9> magnetic shelf adsorb beads for 5 minutes until the solution is clear. The supernatant 14ul was transferred to a new 0.2ml PCR tube for subsequent experiments.
Second round PCR amplification System configuration
In this step, Illumina sequencing corresponding joint labels are introduced, and different label primers are selected from different samples.
A second round of PCR amplification was prepared in 0.2ml PCR tubes according to Table 3. Then, the prepared PCR amplification system is vortexed, mixed evenly and centrifuged for a short time.
TABLE 3 second round PCR amplification System
The tag primers must be different for different samples. Making a sample and label primer control record.
Second round of PCR amplification
The PCR conditions were set according to the parameters in Table 4, and the system prepared in the previous step was placed in a PCR apparatus to start the procedure.
TABLE 4 second round PCR amplification conditions
And (4) putting the PCR system in the previous step into a PCR instrument, and starting the program. This step takes about 20 minutes.
Purification and recovery of the product
7.1> first round recovery:
① PCR product was added with 21ul AMPure XP magnetic beads, and the mixture was pipetted up and down 10 times with a50 ul pipette to mix well and left at room temperature for 2 minutes.
② magnetic rack adsorbs the beads for 5 minutes until the solution is clear.
③ pipette carefully aspirate the supernatant, discard the supernatant and leave the beads.
④ 100ul of 70% ethanol was added and the beads were repeatedly adsorbed back and forth on different sides using a magnetic rack to suspend the beads sufficiently for washing.
⑤ the magnetic stand adsorbs the beads for 2 minutes until the solution is clear the supernatant is carefully removed with a pipette to avoid attracting the beads.
⑥ standing at room temperature until the ethanol is completely volatilized.
⑦ 20ul of eluent was added to the mixture, the beads were suspended sufficiently, and the mixture was allowed to stand at room temperature for 2min to elute the DNA.
⑧ the magnetic beads were adsorbed by a magnetic holder, and the resulting DNA solution supernatant was pipetted into a new 0.2ml PCR tube
7.2> second round recovery:
the procedure was the same as the first round, but in the first step AMPure XP beads were added in a volume of 12 ul. The resulting 20ul DNA solution can be used directly for subsequent experiments or stored at-20 ℃.
Experimental data
This experiment performed high throughput sequencing of the human whole exon regions of tumor patients and of the population with a family history of tumors. The experiment aims at detecting the coding gene region of the human genome, and detects the mutation types such as single nucleotide site variation (SNV), small fragment insertion/deletion (INDEL) and the like of the coding region of the gene.
List of relevant test genes: ADH1B, AIP, ALDH2, ALK, APC, ARID1A, ATM, AXIN2, BAP1, BARD1, BLM, BMPR1A, BRCA1, BRCA2, BRIP RAD 1, BUB1, BUB1B, BUB3, CDC73, CDH1, CDK 1, CDKN 11, CDKN2 RAD 1, CEBPA, CEP 1, CHEK 1, CTNNB1, CYLD, DDB 1, DICER1, DIS3L 1, EGFR, EPWRRB, EPHB 1, ERBB 1, ERCC1, FANCFLNCFLNCFLNCH 1, PSNFR 36, WT1, XPA, XPC.
The experimental result of the invention is as follows:
the first experimental subject was a brother and a sister, and the family information was as follows:
two subjects detected 8 suspected mutations in total: as shown in the table below. It is known from the ClinVar and OMIM [1] databases that patients carry 2 POLE gene mutations of hereditary tumor susceptibility syndrome and related mutations of liver cirrhosis, immunodeficiency hepatic vein occlusion and hereditary intrahepatic bile obstruction disease, thus possibly leading to the occurrence and development of clinical indications.
2) In the second example, the clinical diagnosis of medulloblastoma is performed on the examined person, the pathogenic site is found after the examination of the invention, and the parents are verified, and the examination result and analysis are as follows:
1> the subject co-detected 3 suspected mutations, respectively c.a505g point mutation (MSH2 gene, NM — 000251 transcript), c.a6139g point mutation (MED12 gene, NM — 005120 transcript) and c.765 — 779del deletion mutation (PHOX2B gene, NM — 003924 transcript), wherein c.a505g point mutation and c.765 — 779del deletion mutation are heterozygous mutations and c.a6139g point mutation is homozygous mutation; the MSH2 gene and the PHOX2B gene are tumor genetic risk genes; all 3 mutations have been recorded in ClinVar database [3] [4] [5], which judges c.A505G point mutation as a possibly Benign mutation (Likely Benign), c.A6139G point mutation as a clinically insignificant mutation (Uncertainicicane), and c.765-779 del deletion mutation as a Benign/possibly Benign mutation (Benign/Likelybign) according to ACMG [2] gene mutation guide.
2> ClinVar and OMIM databases indicate:
① c. A505G point mutation (MSH2 gene, NM-000251 transcript) is related to Lynch syndrome (Lynch syndrome, namely) and Rockot syndrome/Mismatch reppair cancer, Lynch syndrome is hereditary nonpolyposis colorectal cancer, and the Tryp syndrome, also known as glioma polyposis syndrome, is mainly characterized by the occurrence of tumors of the central nervous system, commonly expressed as gliomas, associated with patients with familial hereditary large intestine polyposis (the medulloblastoma from which patients are clinically diagnosed is a pathological classification of gliomas [6 ]).
