CN107083442B - BRCA1/2 gene variation combined detection kit and application thereof - Google Patents

BRCA1/2 gene variation combined detection kit and application thereof Download PDF

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CN107083442B
CN107083442B CN201710447510.7A CN201710447510A CN107083442B CN 107083442 B CN107083442 B CN 107083442B CN 201710447510 A CN201710447510 A CN 201710447510A CN 107083442 B CN107083442 B CN 107083442B
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CN107083442A (en
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朱凯
陈悦科
陈成
刘璐
吴燕灵
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Zhejiang Shaoxing Dingjing Biomedical Technology Co ltd
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上海鼎晶生物医药科技股份有限公司
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Abstract

The invention provides a BRCA1/2 gene variation combined detection kit, and belongs to the field of gene variation detection. The kit comprises the following reagents: BRCA1 gene primer group, BRCA2 gene primer group, polymerase, PCR reaction liquid, digestive enzyme, ligase buffer, adapter, fluorescent probe, Taq enzyme for qPCR reaction, qPCR primer and qPCR reaction liquid. The combined detection kit provided by the invention can simultaneously detect mutation sites of BRCA1/2 genes of breast cancer, so that the sensitivity of an experimental result is high, the mutation rate of less than 5% can be detected, the detection sensitivity is greatly improved, the problems in the prior art are overcome, and meanwhile, the whole experimental process meets the requirement of large-batch and rapid detection.

Description

BRCA1/2 gene variation combined detection kit and application thereof
Technical Field
The invention belongs to the technical field of gene variation detection, and particularly relates to a BRCA1/2 gene variation combined detection kit and application thereof.
Background
In 1990, researchers discovered a gene closely related to hereditary breast cancer, named as breast cancer gene No. 1 (BRCA1), and located on chromosome 17; after 4 years, researchers have found another gene associated with breast cancer on chromosome 13, called BRCA2, both of which are commonly referred to collectively as BRCA 1/2.
BRCA is a cancer suppressor gene, and plays an important role in regulating cell replication, DNA damage repair and normal cell growth; if the BRCA gene is mutated, the function of inhibiting tumorigenesis is lost. The BRCA mutation types are hundreds of types, and are associated with the occurrence of many cancers in humans, the most closely related being breast cancer, followed by ovarian cancer.
Breast cancer is one of the common malignant tumors of women, the incidence rate of which is the first female tumor, and about 50 ten thousand women die of breast cancer every year worldwide. Breast cancer is the result of multifactorial, polygenic and environmental co-action, and its familial inheritance has long been recognized. Studies have found that 5% to 10% of breast cancer patients have familial aggregative and genetic predisposition. Numerous studies have shown that families with the germline BRCA1 mutation exhibit a higher susceptibility to breast cancer: the female has a lifetime risk of breast cancer of 80% to 90% and the age of the patient is significantly reduced. The risk of breast cancer in carriers of BRCA2 gene mutation is similar to that of BRCA1 mutant, and is also 80% -90%.
Presumably, about 25 to 50 million carriers of BRCA1/2 gene mutation are among more than 3 hundred million Americans. The BRCA gene mutation carrying rate in the general population is 1/400-600, while in some specific populations the BRCA mutation carrying rate is as high as 1/40-50, such as German Uygur, Iceland and Fancao. The BRCA1/2 gene mutation carrying situation in Chinese population is not clear, and is generally considered to be lower than that in the United states and northern Europe countries.
According to the BIC database (Breast Cancer Information Core, http:// research. nhgri. nih. gov/BIC /), the currently reported types of mutations of BRCA1 and BRCA2 genes include nonsense mutations, frameshift mutations, non-frameshift insertions, non-frameshift deletions, missense mutations, synonymous mutations, splice site mutations, and deletion or rearrangement of large fragments, and these mutations are spread over nearly 4000 mutation sites, and there is no evidence that there are regions of mutation hotspots, and only a few relatively high frequency mutation sites. Although the BRCA1/2 gene has variation and cannot be judged to have breast cancer, the detection of the BRCA gene of the breast cancer can indicate that individuals have risks of breast cancer and ovarian cancer, and the physical conditions of the individuals are known in advance. The targeted professional prevention is carried out on high-risk individuals, for example, the intervention is carried out by taking some targeted medicines, and the preventive stripping/removing operation is carried out. And carrying out accurate treatment on the diagnosed cancer. At present, Sanger sequencing, qPCR (quantitative polymerase chain reaction) or MLPA (MLPA) are mainly used as methods for detecting BRCA1/2 gene mutation, but the methods have low sequencing flux and low sensitivity, and need a large amount of time and are not expensive.
Disclosure of Invention
In view of this, the invention aims to provide a BRCA1/2 gene mutation joint detection kit, which can simultaneously detect different mutation sites of embryonic lines BRCA1 and BRCA2 genes in a blood sample, and has the characteristics of high detection sensitivity and good repeatability.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a BRCA1/2 gene variation combined detection kit, which comprises the following reagents: BRCA1 gene primer group, BRCA2 gene primer group, polymerase, PCR reaction liquid, digestive enzyme, ligase buffer solution, a connecting joint, a fluorescent probe, Taq enzyme for qPCR reaction, qPCR primer and qPCR reaction liquid;
the BRCA1 gene primer group comprises 52 pairs of primers, and the nucleic acid sequence of the BRCA1 gene primer is shown as Seq ID No 1-Seq ID No 104;
the BRCA2 gene primer group comprises 84 pairs of primers; the nucleic acid sequences of the primers for BRCA2 gene are shown in Seq ID No105 to Seq ID No 272.
Preferably, the nucleotide sequence of the qPCR primer is shown in Seq ID No273 to Seq ID No 274.
Preferably, the nucleotide sequence of the fluorescent probe is shown in Seq ID No 275.
Preferably, the nucleotide sequence of the linker is shown in Seq ID No276 to Seq ID No 286.
Preferably, the digestive enzyme is phosphodiesterase; the enzyme activity of the phosphodiesterase is 2U/mu L-8U/mu L.
Preferably, the ligase is T4 ligase; the enzyme activity of the T4 ligase is 0.5U/mu L-2U/mu L.
Preferably, the PCR reaction solution and the qPCR reaction solution comprise the following components in content: a 5 Xamplification enzyme reaction solution containing a dNTP mixture at a molar concentration of 10 mmol/L; the 5 Xamplification enzyme reaction solution comprises Tris-HCl with the molar concentration of 25 mmol/L-75 mmol/L, KCl with the molar concentration of 25 mmol/L-75 mmol/L and Mg with the molar concentration of 5 mmol/L-10 mmol/L2+And glycerol with the mass concentration of 25-70 percent.
Preferably, the enzyme activity of the polymerase is 2.5U/. mu.L to 7U/. mu.L.
Preferably, the ligase buffer comprises the following components in amounts: a 5 Xamplification enzyme reaction solution containing a dNTP mixture at a molar concentration of 10 mmol/L; the 5 Xamplification enzyme reaction solution contains 25 mmol/L-75 mmol/L LTris-HCl and 25 mmol/L-75 mmol/L Mg2+2.5 mmol/L-7.5 mmol/L ATP, 2.5 mmol/L-7.5 mmol/L LDTT and polyethylene glycol with mass concentration of 15-35%.
The invention also provides application of the kit in detection of BRCA1 and/or BRCA2 gene mutation.
The invention provides a BRCA1/2 gene variation combined detection kit, which comprises the following reagents: BRCA1 gene primer group, BRCA2 gene primer group, polymerase, PCR reaction liquid, digestive enzyme, ligase buffer solution, a connecting joint, a fluorescent probe, Taq enzyme for qPCR reaction, qPCR primer and qPCR reaction liquid; the BRCA1 gene primer group comprises 52 pairs of primers, and the nucleic acid sequence of the BRCA1 gene primer is shown as Seq ID No 1-Seq ID No 104; the BRCA2 gene primer group comprises 84 pairs of primers; the nucleic acid sequences of the primers for BRCA2 gene are shown in Seq ID No105 to Seq ID No 273. The combined detection kit provided by the invention can simultaneously detect mutation sites of two driving genes of breast cancer, including a plurality of mutation sites of BRCA1 and BRCA2 genes, so that the sensitivity of an experimental result is high, the mutation rate of less than 5% can be detected, the detection sensitivity is greatly improved, and the problems in the prior art are overcome.
Meanwhile, the kit provided by the invention can simultaneously detect dozens of or even hundreds of samples by one-time detection through the application of the connecting joint, so that the whole experimental process is simple and rapid, and the cost is low; in addition, the kit has low demand for each sample, and each sample only needs 10-100 ng of DNA. The kit provided by the invention provides an effective means for detecting the driving mutant gene.
Drawings
FIG. 1 is a flow chart of library construction according to the present invention;
FIG. 2 shows the result of Ion Torrent Proton sequencing in example 1.
Detailed Description
The invention provides a BRCA1/2 gene variation combined detection kit, which comprises the following parts: BRCA1 gene primer group, BRCA2 gene primer group, polymerase, PCR reaction liquid, digestive enzyme, ligase buffer solution, a connecting joint, a fluorescent probe, Taq enzyme for qPCR reaction, qPCR primer and qPCR reaction liquid;
the BRCA1 gene primer group comprises 52 pairs of primers, and the nucleic acid sequence of the BRCA1 gene primer is shown as Seq ID No 1-Seq ID No 104;
the BRCA2 gene primer group comprises 84 pairs of primers; the nucleic acid sequences of the BRCA2 gene primers are shown in Seq ID No105 to Seq ID No 272.
The sources of the forward sequence and the reverse sequence of each gene primer set were synthesized by Thermo Fisher. The molar concentration of the primer is preferably 2-10 mu mol/L, and more preferably 5 mu mol/L. The volume of the primer is preferably 5-20 mu L, and more preferably 10 mu L.
In the present invention, the nucleotide sequences of the forward and reverse primers of qPCR are preferably as shown in Seq ID No273 to Seq ID No 274. The sources of the qPCR forward and reverse primers were entrusted to nucleotide synthesis. The molar concentration of the forward primer or the reverse primer is preferably 5-20 mu mol/L, and more preferably 10 mu mol/L. The volume of the forward primer or the reverse primer is preferably 5-20 mu L, and more preferably 10 mu L.
In the present invention, the nucleotide sequence of the fluorescent probe is preferably as shown in Seq ID No 275. The molar concentration of the DNA probe is preferably 2.5-10 mu mol/L, and more preferably 5 mu mol/L. The volume of the DNA probe is preferably 2.5-10 mu L, and more preferably 5 mu L. The fluorescent probe was obtained from a source entrusted to Thermo Fisher corporation.
In the present invention, the nucleotide sequence of the linker is preferably represented by Seq ID No276 to Seq ID No 286. Each type of the connecting joint corresponds to one sample to be detected, so that the detection results of different samples are distinguished through different connecting joints. The molar concentration of the connecting joint is preferably 5-20 mu mol/L, and more preferably 10 mu mol/L. The volume of the connecting joint is preferably 2-20 mu L, and more preferably 10 mu L.
In the present invention, the digestive enzyme is preferably phosphodiesterase; the enzyme activity of the phosphodiesterase is preferably 2U/mu L-8U/mu L, and more preferably 5U/mu L. The source of the phosphodiesterase is not particularly limited in the present invention, and a source of the phosphodiesterase known to those skilled in the art may be used. In the examples of the present invention, the phosphodiesterase is purchased from sigma.
In the invention, the ligase is T4 ligase; the enzyme activity of the T4 ligase is preferably 0.5U/mu L-2U/mu L, and more preferably 1U/mu L. The source of the ligase in the present invention is not particularly limited, and any ligase known to those skilled in the art may be used. In the examples of the present invention, the ligase was purchased from Thermo Fisher.
In the present invention, the PCR reaction solution preferably contains the following components in amounts: 10mM of a 5 Xamplification enzyme reaction mixture of dNTP; the 5 x amplification enzyme reaction solution comprises the following components in percentage by weight: 25 mmol/L-75 mmol/L Tris-HCl, 25 mmol/L-75 mmol/L KCl, 5 mmol/L-10 mmol/L Mg2+And 25 to 70 mass percent of glycerol; more preferably:
Figure BDA0001321589120000041
Figure BDA0001321589120000051
the volume of the PCR reaction solution is preferably 10-100. mu.L, and more preferably 50. mu.L.
In the present invention, the qPCR reaction solution preferably comprises the following components: 10mmol/L of a 5 Xamplification enzyme reaction solution of dNTP mixture; the 5 x amplification enzyme reaction solution comprises the following components in percentage by weight: 25 mmol/L-75 mmol/L Tris-HCl, 25 mmol/L-75 mmol/L KCl, 5 mmol/L-10 mmol/L Mg2+And 25 to 70 mass percent of glycerol; more preferably:
composition (I) Concentration of
Tris-HCl(pH8.2) 50mmol/L
KCl 50mmol/L
MgCl2 8mmol/L
Glycerol
50％
The volume of the qPCR reaction solution is preferably 10-100 mu L, and more preferably 25 mu L.
In the present invention, the source of Taq enzyme for the qPCR reaction was purchased from Thermo Fisher.
In the present invention, the enzyme activity of the polymerase is preferably 5U/. mu.L. The volume of the polymerase is preferably 10. mu.L. The source of the polymerase is not particularly limited in the present invention, and any source of polymerase known to those skilled in the art may be used. In the examples of the present invention, the polymerase was purchased from Thermo Fisher.