② c. A6139G point mutation (MED12 gene, NM-005120 transcript) related diseases are intellectual disturbance diseases (Lujan-Fryns syndrome, Ohdo syndrome, Opitz-Kaveggia syndrome);
3> c.765_779del deletion mutation (PHOX2B gene, NM _003924 transcript) associated diseases are Neuroblastoma susceptibility type 2 (Neuroblastoma, subactivity to,2) and Neuroblastoma with giant colon (Neuroblastoma with Hirschsprushing disease).
3) The third experimental subject is a fetus B-ultrasonic display polycystic kidney, and pathogenic sites are found after the birth tissue is detected by using the invention, and the detection result and analysis are as follows:
1> the test person detects c.G10102A and c.G3931A heterozygous mutation on PKD1 gene, and the heterozygous mutation is not recorded by ClinVar database, and InterVar software judges that two point mutations are unknown in clinical significance according to ACMG [2] gene mutation guide. The OMIM database indicated that the disease associated with both mutations was Polycystic kidney disease type 1 (Polycystic kidney disease 1).
2> the test subject detects c.C460G heterozygous mutation on MUC1 gene, and the heterozygous mutation is not recorded by ClinVar database, and InterVar software judges that two point mutations are of unknown clinical significance according to ACMG [2] gene mutation guide. The OMIM database indicated that the disease associated with these two mutations was myeloid polycystic kidney disease type 1 (Memullary cystic kidney disease 1).
3> the test person detects c.T8335G and c.G325A heterozygous mutation on PKHD1 gene, and the heterozygous mutation is recorded by ClinVar database, and the database judges that the two point mutations are clinically unknown and disputed pathogenicity respectively according to ACMG 2 gene mutation guide. The Clinvar database indicated that the disease associated with these two mutations was Polycystic kidney disease type 4 (Polycystic kidney disease 4).
The experimental results show that the invention detects all exon regions of 152 core CPG oncogenes (including 468 genes in MSK-IMPACT) inherited by the germ line at one time and the frequently mutated intron regions, detects germ line pathogenic mutation of the hereditary tumor related genes, and the detection result accords with clinical manifestations of the examinee and clinical diagnosis of other laboratory examination results.
<110> Guangzhou Wande Gene medicine science and technology Co., Ltd
<120> multiplex PCR primer group and kit for genetic tumor germ line mutation detection
<160>276
<210>1<211>22<212> DNA <213> Artificial primer <400>1 cctggaaaggccactttgtaag22
<210>2<211>25<212> DNA <213> Artificial primer <400>2 gtcgattgattagagcctagtccag25
<210>3<211>22<212> DNA <213> Artificial primer <400>3 cctggaaaggccactttgtaag22
<210>4<211>25<212> DNA <213> Artificial primer <400>4 gtcgattgattagagcctagtccag25
<210>5<211>21<212> DNA <213> Artificial primer <400>5 tggccagaaccaccatctttc21
<210>6<211>25<212> DNA <213> Artificial primer <400>6 cttaacttgtttacagcgatgccaa25
<210>7<211>29<212> DNA <213> Artificial primer <400>7 tcaagttcactttcttccatttctatgct29
<210>8<211>25<212> DNA <213> Artificial primer <400>8 tacctggtactgattatggcactca25
<210>9<211>33<212> DNA <213> Artificial primer <400>9 gaccaagatttttggcaaaactataagataagg33
<210>10<211>26<212> DNA <213> Artificial primer <400>10 caagtttctcttcaggaggaaaagca26
<210>11<211>32<212> DNA <213> Artificial primer <400>11 ctgaacataaaaacaacaattacgaaccaaac32
<210>12<211>29<212> DNA <213> Artificial primer <400>12 ggtttgcctaaattcctagtttgtagttc29
<210>13<211>28<212> DNA <213> Artificial primer <400>13 cttcagagaattctttgccacgtatttc28
<210>14<211>32<212> DNA <213> Artificial primer <400>14 tgaaagtctctttaggtgattctcttattctg32
<210>15<211>32<212> DNA <213> Artificial primer <400>15 caggaagtcagtttgaatttactcagtttaga32
<210>16<211>23<212> DNA <213> Artificial primer <400>16 gtcatttttcaacaggccagcaa23
<210>17<211>25<212> DNA <213> Artificial primer <400>17 atttgcgttgaggaacttgtgacta25
<210>18<211>22<212> DNA <213> Artificial primer <400>18 catccaatgcctcgtaacaacc22
<210>19<211>26<212> DNA <213> Artificial primer <400>19 caaccaaagtctttgttccacctttt26
<210>20<211>27<212> DNA <213> Artificial primer <400>20 aactgaaaggcaaaaattcatcacaca27
<210>21<211>27<212> DNA <213> Artificial primer <400>21 ttaaccacacccttaagatgagctcta27
<210>22<211>26<212> DNA <213> Artificial primer <400>22 gttgtgacatcccttgataaaccttg26
<210>23<211>20<212> DNA <213> Artificial primer <400>23 atccactaggactgctccca20
<210>24<211>29<212> DNA <213> Artificial primer <400>24 tgcaacataagtactaatgtgtggtttga29
<210>25<211>27<212> DNA <213> Artificial primer <400>25 agatatattcctccaattcaggaccca27
<210>26<211>28<212> DNA <213> Artificial primer <400>26 agcaaaacacctgcagatctaatagaaa28
<210>27<211>35<212> DNA <213> Artificial primer <400>27 taacaataaaaacatcaaaaagacattttagccat35
<210>28<211>26<212> DNA <213> Artificial primer <400>28 acagttccagtagtcctactttgaca26
<210>29<211>32<212> DNA <213> Artificial primer <400>29 ggaaggaaagaattttgcttaagatatcagtg32
<210>30<211>30<212> DNA <213> Artificial primer <400>30 agcttctcaaagtatttcattttcttggtg30
<210>31<211>25<212> DNA <213> Artificial primer <400>31 ccctagagtgctaacttccagtaac25
<210>32<211>28<212> DNA <213> Artificial primer <400>32 acaaatgcacctggttcttttactaagt28
<210>33<211>22<212> DNA <213> Artificial primer <400>33 taaggcaggaggactgcttcta22
<210>34<211>35<212> DNA <213> Artificial primer <400>34 gaaaataactctcctgaacatctaaaagatgaagt35
<210>35<211>29<212> DNA <213> Artificial primer <400>35 atcagcaaactgaaaaacctcttcttaca29
<210>36<211>29<212> DNA <213> Artificial primer <400>36 ggaaacatcatctgcttgatccattttag29
<210>37<211>25<212> DNA <213> Artificial primer <400>37 gtggcttcttcatttcagggtatca25
<210>38<211>25<212> DNA <213> Artificial primer <400>38 tcaaagctacagaattctgtgtggt25
<210>39<211>26<212> DNA <213> Artificial primer <400>39 gcaagcaatttgaaggtacagttgaa26
<210>40<211>26<212> DNA <213> Artificial primer <400>40 acttattggatgtacctctgcagaag26
<210>41<211>30<212> DNA <213> Artificial primer <400>41 aacgagaataaatcaaaaatttgccaaacg30
<210>42<211>28<212> DNA <213> Artificial primer <400>42 tgaagtttccaaactaacatcacaaggt28
<210>43<211>35<212> DNA <213> Artificial primer <400>43 ggttgtgctttttaaatttcaattttatttttgct35
<210>44<211>31<212> DNA <213> Artificial primer <400>44 gtcataaaagccatcagtattgtagacaaac31
<210>45<211>20<212> DNA <213> Artificial primer <400>45 ggaaggccatggaatctgct20
<210>46<211>33<212> DNA <213> Artificial primer <400>46 gtggattttgcttctctgatataaactaacttt33
<210>47<211>31<212> DNA <213> Artificial primer <400>47 gttgactttttgcaaatgtttaacataggtg31
<210>48<211>28<212> DNA <213> Artificial primer <400>48 ctgcaaatgctatcgatttcttgatcac28
<210>49<211>24<212> DNA <213> Artificial primer <400>49 gaagacttctgaggctacagtagg24
<210>50<211>21<212> DNA <213> Artificial primer <400>50 agccttcatccggagagtgta21
<210>51<211>27<212> DNA <213> Artificial primer <400>51 agatgatgtcagcaaacctaagaatgt27
<210>52<211>22<212> DNA <213> Artificial primer <400>52 ccagtcctgccaatgagaagaa22
<210>53<211>30<212> DNA <213> Artificial primer <400>53 ggctaggattgacaaattctttaagttcac30
<210>54<211>29<212> DNA <213> Artificial primer <400>54 gcaaattgatagttgttctagcagtgaag29
<210>55<211>23<212> DNA <213> Artificial primer <400>55 cacggtttctgtagcccatactt23
<210>56<211>29<212> DNA <213> Artificial primer <400>56 caggtaaccttaatgcattgtcttaacac29
<210>57<211>32<212> DNA <213> Artificial primer <400>57 aaggaatgttcccaatagtagacataaaagtc32
<210>58<211>23<212> DNA <213> Artificial primer <400>58 atcttctaccaggctcttagcca23
<210>59<211>33<212> DNA <213> Artificial primer <400>59 ggactccttatgtccaaatttaattgataatgg33
<210>60<211>26<212> DNA <213> Artificial primer <400>60 atttgcaaatgtaagtggtgcttcaa26
<210>61<211>30<212> DNA <213> Artificial primer <400>61 agctgtgaaactgtttagtgatattgagaa30
<210>62<211>32<212> DNA <213> Artificial primer <400>62 ggcagcagtatatttgttatcttcattttcag32
<210>63<211>28<212> DNA <213> Artificial primer <400>63 accaaaatatgtctggattggagaaagt28
<210>64<211>32<212> DNA <213> Artificial primer <400>64 acttgcttggtactatcttctatttcagaaaa32
<210>65<211>23<212> DNA <213> Artificial primer <400>65 acagcagactgtggaatgtatgg23
<210>66<211>29<212> DNA <213> Artificial primer <400>66 tgaaaagactctgcatttttgctgttaat29
<210>67<211>27<212> DNA <213> Artificial primer <400>67 acttcttccattgcatctttctcatct27
<210>68<211>33<212> DNA <213> Artificial primer <400>68 gattccataaactaacaagcacttatcaaaact33
<210>69<211>28<212> DNA <213> Artificial primer <400>69 ccagaggaaacctcagaaaaagtagaaa28