In the invention, the ligase buffer solution mainly comprises the following components: 25-75 mmol/L Tris-HCl, 25-75 mmol/L Mg2+2.5 mmol/L-7.5 mmol/L ATP, 2.5 mmol/L-7.5 mmol/L Dithiothreitol (DTT) and polyethylene glycol with mass concentration of 15% -35%; more preferably:
composition (I) Concentration of
Tris-HCl(pH7.6) 50mmol/L
MgCl2 50mmol/L
ATP 5mmol/L
DTT 5mmol/L
Polyethylene glycol 25％(w/v)
The invention also provides application of the kit in detection of BRCA1 and/or BRCA2 gene mutation.
In the present invention, the detection method of the kit preferably comprises the following steps:
(1) amplifying the DNA of a sample to be detected by using the BRCA1 primer group or the BRCA2 primer group in the kit in the technical scheme to obtain a PCR amplification product;
(2) mixing the PCR amplification product obtained in the step (1) with digestive enzyme, and shearing primers at two ends of a target fragment to obtain a blunt-end DNA fragment;
(3) mixing and connecting the blunt-end DNA fragment obtained in the step (2) with a connecting joint, ligase and a ligase buffer solution to obtain a DNA fragment connected with the joint;
(4) purifying the DNA fragment obtained in the step (3) by using a magnetic bead purification kit according to an operation instruction to obtain a detection library;
(5) carrying out quantitative detection on the detection library obtained in the step (4) by using a fluorescent quantitative PCR method;
(6) mixing the quantitative libraries prepared from different samples, connecting ion sphere particles, performing water-in-oil PCR on the library connected with the ion sphere particles on an OT2 instrument, and sequencing the obtained template to obtain a sequencing result;
(7) and (3) comparing the sequence obtained in the step (6) after screening with a database hg19 of a human gene library to obtain a mutation site, and annotating the mutation site by combining bioinformatics analysis software to determine the mutant gene.
The invention uses the primer group in the kit of the technical proposal to carry out PCR amplification on the DNA of a sample to be detected.
In the invention, the amplification system of the PCR amplification is as follows:
Figure BDA0001321589120000061
in the invention, the amplification procedure of the PCR amplification is as follows:
Figure BDA0001321589120000062
Figure BDA0001321589120000071
after the PCR amplification is finished, two groups of amplification products of the same template DNA sample are mixed.
In the present invention, after obtaining the PCR amplification product, agarose gel electrophoresis is preferably performed to detect the amplification condition. The method of agarose gel electrophoresis is not particularly limited, and may be performed using a protocol of agarose gel electrophoresis well known to those skilled in the art.
After obtaining the PCR product, the invention adds digestive enzyme into the PCR product, and carries out shearing treatment on the primers at two ends of the target fragment to obtain the blunt-end DNA fragment.
In the invention, the addition volume of the digestive enzyme is preferably 0.08-0.15 times, and more preferably 0.1 times of the volume of the PCR product.
In the present invention, the procedure for the digestion is as follows:
temperature of Time of day
50℃ 10min
55℃ 15min
60℃ 20min
After the blunt-end DNA fragment is obtained, the blunt-end DNA fragment, the linker, the ligase and the ligase buffer are mixed and ligated to obtain the linker-ligated DNA fragment.
In the invention, the mixing ratio of the blunt-ended DNA fragment, the ligation linker, the ligase and the ligase buffer is as follows:
components Volume of
5 Xligase buffer 4μL
Ligase (Ligase) 2μL
Connecting joint (100pmol/L) 2μL
Digestion products 22μL
Total volume 30μL
The method of the connection is not particularly limited as long as the connection scheme known to those skilled in the art is employed. In the present invention, the ligation is preferably performed in a PCR apparatus. The procedure for the ligation is as follows:
Figure BDA0001321589120000072
Figure BDA0001321589120000081
after the DNA fragment connected with the joint is obtained, the magnetic bead purification kit is used for purifying the DNA fragment according to the operation instruction to obtain the detection library.
In the present invention, the magnetic bead purification kit is purchased from Beckmann corporation.
After obtaining the detection library, the invention uses the fluorescence quantitative PCR method to carry out quantitative detection on the detection library.
In the invention, the reaction volume of the fluorescent quantitative PCR is as follows:
components Volume of
Taq enzyme for qPCR 1μL
5 XqPCR buffer 5μL
qPCR primer (100pmol/L) 2μL
Fluorescent probe (100pmol/L) 2μL
Form panel 4μL
ddH2O Make up to 20 mu L
In the invention, the reaction procedure of the fluorescent quantitative PCR is as follows:
Figure BDA0001321589120000082
after obtaining the quantitative library, the invention mixes the quantitative libraries prepared by different samples, connects ion sphere particles, carries out water-in-oil PCR on the library connected with the ion sphere particles on an OT2 instrument, and carries out sequencing on the obtained template to obtain a sequencing result.
According to the invention, the concentration of the library is obtained according to the qPCR result, the prepared library is diluted to the concentration of 100pM, the libraries of different samples are mixed in equal volume, and then water-in-oil PCR is carried out on a matched instrument Ion OneTouch2 and an ES instrument, so as to enrich PCR products.
In the present invention, it is preferable to use the IonOneTouch200template Kit v2 Kit manufactured by Thermo Fisher.
After the sequencing result is obtained, the invention compares the sequence of the screened sequencing result with the database hg19 of the human gene library to obtain a mutation site, and annotates the mutation site by combining with bioinformatics analysis software, thereby determining the mutant gene.
In the invention, the screening method is to use default quality control parameters carried by an Ion torrent Proton instrument to remove the polyclonal data and the low-quality data.
In the invention, the comparison preferably adopts a server carried by an Ion torrent Proton instrument to compare the sequence data after screening with the sequence of the hg19 database of the human gene library so as to obtain the mutation sites.
According to the invention, when the sequencing depth data obtained after sequencing is more than 500% and the coverage data reaches more than 90%, the sequencing quality reliability is high, and the obtained base variation condition obtained after comparison is proved.
In the invention, each sample of the sequencing depth is more than 3 thousands, and the coverage of sequencing reads is more than 90%.
In the present invention, the mutation sites to be detected are annotated in combination with bioinformatics analysis software. The annotation process is preferably performed with reference to a database of alignments. The databases for the reference alignments are preferably the clinvar database and the REFSEQ database.
The BRCA1/2 gene variation combined detection kit and the application thereof provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The HORIZON BRCA solar Multiplex Reference Standard was used for the combined detection of multiple gene-driven mutations.
1. The gDNA was diluted according to the Kit and method of the present invention and the diluted gDNA was accurately quantified using a qubit dsDNA HS Assay Kit quantification Kit to a DNA concentration of about 3 ng/uL.