<210>70<211>33<212> DNA <213> Artificial primer <400>70 ctcctagaattaaacacacatcacatacataca33
<210>71<211>28<212> DNA <213> Artificial primer <400>71 ccatcgtgggatcttgcttataatactc28
<210>72<211>30<212> DNA <213> Artificial primer <400>72 aaagctcttcctttttgaaagtctgttttt30
<210>73<211>21<212> DNA <213> Artificial primer <400>73 tgacgtcctagctgtgtgaag21
<210>74<211>28<212> DNA <213> Artificial primer <400>74 gacaaggaattggtttcagatgatgaag28
<210>75<211>26<212> DNA <213> Artificial primer <400>75 ctgactggcatttggttgtacttttt26
<210>76<211>29<212> DNA <213> Artificial primer <400>76 tcactcgaaaaagaatctgctttcaaaac29
<210>77<211>24<212> DNA <213> Artificial primer <400>77 acttcctgagttttcatggacagc24
<210>78<211>24<212> DNA <213> Artificial primer <400>78 gaggttttctactgttgctgcatc24
<210>79<211>32<212> DNA <213> Artificial primer <400>79 tacatatgtgttggcattttaaacatcacttg32
<210>80<211>33<212> DNA <213> Artificial primer <400>80 taatcatacctgacttatctctttgtggtgtta33
<210>81<211>28<212> DNA <213> Artificial primer <400>81 gaactaattaactgttcagcccagtttg28
<210>82<211>32<212> DNA <213> Artificial primer <400>82 tgacctgattctaaacactggtaattaaagac32
<210>83<211>30<212> DNA <213> Artificial primer <400>83 tggcacttttgttgaagaaattactgaaaa30
<210>84<211>26<212> DNA <213> Artificial primer <400>84 ctgagtgtttccctccttcataaact26
<210>85<211>28<212> DNA <213> Artificial primer <400>85 ccaggtatcagatgcttcattacaaaac28
<210>86<211>25<212> DNA <213> Artificial primer <400>86 acttgctttccacttgctgtactaa25
<210>87<211>25<212> DNA <213> Artificial primer <400>87 tgtagctgtatacgtatggcgtttc25
<210>88<211>32<212> DNA <213> Artificial primer <400>88 tgagggaatacataaaagttaacacacaatct32
<210>89<211>25<212> DNA <213> Artificial primer <400>89 aacaactaccggtacaaacctttca25
<210>90<211>28<212> DNA <213> Artificial primer <400>90 acaacagaaacgacaaatcctattaggt28
<210>91<211>21<212> DNA <213> Artificial primer <400>91 tccagagccatttccatcctg21
<210>92<211>22<212> DNA <213> Artificial primer <400>92 cccaaactacggacattttcgc22
<210>93<211>29<212> DNA <213> Artificial primer <400>93 acctagcaagaaagaaaatgttgaacatc29
<210>94<211>22<212> DNA <213> Artificial primer <400>94 tctggatttgacggctcctcta22
<210>95<211>21<212> DNA <213> Artificial primer <400>95 aaaatgaagcggcccatctct21
<210>96<211>27<212> DNA <213> Artificial primer <400>96 ctgaatgccttaaatatgacgtgtctg27
<210>97<211>28<212> DNA <213> Artificial primer <400>97 tgctcttcttgattattttcttccaagc28
<210>98<211>26<212> DNA <213> Artificial primer <400>98 agtaaccaggtaatattggcaaaggc26
<210>99<211>31<212> DNA <213> Artificial primer <400>99 gagttccatattgcttatactgctgcttata31
<210>100<211>23<212> DNA <213> Artificial primer <400>100 caggccttcatcctgaggatttt23
<210>101<211>30<212> DNA <213> Artificial primer <400>101 tctgatgaatggttttataggaacgctatg30
<210>102<211>27<212> DNA <213> Artificial primer <400>102 tcttttcatggctatttgccttttgag27
<210>103<211>30<212> DNA <213> Artificial primer <400>103 tctgatgaatggttttataggaacgctatg30
<210>104<211>27<212> DNA <213> Artificial primer <400>104 tcttttcatggctatttgccttttgag27
<210>105<211>32<212> DNA <213> Artificial primer <400>105 tgctttgttttattttagtcctgttgttctac32
<210>106<211>30<212> DNA <213> Artificial primer <400>106 gcaaaggtataacgctattgtcaaattctc30
<210>107<211>32<212> DNA <213> Artificial primer <400>107 tgctttgttttattttagtcctgttgttctac32
<210>108<211>30<212> DNA <213> Artificial primer <400>108 gcaaaggtataacgctattgtcaaattctc30
<210>109<211>33<212> DNA <213> Artificial primer <400>109 aaggcttttcatataatgtggtaaattcatctg33
<210>110<211>24<212> DNA <213> Artificial primer <400>110 ttctgagtttacacagtgctctgg24
<210>111<211>33<212> DNA <213> Artificial primer <400>111 attgatggtactttaattttgtcactttgtgtt33
<210>112<211>22<212> DNA <213> Artificial primer <400>112 ctgcaggcatgacagagaatca22
<210>113<211>32<212> DNA <213> Artificial primer <400>113 gctatttactgatcagcacaacatatgtctta32
<210>114<211>28<212> DNA <213> Artificial primer <400>114 actaatatttttcccacttgcagtctga28
<210>115<211>33<212> DNA <213> Artificial primer <400>115 atgatcttgaacaatgtagtttttgtacagaga33
<210>116<211>20<212> DNA <213> Artificial primer <400>116 agcaccctttctgggcttag20
<210>117<211>29<212> DNA <213> Artificial primer <400>117 atttttagatccagactttcagccatctt29
<210>118<211>27<212> DNA <213> Artificial primer <400>118 aatcagaggttcaaagaggcttacttt27