A. Amplifying target DNA;
a PCR system was prepared for the amplification of the target fragment with the following composition.
Figure BDA0001321589120000091
Figure BDA0001321589120000101
PCR amplification was performed under the following amplification conditions:
Figure BDA0001321589120000102
after the PCR amplification is finished, two groups of amplification products of the same template DNA sample are mixed.
B. Partial digestion of primer sequences:
to the amplified product, 2. mu.L of a digesting enzyme was added, and the digestion reaction was carried out under the following conditions. Can be carried out in a PCR instrument.
Temperature of Time of day
50℃ 10min
55℃ 15min
60℃ 20min
C. Connecting a joint:
adding a linker sequence and a ligase system into the system after digestion. The system is as follows:
components Volume of
5 Xligase buffer 4μL
Ligase (Ligase) 2μL
Connecting joint (100pmol/L) 2μL
Digestion products 22μL
Total volume 30μL
The reaction conditions were as follows, and the reaction was carried out in a PCR apparatus:
temperature of Time of day
22℃ 30min
68℃ 2min
72℃ 5min
D. And (3) cDNA purification:
DNA was purified using the magnetic beads of the present invention (available from Beckmann). Adsorbing a target DNA fragment by using magnetic beads, washing DNA for 1 time by using 75% alcohol on a magnetic frame, and then eluting by using a Tris-EDTA solution prepared in a kit to obtain a purified library required by people.
E. Library quantification:
and (3) carrying out fluorescent quantitative PCR quantification on the library obtained in the step. Using reference substance as control, making standard curve, diluting each library by 100 times, and configuring reaction solution according to the following system:
components Volume of
Taq enzyme for qPCR 1μL
5 XqPCR buffer 5μL
qPCR primer (100pmol/L) 2μL
Fluorescent probe (100pmol/L) 2μL
Form panel 4μL
ddH2O Make up to 20 mu L
qPCR was performed according to the following reaction program:
Figure BDA0001321589120000111
according to the results, the library was diluted to a concentration of 100pM and the libraries were mixed in equal volumes, by which time library preparation was complete.
2. On an Ion OneTouch2 experiment platform, water-in-oil PCR was performed with an Ion OneTouch200template Kit v2 Kit, and PCR products were enriched. The operation was performed according to the instructions for the use of the instrument.
3. The enriched PCR product can be subjected to sample treatment according to the Ion Proton200sequencing Kit specification, and is loaded to a P1 chip matched with a sequencer, and the Ion torent Proton is subjected to high-throughput sequencing.
4. And (6) analyzing results.
And screening the sequencing result, and removing the polyclonal data and the low-quality data in a default quality control link of a program carried by the instrument. The obtained data are compared with the database of the human gene library hg19 by the server of the Ion torrent platform (as shown in fig. 2), and the coincidence rate reaches 95%. A sequencing depth data map (shown in Table 1) and a coverage data map (shown in Table 1) as well as base variation (shown in Table 2) are obtained, wherein the sequencing depth is more than 3 ten thousand for each sample, and the coverage of sequencing reads is more than 90%. And (3) annotating the mutation sites by combining bioinformatics analysis software on the detected mutation, wherein databases mainly used for comparison are a clinvar database and a REFSEQ database.
TABLE 1 sequencing depth data and coverage data results for samples tested in example 1
Sample(s) Number of detected repeats Mean depth of sequencing Coverage degree
Standard article 2,000,851 8,186 98.31％
The results of the combined detection of gene mutations are shown in Table 2.
TABLE 2 base mutation in the present invention
Chromosome Position of Name of Gene Amino acid mutations Ref (Ref) Mutations
17 41223094 BRCA1 S1613G A G
17 41244000 BRCA1 K1183R A G
17 41245090 BRCA1 K820E G A
17 41234451 BRCA1 R1443Stop G A
17 41246245 BRCA1 D435Y G T
17 41244936 BRCA1 P871L C T
13 32906480 BRCA2 N289H A C
13 32929387 BRCA2 V2466A C T
13 32911463 BRCA2 N991D A G
13 32913558 BRCA2 K1691fs / DelA
13 32913836 BRCA2 N1784fs / DelA
13 32912750 BRCA2 D1420Y G T
13 32937354 BRCA2 I2675fs / InsA
As can be seen from Table 2, in one sequencing experiment, multiple mutation sites of different gene mutations including BRCA1 and BRCA2 can be detected simultaneously, and the operation process is simple and short in time consumption.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai Ding Jing biomedical science and technology Co., Ltd
<120> BRCA1/2 gene variation combined detection kit and application thereof
<130>2017
<160>286
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<213> Artificial sequence
<400>14
caattggtgg cgatggtttt ctc 23
<210>15
<211>27
<212>DNA
<213> Artificial sequence
<400>15
agatgatgtc agcaaaccta agaatgt 27
<210>16