<210>119<211>28<212> DNA <213> Artificial primer <400>119 cattgaccaccttttattactccagcta28
<210>120<211>33<212> DNA <213> Artificial primer <400>120 aaatcaaagcattcttaccttactacatcatca33
<210>121<211>33<212> DNA <213> Artificial primer <400>121 ttaaattttctttctctaggtgaagctgtactt33
<210>122<211>32<212> DNA <213> Artificial primer <400>122 caagtttattttcatggtgttttatccctctt32
<210>123<211>29<212> DNA <213> Artificial primer <400>123 tggaaaatttgtgcattgttaaggaaagt29
<210>124<211>22<212> DNA <213> Artificial primer <400>124 tcctctgtcattcttcctgtgc22
<210>125<211>28<212> DNA <213> Artificial primer <400>125 agaacattttgtttcctcactaaggtga28
<210>126<211>25<212> DNA <213> Artificial primer <400>126 ggccaagaaattagagtcctcagaa25
<210>127<211>25<212> DNA <213> Artificial primer <400>127 caggagtcttttgaactgccaaatc25
<210>128<211>28<212> DNA <213> Artificial primer <400>128 gtgaaagagttcactccaaatcagtaga28
<210>129<211>33<212> DNA <213> Artificial primer <400>129 aaagtaaagcttctataaagttaggtgtttcct33
<210>130<211>27<212> DNA <213> Artificial primer <400>130 actcagtcataacagctcaaagttgaa27
<210>131<211>23<212> DNA <213> Artificial primer <400>131 tagcgttatacctttgccctgag23
<210>132<211>22<212> DNA <213> Artificial primer <400>132 agtagaactaagggtgggtggt22
<210>133<211>32<212> DNA <213> Artificial primer <400>133 gacaaaatgtatcaaaaatacttcctcgtgtt32
<210>134<211>27<212> DNA <213> Artificial primer <400>134 tcccaaaacatgaatgttctcaacaag27
<210>135<211>33<212> DNA <213> Artificial primer <400>135 aggaaaaactacagttatttattaccccagaag33
<210>136<211>28<212> DNA <213> Artificial primer <400>136 aaataagagtgctggcattttcatgatc28
<210>137<211>32<212> DNA <213> Artificial primer <400>137 actgctactaaaacggagcaaaatataaaaga32
<210>138<211>28<212> DNA <213> Artificial primer <400>138 ctgggacactttctttcagtattttgtg28
<210>139<211>28<212> DNA <213> Artificial primer <400>139 tatggaatgtgcctttcctaaggaattt28
<210>140<211>22<212> DNA <213> Artificial primer <400>140 ctagggatgacaggagaacagc22
<210>141<211>28<212> DNA <213> Artificial primer <400>141 ttgaaattttagcactgtaagcaacagg28
<210>142<211>32<212> DNA <213> Artificial primer <400>142 ggtctatccaaaactttattgccagtaaattg32
<210>143<211>25<212> DNA <213> Artificial primer <400>143 ctgcagaaagacttgaaggcgtata25
<210>144<211>22<212> DNA <213> Artificial primer <400>144 actgtatccccctgaagtccat22
<210>145<211>23<212> DNA <213> Artificial primer <400>145 tggtaactcagactcagcatcag23
<210>146<211>26<212> DNA <213> Artificial primer <400>146 aaactgaggctctttagcttcttagg26
<210>147<211>22<212> DNA <213> Artificial primer <400>147 cagggaagctcttcatcctcac22
<210>148<211>24<212> DNA <213> Artificial primer <400>148 gtcatgcatctcaggtttgttctg24
<210>149<211>30<212> DNA <213> Artificial primer <400>149 ttccgataggttttcccaaatattttgtct30
<210>150<211>29<212> DNA <213> Artificial primer <400>150 aaagttaatgagtggttttccagaagtga29
<210>151<211>31<212> DNA <213> Artificial primer <400>151 agcctaatcttactagacatgtcttttcttc31
<210>152<211>33<212> DNA <213> Artificial primer <400>152 ttctaatgtgttaaagttcattggaacagaaag33
<210>153<211>27<212> DNA <213> Artificial primer <400>153 ggatcctgatatgtcttggtcaagttc27
<210>154<211>22<212> DNA <213> Artificial primer <400>154 cttgacaccactggactaccac22
<210>155<211>29<212> DNA <213> Artificial primer <400>155 aacaagacaaacaacagttggtattagga29
<210>156<211>29<212> DNA <213> Artificial primer <400>156 tctgtcagttcatcatcttccataaaagc29
<210>157<211>28<212> DNA <213> Artificial primer <400>157 cagtgatactgactttcaatcccagaaa28
<210>158<211>28<212> DNA <213> Artificial primer <400>158 tggcaacagctcaacgtttttataattt28
<210>159<211>32<212> DNA <213> Artificial primer <400>159 acaaaatggacattctaagttatgaggaaaca32
<210>160<211>27<212> DNA <213> Artificial primer <400>160 ggtgatttcactagtaccttgctcttt27
<210>161<211>26<212> DNA <213> Artificial primer <400>161 acagtggaattctagagtcacacttc26
<210>162<211>23<212> DNA <213> Artificial primer <400>162 acagcataccacccatctgtaag23
<210>163<211>23<212> DNA <213> Artificial primer <400>163 acttgcccctttcgtctatttgt23
<210>164<211>25<212> DNA <213> Artificial primer <400>164 atgtgtggtgatgctgaaaagtaac25
<210>165<211>29<212> DNA <213> Artificial primer <400>165 ttagctcatttttgttaatggtggctttt29
<210>166<211>29<212> DNA <213> Artificial primer <400>166 ggacttcttgacttaatcggtttaggaat29
<210>167<211>22<212> DNA <213> Artificial primer <400>167 tcgcctcatgtggttttatgca22
<210>168<211>20<212> DNA <213> Artificial primer <400>168 ccaggacacgtgtagaacgt20
<210>169<211>30<212> DNA <213> Artificial primer <400>169 cttcctttatttcaccatcatctaacaggt30
<210>170<211>23<212> DNA <213> Artificial primer <400>170 aacagagggccaaaattgaatgc23
<210>171<211>23<212> DNA <213> Artificial primer <400>171 ccccatcatgtgagtcatcagaa23
<210>172<211>23<212> DNA <213> Artificial primer <400>172 gcttagcaaggagccaacataac23
<210>173<211>31<212> DNA <213> Artificial primer <400>173 agtttattcactgtgttgattgacctttcta31
<210>174<211>31<212> DNA <213> Artificial primer <400>174 tgttagcaatttcaacagtctaatcaatgtc31
<210>175<211>31<212> DNA <213> Artificial primer <400>175 ttcagaaaactactttgaaacagaagcagta31
<210>176<211>23<212> DNA <213> Artificial primer <400>176 agtgattggcaacacgaaaggta23
<210>177<211>29<212> DNA <213> Artificial primer <400>177 ttcccatggaaaagaatcaagatgtatgt29
<210>178<211>30<212> DNA <213> Artificial primer <400>178 tcctttcattagctacttggaagacaaaat30
<210>179<211>29<212> DNA <213> Artificial primer <400>179 caaaggaatctttggacaaagtgaaaaac29
<210>180<211>26<212> DNA <213> Artificial primer <400>180 caccacagtctcaatagaaacaaggt26
<210>181<211>31<212> DNA <213> Artificial primer <400>181 < 181 taaaactagtagtgcagatacccaaaaagtg31
<210>182<211>29<212> DNA <213> Artificial primer <400>182 caatgactgatttttaccaagagtgcaaa29
<210>183<211>25<212> DNA <213> Artificial primer <400>183 ggttttgtaattttgcatcggcatg25
<210>184<211>25<212> DNA <213> Artificial primer <400>184 gccctgaagtacagtctttagttgg25
<210>185<211>35<212> DNA <213> Artificial primer <400>185 cacagcataatatgtgtcacattataaagattcag35
<210>186<211>30<212> DNA <213> Artificial primer <400>186 tatctcactcgataatctggatgactcatt30
<210>187<211>33<212> DNA <213> Artificial primer <400>187 gacaatacctacataaaactctttccagaatgt33
<210>188<211>24<212> DNA <213> Artificial primer <400>188 aagttccccaattgaaagttgcag24
<210>189<211>25<212> DNA <213> Artificial primer <400>189 ttatagacctcaggttgcaaaaccc25
<210>190<211>27<212> DNA <213> Artificial primer <400>190 acctgaaagagaaatgggaaatgagaa27
<210>191<211>27<212> DNA <213> Artificial primer <400>191 ccgcctatcattacatgtttccttact27
<210>192<211>23<212> DNA <213> Artificial primer <400>192 gatttgaacaccactgagaagcg23
<210>193<211>27<212> DNA <213> Artificial primer <400>193 ccgcctatcattacatgtttccttact27
<210>194<211>23<212> DNA <213> Artificial primer <400>194 gatttgaacaccactgagaagcg23
<210>195<211>27<212> DNA <213> Artificial primer <400>195 gggaatcaggctttactagaagaacag27
<210>196<211>26<212> DNA <213> Artificial primer <400>196 ccaatgtggtctttgcagctatttac26
<210>197<211>28<212> DNA <213> Artificial primer <400>197 tcaatccagactctgaagaacttttctc28
<210>198<211>27<212> DNA <213> Artificial primer <400>198 tcctctgcaagaacataaaccaaatct27
<210>199<211>29<212> DNA <213> Artificial primer <400>199 cccaaagtgtaaagaaatgcagaattctc29
<210>200<211>28<212> DNA <213> Artificial primer <400>200 aggctgaattttcaatgactgaataagg28
<210>201<211>34<212> DNA <213> Artificial primer <400>201 aatggtctatagacttttgagaaataaaactgat34
<210>202<211>33<212> DNA <213> Artificial primer <400>202 attatataccatacctatagagggagaacagat33
<210>203<211>32<212> DNA <213> Artificial primer <400>203 tttaaggcagttctagaagaatgaaaactctt32
<210>204<211>32<212> DNA <213> Artificial primer <400>204 atcatacctgtatagggtatgctctttgaata32
<210>205<211>25<212> DNA <213> Artificial primer <400>205 gcatatactgcatgcaaatgatccc25
<210>206<211>31<212> DNA <213> Artificial primer <400>206 agccacataacaaccacattttctaattaag31
<210>207<211>33<212> DNA <213> Artificial primer <400>207 tacctgttaagtttgtatgcaacatttctaaag33
<210>208<211>27<212> DNA <213> Artificial primer <400>208 atatcattacaccagttcgtccctttc27
<210>209<211>22<212> DNA <213> Artificial primer <400>209 gtagtatgagcagcagctggac22
<210>210<211>32<212> DNA <213> Artificial primer <400>210 aacattcatcgttgtgtaaattaaacttctcc32
<210>211<211>29<212> DNA <213> Artificial primer <400>211 taatgttattacggctaattgtgctcact29
<210>212<211>27<212> DNA <213> Artificial primer <400>212 ggctctaggttttgtctatcatctcag27
<210>213<211>27<212> DNA <213> Artificial primer <400>213 gaactaccctgatacttttctggatgc27
<210>214<211>29<212> DNA <213> Artificial primer <400>214 ctctaatatagccagttggttgatttcca29