<211>22
<212>DNA
<213> Artificial sequence
<400>16
ccagtcctgc caatgagaag aa 22
<210>17
<211>23
<212>DNA
<213> Artificial sequence
<400>17
gtaactcaga ctcagcatca gca 23
<210>18
<211>27
<212>DNA
<213> Artificial sequence
<400>18
aaaactgagg ctctttagct tcttagg 27
<210>19
<211>29
<212>DNA
<213> Artificial sequence
<400>19
gcctaccaca aatacaaatt atgaccaag 29
<210>20
<211>24
<212>DNA
<213> Artificial sequence
<400>20
ttctcttcag gaggaaaagc acag 24
<210>21
<211>22
<212>DNA
<213> Artificial sequence
<400>21
cccatctgtc agcttcggaa at 22
<210>22
<211>30
<212>DNA
<213> Artificial sequence
<400>22
gggaattaca gataaattaa aactgcgact 30
<210>23
<211>28
<212>DNA
<213> Artificial sequence
<400>23
gagacttcaa gcagaaaatc tttaaggg 28
<210>24
<211>23
<212>DNA
<213> Artificial sequence
<400>24
ggacagtgta gcactctacc agt 23
<210>25
<211>27
<212>DNA
<213> Artificial sequence
<400>25
atttggagta atgagtccag tttcgtt 27
<210>26
<211>26
<212>DNA
<213> Artificial sequence
<400>26
agaagaggaa tgtgcaacat tctctg 26
<210>27
<211>22
<212>DNA
<213> Artificial sequence
<400>27
ggaagcaggg aagctcttca tc 22
<210>28
<211>24
<212>DNA
<213> Artificial sequence
<400>28
tcatgcatct caggtttgtt ctga 24
<210>29
<211>33
<212>DNA
<213> Artificial sequence
<400>29
attaaattac acagaactgt gattgttttc tag 33
<210>30
<211>26
<212>DNA
<213> Artificial sequence
<400>30
gctcatacta ctgatactgc tgggta 26
<210>31
<211>28
<212>DNA
<213> Artificial sequence
<400>31
tccaatacct aagtttgaat ccatgctt 28
<210>32
<211>23
<212>DNA
<213> Artificial sequence
<400>32
caaaggcatc tcaggaacat cac23
<210>33
<211>28
<212>DNA
<213> Artificial sequence
<400>33
tccaggaaga ctttgtttat agacctca 28
<210>34
<211>30
<212>DNA
<213> Artificial sequence
<400>34
ctgaaagaga aatgggaaat gagaacattc 30
<210>35
<211>29
<212>DNA
<213> Artificial sequence
<400>35
tggatactta aagccttctg tgtcatttc 29
<210>36
<211>28
<212>DNA
<213> Artificial sequence
<400>36
cccaaagatc tcatgttaag tggagaaa 28
<210>37
<211>28
<212>DNA
<213> Artificial sequence
<400>37
attcgagttc catattgctt atactgct 28
<210>38
<211>30
<212>DNA
<213> Artificial sequence
<400>38
tcctgaggat tttatcaaga aagcagattt 30
<210>39
<211>29
<212>DNA
<213> Artificial sequence
<400>39
ccttcttccg ataggttttc ccaaatatt 29
<210>40
<211>29
<212>DNA
<213> Artificial sequence
<400>40
agaaagttaa tgagtggttt tccagaagt 29
<210>41
<211>22
<212>DNA
<213> Artificial sequence
<400>41
gctgggagtc cgcctatcat ta 22
<210>42
<211>25
<212>DNA
<213> Artificial sequence
<400>42
gttctgtttc aaacttgcat gtgga 25
<210>43
<211>25
<212>DNA
<213> Artificial sequence
<400>43
tgttccactc caacatttga gaact 25
<210>44
<211>30
<212>DNA
<213> Artificial sequence
<400>44
gagcaagatg ctgattcatt atttatcagc 30
<210>45
<211>23
<212>DNA
<213> Artificial sequence
<400>45
gggagactga agcacagtga aaa 23
<210>46
<211>29
<212>DNA
<213> Artificial sequence
<400>46
tttcataaac ccattatcca ggactgttt 29
<210>47
<211>27
<212>DNA
<213> Artificial sequence
<400>47
acttaccaga tgggacactc taagatt 27
<210>48
<211>29
<212>DNA
<213> Artificial sequence
<400>48
gacgttgtca ttagttcttt ggtttgtat 29
<210>49
<211>26
<212>DNA
<213> Artificial sequence
<400>49
catttgttaa cttcagctct gggaaa 26
<210>50
<211>30
<212>DNA
<213> Artificial sequence
<400>50
caaattgata gttgttctag cagtgaagag 30
<210>51
<211>27
<212>DNA
<213> Artificial sequence
<400>51
cttcctggtg ggatctgtca ttttata 27
<210>52
<211>32
<212>DNA
<213> Artificial sequence
<400>52
gaaactagaa gagatttcta aaagtctgag at 32
<210>53
<211>30
<212>DNA
<213> Artificial sequence
<400>53
agcctaatct tactagacat gtcttttctt 30
<210>54
<211>30
<212>DNA
<213> Artificial sequence
<400>54
cgcgttgaag aagtacaaaa tgtcattaat 30
<210>55
<211>29
<212>DNA
<213> Artificial sequence
<400>55
tgatgaatgg ttttatagga acgctatgt 29
<210>56
<211>30
<212>DNA
<213> Artificial sequence
<400>56
cagttgtgag attatctttt catggctatt 30
<210>57
<211>23
<212>DNA
<213> Artificial sequence
<400>57
cttctccctg ctcacacttt ctt 23
<210>58
<211>30
<212>DNA
<213> Artificial sequence
<400>58
cgttgtgtaa attaaacttc tcccattcct 30
<210>59
<211>22
<212>DNA
<213> Artificial sequence
<400>59
cccatcgtgg gatcttgctt at 22
<210>60
<211>30
<212>DNA
<213> Artificial sequence
<400>60
aaagctcttc ctttttgaaa gtctgttttt 30
<210>61
<211>30
<212>DNA
<213> Artificial sequence
<400>61
aaataaagat gtcagatacc acagcatctt 30
<210>62
<211>30
<212>DNA
<213> Artificial sequence
<400>62
cctgaattat cactatcaga acaaagcagt 30
<210>63
<211>22
<212>DNA
<213> Artificial sequence
<400>63
gcctcgcctc atgtggtttt at 22
<210>64
<211>28
<212>DNA
<213> Artificial sequence
<400>64
taactagtat tctgagctgt gtgctaga 28
<210>65
<211>30
<212>DNA
<213> Artificial sequence
<400>65
gggaaggaaa gaattttgct taagatatca 30
<210>66
<211>29
<212>DNA
<213> Artificial sequence
<400>66
ctcaaagtat ttcattttct tggtgccat 29
<210>67
<211>30
<212>DNA
<213> Artificial sequence
<400>67
aaatatttaa aatgtgccaa gaactgtgct 30
<210>68
<211>29
<212>DNA
<213> Artificial sequence
<400>68
aaaaatgatg aagtgacagt tccagtagt 29
<210>69
<211>30
<212>DNA
<213> Artificial sequence
<400>69
atcatggaaa atttgtgcat tgttaaggaa 30
<210>70
<211>24
<212>DNA
<213> Artificial sequence
<400>70
cctctcctct gtcattcttc ctgt 24
<210>71
<211>30
<212>DNA
<213> Artificial sequence
<400>71
ataggaaaat accagcttca tagacaaagg 30
<210>72
<211>30
<212>DNA
<213> Artificial sequence
<400>72
gccacagtag atgctcagta aatatttcta 30
<210>73
<211>30
<212>DNA
<213> Artificial sequence
<400>73
cttcctttat ttcaccatca tctaacaggt 30
<210>74
<211>30
<212>DNA
<213> Artificial sequence
<400>74
ccaaaattga atgctatgct tagattaggg 30
<210>75
<211>23
<212>DNA
<213> Artificial sequence
<400>75
cctcaaggca tattccagtt cct 23
<210>76
<211>25
<212>DNA
<213> Artificial sequence
<400>76
tgctctgggt aaaggtagta gagtc 25
<210>77
<211>29
<212>DNA
<213> Artificial sequence
<400>77
aggatgttaa agctcattca gtcaaagat 29
<210>78
<211>25
<212>DNA
<213> Artificial sequence
<400>78
aggaacgggc ttggaagaaa ataat 25
<210>79
<211>29
<212>DNA
<213> Artificial sequence
<400>79
aagaaaacaa gctagcagaa cattttgtt 29
<210>80
<211>29
<212>DNA
<213> Artificial sequence
<400>80
caagaaatta gagtcctcag aagagaact 29
<210>81
<211>30
<212>DNA
<213> Artificial sequence
<400>81
ttctctaatg ttattacggc taattgtgct 30
<210>82
<211>27
<212>DNA
<213> Artificial sequence
<400>82
gaggctctag gttttgtcta tcatctc 27
<210>83
<211>29
<212>DNA
<213> Artificial sequence
<400>83
gtgacttttg gactttgttt ctttaagga 29
<210>84
<211>25
<212>DNA
<213> Artificial sequence
<400>84
ccaagggact aattcatggt tgttc 25
<210>85
<211>24
<212>DNA
<213> Artificial sequence
<400>85
ctgactggca tttggttgta cttt 24
<210>86
<211>26
<212>DNA
<213> Artificial sequence
<400>86
agaatctgct ttcaaaacga aagctg 26
<210>87
<211>28
<212>DNA
<213> Artificial sequence
<400>87
gttccctgat ttatcatttc aggagtct 28
<210>88
<211>30
<212>DNA
<213> Artificial sequence
<400>88
agagttcact ccaaatcagt agagagtaat 30
<210>89
<211>24
<212>DNA
<213> Artificial sequence
<400>89
ccatcatgtg agtcatcaga acct 24
<210>90
<211>24
<212>DNA
<213> Artificial sequence
<400>90
caacataaca gatgggctgg aagt 24
<210>91
<211>26
<212>DNA
<213> Artificial sequence
<400>91
ctgtaatgag ctggcatgag tatttg 26
<210>92
<211>30
<212>DNA
<213> Artificial sequence
<400>92
cacctccaag gtgtatgaag tatgtatttt 30
<210>93
<211>20
<212>DNA
<213> Artificial sequence
<400>93
accatgccca ggtttcaagt 20
<210>94
<211>30
<212>DNA
<213> Artificial sequence
<400>94
gggatcgatt atgtgactta aagtcagaat 30
<210>95
<211>22
<212>DNA
<213> Artificial sequence
<400>95
ctaagcagca gcctctcaga at 22
<210>96
<211>25
<212>DNA
<213> Artificial sequence
<400>96
tccgtctctt tccttttacg tcatc 25
<210>97
<211>24
<212>DNA
<213> Artificial sequence
<400>97
cctaagccaa caacagcctg aata 24
<210>98
<211>30
<212>DNA
<213> Artificial sequence
<400>98
tccttctact gtcctggcta ctaaatattt 30
<210>99
<211>30
<212>DNA
<213> Artificial sequence
<400>99
aatactgcta ctctctacag atctttcagt 30
<210>100
<211>28
<212>DNA
<213> Artificial sequence
<400>100
gccaaatgaa cagacaagta aaagacat 28
<210>101
<211>22
<212>DNA
<213> Artificial sequence
<400>101
ggctagggaa gacctagtcc tt 22
<210>102
<211>30
<212>DNA
<213> Artificial sequence
<400>102
cctcttaaaa tataagacct ctggcatgaa 30
<210>103
<211>32
<212>DNA
<213> Artificial sequence
<400>103
gaagaagaag aagaaaacaa atggttttac ca 32
<210>104
<211>31
<212>DNA
<213> Artificial sequence
<400>104
aactgcaaac ataatgtttt cccttgtatt t 31
<210>105
<211>30
<212>DNA
<213> Artificial sequence
<400>105
tgataatcac ttcttccatt gcatctttct 30
<210>106
<211>29
<212>DNA
<213> Artificial sequence
<400>106
atggagattc cataaactaa caagcactt 29
<210>107
<211>28
<212>DNA
<213> Artificial sequence
<400>107
gcaataaaac tagtagtgca gataccca 28
<210>108
<211>30
<212>DNA
<213> Artificial sequence
<400>108
aatgactgat ttttaccaag agtgcaaatt 30
<210>109
<211>30
<212>DNA
<213> Artificial sequence
<400>109
tgacattgaa gactgacttt actctttcaa 30
<210>110
<211>30
<212>DNA
<213> Artificial sequence
<400>110
gctttttgaa gcctttaagg atttttcttg 30
<210>111
<211>30
<212>DNA
<213> Artificial sequence
<400>111
gggatttgct ttgttttatt ttagtcctgt 30
<210>112
<211>25
<212>DNA
<213> Artificial sequence
<400>112
atgaaacaaa ctcccacata ccact 25
<210>113
<211>28
<212>DNA
<213> Artificial sequence
<400>113
cccttcactt cagcaaattt ttagatcc 28
<210>114
<211>30
<212>DNA
<213> Artificial sequence
<400>114
ctggtagctc caactaatca taagagattt 30
<210>115
<211>30
<212>DNA
<213> Artificial sequence
<400>115
caaatagtag atgtgctttt tgatgtctga 30
<210>116
<211>29
<212>DNA
<213> Artificial sequence
<400>116
ggtttagaga ctttctcaaa ggcttagat 29
<210>117
<211>24
<212>DNA
<213> Artificial sequence
<400>117
gcttttattc tgctcatggc acaa 24
<210>118
<211>30
<212>DNA
<213> Artificial sequence
<400>118
caacaaaagt gccagtagtc atttcaatat 30
<210>119
<211>30
<212>DNA
<213> Artificial sequence
<400>119
gttgttgaat tcagtatcat cctatgtggt 30
<210>120
<211>29
<212>DNA
<213> Artificial sequence
<400>120
tgccgtatat gattacgtaa tgtaatgct 29
<210>121
<211>22
<212>DNA
<213> Artificial sequence
<400>121
atttcctttc gccacactga ga 22
<210>122
<211>22
<212>DNA
<213> Artificial sequence
<400>122
cggagtaagc tgacaaaaac cg 22
<210>123
<211>22
<212>DNA
<213> Artificial sequence
<400>123