<210>215<211>27<212> DNA <213> Artificial primer <400>215 gaactaccctgatacttttctggatgc27
<210>216<211>29<212> DNA <213> Artificial primer <400>216 ctctaatatagccagttggttgatttcca29
<210>217<211>29<212> DNA <213> Artificial primer <400>217 actttaacaggatttggaaaaacatcagg29
<210>218<211>24<212> DNA <213> Artificial primer <400>218 acattcatcagcgtttgcttcatg24
<210>219<211>24<212> DNA <213> Artificial primer <400>219 caggtgataaacaagcaacccaag24
<210>220<211>24<212> DNA <213> Artificial primer <400>220 ctgaagctacctccaaaactgtga24
<210>221<211>25<212> DNA <213> Artificial primer <400>221 cagcaaaaagtcctgcaacttgtta25
<210>222<211>28<212> DNA <213> Artificial primer <400>222 tcggagagatgatttttgtcattttcag28
<210>223<211>34<212> DNA <213> Artificial primer <400>223 ttttgtgtatttacagtaacatggatattctctt34
<210>224<211>29<212> DNA <213> Artificial primer <400>224 gggaagtgttaacttcttaacgttagtgt29
<210>225<211>24<212> DNA <213> Artificial primer <400>225 cagtgatggaggaaatgttggttg24
<210>226<211>21<212> DNA <213> Artificial primer <400>226 ggtggctcagctacttgagag21
<210>227<211>26<212> DNA <213> Artificial primer <400>227 ggagggagactgtgtgtaatatttgc26
<210>228<211>28<212> DNA <213> Artificial primer <400>228 ctctttctcccctttacaagactttgaa28
<210>229<211>25<212> DNA <213> Artificial primer <400>229 gaccaatggctaagtgaagatgaca25
<210>230<211>29<212> DNA <213> Artificial primer <400>230 ttccaataaattctcagatccaggaagag29
<210>231<211>28<212> DNA <213> Artificial primer <400>231 aaatcaaagtgtttgttccaatacagca28
<210>232<211>21<212> DNA <213> Artificial primer <400>232 ggcgatggttttctccttcca21
<210>233<211>31<212> DNA <213> Artificial primer <400>233 catgtttatttggagtaatgagtccagtttc31
<210>234<211>26<212> DNA <213> Artificial primer <400>234 tctgggtccttaaagaaacaaagtcc26
<210>235<211>26<212> DNA <213> Artificial primer <400>235 ccaagagattttgtgggttgtaaagg26
<210>236<211>29<212> DNA <213> Artificial primer <400>236 aatgtttgatcttggtcatttgacagttc29
<210>237<211>29<212> DNA <213> Artificial primer <400>237 tacctcagtcacataataaggaatgcatc29
<210>238<211>24<212> DNA <213> Artificial primer <400>238 ggttctaagcaacactgtgacgta24
<210>239<211>32<212> DNA <213> Artificial primer <400>239 atctacaaaaagtaagaactagcaagactagg32
<210>240<211>24<212> DNA <213> Artificial primer <400>240 agggttagttgagaccattcacag24
<210>241<211>22<212> DNA <213> Artificial primer <400>241 acaaatgggcaggactcttagg22
<210>242<211>30<212> DNA <213> Artificial primer <400>242 aactacactactctgtaaatgtgcagatac30
<210>243<211>29<212> DNA <213> Artificial primer <400>243 ggtcaaccagaaagaataaatactgcaga29
<210>244<211>31<212> DNA <213> Artificial primer <400>244 tactggctcaataccagaatcaagtttattt31
<210>245<211>32<212> DNA <213> Artificial primer <400>245 atgtgttatgtgaggtagattgtaaagtcaaa32
<210>246<211>24<212> DNA <213> Artificial primer <400>246 ccaggtgcggtaaaatttggattc24
<210>247<211>35<212> DNA <213> Artificial primer <400>247 ctggcctgatacaattaacttgaatgttatatatg35
<210>248<211>35<212> DNA <213> Artificial primer <400>248 gtctctaagactttgttctcatattagaaataaca35
<210>249<211>22<212> DNA <213> Artificial primer <400>249 agtctgtttccacacctgtctc22
<210>250<211>25<212> DNA <213> Artificial primer <400>250 tggagtcatctgaggagaattcagt25
<210>251<211>21<212> DNA <213> Artificial primer <400>251 aatggctacgacccagttacc21
<210>252<211>29<212> DNA <213> Artificial primer <400>252 tccaatacatggaaggatgagaatttcaa29
<210>253<211>27<212> DNA <213> Artificial primer <400>253 tgtcagataccacagcatctttacatt27
<210>254<211>33<212> DNA <213> Artificial primer <400>254 tgaattatcactatcagaacaaagcagtaaagt33
<210>255<211>27<212> DNA <213> Artificial primer <400>255 tgtcagataccacagcatctttacatt27
<210>256<211>33<212> DNA <213> Artificial primer <400>256 tgaattatcactatcagaacaaagcagtaaagt33
<210>257<211>30<212> DNA <213> Artificial primer <400>257 ccttgattttcttccttttgttcacattca30
<210>258<211>25<212> DNA <213> Artificial primer <400>258 tccattgggacatgaagttaaccac25
<210>259<211>28<212> DNA <213> Artificial primer <400>259 aggaaaataccagcttcatagacaaagg28
<210>260<211>33<212> DNA <213> Artificial primer <400>260 ctttaaccacttctctgtattacatactagctt33
<210>261<211>31<212> DNA <213> Artificial primer <400>261 tttttccttatgatctttaactgttctgggt31
<210>262<211>28<212> DNA <213> Artificial primer <400>262 gctgattataagatggtttcctttgtgg28