tccagcgctt ctgagtttta cc 22
<210>124
<211>24
<212>DNA
<213> Artificial sequence
<400>124
gtgacgtact gggtttttag caag 24
<210>125
<211>30
<212>DNA
<213> Artificial sequence
<400>125
cactgaatta ttgtactgtt tcaggaagga 30
<210>126
<211>30
<212>DNA
<213> Artificial sequence
<400>126
caggctctta gccaaaatat tagcataaaa 30
<210>127
<211>30
<212>DNA
<213> Artificial sequence
<400>127
aaaaataccg aaagaccaaa aatcagaact 30
<210>128
<211>30
<212>DNA
<213> Artificial sequence
<400>128
cctaaacaat catgtataca gatgatgcct 30
<210>129
<211>30
<212>DNA
<213> Artificial sequence
<400>129
gaagataaca aatatactgc tgccagtaga 30
<210>130
<211>24
<212>DNA
<213> Artificial sequence
<400>130
ttgagctttc gcaacttcca aaaa 24
<210>131
<211>25
<212>DNA
<213> Artificial sequence
<400>131
gggagggaga ctgtgtgtaa tattt 25
<210>132
<211>29
<212>DNA
<213> Artificial sequence
<400>132
agttcttttg gtcatcaatc tctttctcc 29
<210>133
<211>25
<212>DNA
<213> Artificial sequence
<400>133
ccctttcgtc tatttgtcag acgaa 25
<210>134
<211>27
<212>DNA
<213> Artificial sequence
<400>134
aaatgtgtgg tgatgctgaa aagtaac 27
<210>135
<211>26
<212>DNA
<213> Artificial sequence
<400>135
gggaatcagg ctttactaga agaaca 26
<210>136
<211>27
<212>DNA
<213> Artificial sequence
<400>136
caatgtggtc tttgcagcta tttactt 27
<210>137
<211>30
<212>DNA
<213> Artificial sequence
<400>137
cgctgatgaa tgtgaaaaat ctaaaaacca 30
<210>138
<211>24
<212>DNA
<213> Artificial sequence
<400>138
attttctcca tctgggctcc attt 24
<210>139
<211>30
<212>DNA
<213> Artificial sequence
<400>139
tgaggaaaca gtggtaaata agagagatga 30
<210>140
<211>22
<212>DNA
<213> Artificial sequence
<400>140
tcctccttct gtgagcaaac ag 22
<210>141
<211>25
<212>DNA
<213> Artificial sequence
<400>141
tgaacctgca gaagaatctg aacat 25
<210>142
<211>30
<212>DNA
<213> Artificial sequence
<400>142
gtttgcctaa attcctagtt tgtagttctc 30
<210>143
<211>27
<212>DNA
<213> Artificial sequence
<400>143
gctgatttct gttgtatgct tgtactg 27
<210>144
<211>27
<212>DNA
<213> Artificial sequence
<400>144
gtaatcggct ctaaagaaac atgatgc 27
<210>145
<211>30
<212>DNA
<213> Artificial sequence
<400>145
tgagctctaa ttttgttgta tttgtcctgt 30
<210>146
<211>22
<212>DNA
<213> Artificial sequence
<400>146
acttccacac ggttgtgaca tc 22
<210>147
<211>29
<212>DNA
<213> Artificial sequence
<400>147
gtttattgca ttcttctgtg aaaagaagc 29
<210>148
<211>27
<212>DNA
<213> Artificial sequence
<400>148
tgctttttgg atcattttca cactgtc 27
<210>149
<211>30
<212>DNA
<213> Artificial sequence
<400>149
aaaagtggaa tacagtgata ctgactttca 30
<210>150
<211>28
<212>DNA
<213> Artificial sequence
<400>150
tggcaacagc tcaacgtttt tataattt 28
<210>151
<211>27
<212>DNA
<213> Artificial sequence
<400>151
tccagactct gaagaacttt tctcaga 27
<210>152
<211>25
<212>DNA
<213> Artificial sequence
<400>152
ggttgcttgt ttatcacctg tgtct 25
<210>153
<211>22
<212>DNA
<213> Artificial sequence
<400>153
caaatgggcaggactcttag gt 22
<210>154
<211>24
<212>DNA
<213> Artificial sequence
<400>154
ctgaggcttg ctcagtttct tttg 24
<210>155
<211>27
<212>DNA
<213> Artificial sequence
<400>155
cctcagatgt tattttccaa gcaggat 27
<210>156
<211>30
<212>DNA
<213> Artificial sequence
<400>156
gcattcatta tgacatgaag atcagcatct 30
<210>157
<211>30
<212>DNA
<213> Artificial sequence
<400>157
gaaaaatatt agtgtcgcca aagagtcatt 30
<210>158
<211>29
<212>DNA
<213> Artificial sequence
<400>158
caactgggac actttctttc agtattttg 29
<210>159
<211>24
<212>DNA
<213> Artificial sequence
<400>159
ggttttcata cagctagcgg gaaa 24
<210>160
<211>30
<212>DNA
<213> Artificial sequence
<400>160
accacagtct caatagaaac aaggttttta 30
<210>161
<211>25
<212>DNA
<213> Artificial sequence
<400>161
agcaaaaagt cctgcaactt gttac 25
<210>162
<211>29
<212>DNA
<213> Artificial sequence
<400>162
cttcaagtaa tgaagtctga ctcacagaa 29
<210>163
<211>30
<212>DNA
<213> Artificial sequence
<400>163
atgacaaaaa tcatctctcc gaaaaacaag 30
<210>164
<211>28
<212>DNA
<213> Artificial sequence
<400>164
caagttcctc aacgcaaata tcttcatt 28
<210>165
<211>29
<212>DNA
<213> Artificial sequence
<400>165
cctgcaaaaa taaaaatgca gccattaaa 29
<210>166
<211>22
<212>DNA
<213> Artificial sequence
<400>166
catccaatgc ctcgtaacaa cc 22
<210>167
<211>28
<212>DNA
<213> Artificial sequence
<400>167
acataaggtt tttgctgaca ttcagagt 28
<210>168
<211>26
<212>DNA
<213> Artificial sequence
<400>168
tgctgtgcta aaaatcccac aagtat 26
<210>169
<211>27
<212>DNA
<213> Artificial sequence
<400>169
gaccagctca caagagaaga aaatact 27
<210>170
<211>30
<212>DNA
<213> Artificial sequence
<400>170
ggtttctctt atcaacacga ggaagtattt 30
<210>171
<211>29
<212>DNA
<213> Artificial sequence
<400>171
caacaagaca aacaacagtt ggtattagg 29
<210>172
<211>29
<212>DNA
<213> Artificial sequence
<400>172
tgtcagttca tcatcttcca taaaagctt 29
<210>173
<211>27
<212>DNA
<213> Artificial sequence
<400>173
gtgaggtaga ttgtaaagtc aaaggct 27
<210>174
<211>26
<212>DNA
<213> Artificial sequence
<400>174
aggtgcggta aaatttggat tctgta 26
<210>175
<211>23
<212>DNA
<213> Artificial sequence
<400>175
caaaatatgt ggaggcccaa caa 23
<210>176
<211>29
<212>DNA
<213> Artificial sequence
<400>176
tgttgctatt ctttgtctaa caccaaaaa 29