<210>263<211>26<212> DNA <213> Artificial primer <400>263 agtgacaaaatctccaaggaagttgt26
<210>264<211>26<212> DNA <213> Artificial primer <400>264 ccactgtttcctcatttaatggcttc26
<210>265<211>27<212> DNA <213> Artificial primer <400>265 taatcaaaagaaactgagcaagcctca27
<210>266<211>29<212> DNA <213> Artificial primer <400>266 gcacttcaaatgtactcttctgcaatatg29
<210>267<211>28<212> DNA <213> Artificial primer <400>267 agctattcctaccattctgatgaggtat28
<210>268<211>27<212> DNA <213> Artificial primer <400>268 tgcatttttatttttgcagggtgaaga27
<210>269<211>22<212> DNA <213> Artificial primer <400>269 attgcagcacaactaaggaacg22
<210>270<211>26<212> DNA <213> Artificial primer <400>270 acacactgttcaactctgtgaaaatg26
<210>271<211>27<212> DNA <213> Artificial primer <400>271 tgggtgttttatgcttggttctttagt27
<210>272<211>26<212> DNA <213> Artificial primer <400>272 gagagtctaaaacagcttctcacctt26
<210>273<211>22<212> DNA <213> Artificial primer <400>273 gtggcaccaaatacgaaacacc22
<210>274<211>26<212> DNA <213> Artificial primer <400>274 acaacgtcgtttcagtctgagataat26
<210>275<211>29<212> DNA <213> Artificial primer <400>275 tgatttgcttgagatcaagattgcagata29
<210>276<211>27<212> DNA <213> Artificial primer <400>276 gagggaactcaaagtacatgaacttgt27
Claims (10)
1. A multiplex PCR primer set for genetic tumor germline mutations, the multiplex PCR primer set comprising at least seq id No.: 1 to 276.
2. The set of multiplex PCR primer sequences according to claim 1, wherein: the multiplex PCR primer group also comprises a primer group related to the following genetic diseases:
ADH1B、AIP、ALDH2、ALK、APC、ARID1A、ATM、AXIN2、BAP1、BARD1、BLM、BMPR1A、BRCA1、BRCA2、BRIP1、BUB1、BUB1B、BUB3、CDC73、CDH1、CDK4、CDKN1C、CDKN2A、CEBPA、CEP57、CHEK2、CTNNB1、CYLD、DDB2、DICER1、DIS3L2、EGFR、EPCAM、EPHB2、ERBB4、ERCC1、ERCC2、ERCC3、ERCC4、ERCC5、EXT1、EXT2、EZH2、FANCA、FANCB、FANCC、FANCD2、FANCE、FANCF、FANCG、FANCI、FANCL、FANCM、FH、FLCN、FOXP1、GATA2、GPC3、HDAC9、HNF1A、HOXB13、HRAS、KIT、KLLN、KRAS、LZTR1、MAX、MEN1、MET、MITF、MLH1、MLH3、MPL、MRE11、MRE11A、MSH2、MSH3、MSH6、MUTYH、NAT1、NBN、NF1、NF2、NSD1、PALB2、PBRM1、PDE11A、PHOX2B、PIK3CA、PMS1、PMS2、POLE、POLH、PRF1、PRKAR1A、PRSS1、PTCH1、PTEN、PTPN11、RAD50、RAD51、RAD51C、RAD51D、RB1、RECQL4、RET、RHBDF2、RUNX1、SBDS、SDHA、SDHAF2、SDHB、SDHC、SDHD、SETD2、SLX4、SMAD4、SMARCB1、SPINK1、STK11、SUFU、TGFBR2、TMEM127、TP53、TSC1、TSC2、TSHR、VHL、WRN、WT1、XPA、XPC。
3. a multiplex PCR kit for genetic tumor germline mutation characterized by: the multiplex PCR primer set used therefor is as set forth in claim 1 or 2.
4. A method of multiplex PCR, comprising: the multiplex PCR primer set used therefor is as set forth in claim 1 or 2.
5. The method of claim 4, wherein: in the PCR reaction system, the initial concentration of dNTP was 0.3mM, Mg2+At a concentration of 2.5mM per 50. mu.L reactionThe polymerase in the system was 5U.
6. The method according to claim 4 or 5, characterized in that: in the PCR reaction system, the concentration of the primer was 0.15. mu.M.
7. The method according to claim 4 or 5, characterized in that: the PCR amplification system is as follows:
。
8. The method of claim 7, wherein: the reaction conditions for PCR amplification are as follows:
9. the method according to any one of claims 4 to 8, wherein: further comprising purification of the PCR product, said purification comprising:
1) adding 9 mu L of AMPure XP magnetic beads into each tube in a system after the PCR reaction is finished, uniformly mixing, and standing;
2) adsorbing the magnetic beads until the solution is clarified, and sucking the supernatant into a new 0.2ml PCR tube;
3) adding 15 mu L of magnetic beads into the new PCR tube, uniformly mixing, standing, adsorbing the magnetic beads until the solution is clarified, discarding the supernatant, and leaving the magnetic beads;
4) adding 100 mu L of 70% ethanol into a PCR tube, fully suspending magnetic beads, adsorbing the magnetic beads until the solution is clarified, discarding supernatant, retaining the magnetic beads, and volatilizing the ethanol;
5) adding 15 mu L of eluent, fully suspending the magnetic beads, and eluting the DNA, wherein the eluent is 10mM Tris-HCl, the pH value is 7.5-8.0, and the temperature is 55-65 ℃.
10. The method of claim 9, wherein: the temperature of the eluent was 60 ℃.
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