<210>177
<211>22
<212>DNA
<213> Artificial sequence
<400>177
ggtacagcag actgtggaat gt 22
<210>178
<211>30
<212>DNA
<213> Artificial sequence
<400>178
aaagactctg catttttgct gttaattttt 30
<210>179
<211>24
<212>DNA
<213> Artificial sequence
<400>179
gagttgtggc accaaatacg aaac 24
<210>180
<211>30
<212>DNA
<213> Artificial sequence
<400>180
agtcctagtg gattcactga cagatataaa 30
<210>181
<211>33
<212>DNA
<213> Artificial sequence
<400>181
tcttttagat tgtgtcatta aatggaatga ggt 33
<210>182
<211>32
<212>DNA
<213> Artificial sequence
<400>182
actgagggtt ctatttttct tataatcatg ct 32
<210>183
<211>22
<212>DNA
<213> Artificial sequence
<400>183
aagcatttgc aaaggcgaca at 22
<210>184
<211>20
<212>DNA
<213> Artificial sequence
<400>184
accgagctca gccaaagatg 20
<210>185
<211>25
<212>DNA
<213> Artificial sequence
<400>185
ttcagtgatg gaggaaatgt tggtt 25
<210>186
<211>21
<212>DNA
<213> Artificial sequence
<400>186
gtggtggctc agctacttga g 21
<210>187
<211>27
<212>DNA
<213> Artificial sequence
<400>187
caaccaaagt ctttgttcca cctttta 27
<210>188
<211>30
<212>DNA
<213> Artificial sequence
<400>188
ggcaaaaatt catcacacaa attgtcatac 30
<210>189
<211>33
<212>DNA
<213> Artificial sequence
<400>189
tgatagattt aattacaagt cttcagaatg cca 33
<210>190
<211>31
<212>DNA
<213> Artificial sequence
<400>190
tcataaaagc catcagtatt gtagacaaac a 31
<210>191
<211>30
<212>DNA
<213> Artificial sequence
<400>191
aggagaaccc tcaatcaaaa gaaacttatt 30
<210>192
<211>28
<212>DNA
<213> Artificial sequence
<400>192
tataaagagg tccttgatta ggcacagt 28
<210>193
<211>28
<212>DNA
<213> Artificial sequence
<400>193
caggcaattc agtaaacgtt aagtgaaa 28
<210>194
<211>28
<212>DNA
<213> Artificial sequence
<400>194
acaattatca acctcatctg ctctttct 28
<210>195
<211>27
<212>DNA
<213> Artificial sequence
<400>195
tgggtgtttt atgcttggtt ctttagt 27
<210>196
<211>26
<212>DNA
<213> Artificial sequence
<400>196
agagagtcta aaacagcttc tcacct 26
<210>197
<211>28
<212>DNA
<213> Artificial sequence
<400>197
tatgtgggtt tgcaatttat aaagcagc 28
<210>198
<211>30
<212>DNA
<213> Artificial sequence
<400>198
tatacaacag aatatacgat ggcctccata 30
<210>199
<211>30
<212>DNA
<213> Artificial sequence
<400>199
ttctgcagag gtacatccaa taagtttatc 30
<210>200
<211>30
<212>DNA
<213> Artificial sequence
<400>200
tacttgaatc actgccatca aattctaagt 30
<210>201
<211>25
<212>DNA
<213> Artificial sequence
<400>201
tttttggacc taggttgatt gcaga 25
<210>202
<211>29
<212>DNA
<213> Artificial sequence
<400>202
aaaaacctgt agttcaacta aacagagga 29
<210>203
<211>18
<212>DNA
<213> Artificial sequence
<400>203
gcgcgagctt ctgaaact 18
<210>204
<211>19
<212>DNA
<213> Artificial sequence
<400>204
tcgtcccaac ccactacca 19
<210>205
<211>26
<212>DNA
<213> Artificial sequence
<400>205
atcaagaaaa acatctttgg ctgagc 26
<210>206
<211>33
<212>DNA
<213> Artificial sequence
<400>206
acctcattcc atttaatgac acaatctaaa aga 33
<210>207
<211>28
<212>DNA
<213> Artificial sequence
<400>207
ttgtttccta ggcacaataa aagatcga 28
<210>208
<211>30
<212>DNA
<213> Artificial sequence
<400>208
ggaagtgtta acttcttaac gttagtgtca 30
<210>209
<211>29
<212>DNA
<213> Artificial sequence
<400>209
actttaacag gatttggaaa aacatcagg 29
<210>210
<211>26
<212>DNA
<213> Artificial sequence
<400>210
cagcgtttgc ttcatggaaa attttt 26
<210>211
<211>30
<212>DNA
<213> Artificial sequence
<400>211
atgccttaac aaaagtaatc catagtcaag 30
<210>212
<211>30
<212>DNA
<213> Artificial sequence
<400>212
tgtggagttt taaataggtt tggttcgtaa 30
<210>213
<211>30
<212>DNA
<213> Artificial sequence
<400>213
tggtattgaa attttagcac tgtaagcaac 30
<210>214
<211>30
<212>DNA
<213> Artificial sequence
<400>214
ttaaggtcta tccaaaactt tattgccagt 30
<210>215
<211>24
<212>DNA
<213> Artificial sequence
<400>215
gcctgtgaat ggtctcaact aacc 24
<210>216
<211>28
<212>DNA
<213> Artificial sequence
<400>216
gcaagaatgc agtctgtatg agattcaa 28
<210>217
<211>22
<212>DNA
<213> Artificial sequence
<400>217
ctgaagcctc tgaaagtgga ct 22
<210>218
<211>23
<212>DNA
<213> Artificial sequence
<400>218
catttgcttc aaactgggct gaa 23
<210>219
<211>30
<212>DNA
<213> Artificial sequence
<400>219
agtgacttgt ttaaacagtg gaattctaga 30
<210>220
<211>28
<212>DNA
<213> Artificial sequence
<400>220
gcataccacc catctgtaag ttcaataa 28
<210>221
<211>24
<212>DNA
<213> Artificial sequence
<400>221
gtttccagca gctgaaattt gtga 24
<210>222
<211>30
<212>DNA
<213> Artificial sequence
<400>222
acttatctct ttgtggtgtt acatgtgtac 30
<210>223
<211>30
<212>DNA
<213> Artificial sequence
<400>223
aaatctgaaa gagctaacat acagttagca 30
<210>224
<211>27
<212>DNA
<213> Artificial sequence
<400>224
ctattaggtc cacctcagaa caagatg 27
<210>225
<211>24
<212>DNA
<213> Artificial sequence
<400>225
cctcccagag ccctcaaatt ataa 24
<210>226
<211>27
<212>DNA
<213> Artificial sequence
<400>226
cactgtgcga agacttttat gtctact 27
<210>227
<211>26
<212>DNA
<213> Artificial sequence
<400>227
ggaatctgct gaacaaaagg aacaag 26
<210>228
<211>30
<212>DNA
<213> Artificial sequence
<400>228
gcattagtag tggattttgc ttctctgata 30
<210>229
<211>22
<212>DNA
<213> Artificial sequence
<400>229
actgtgccca aacactacct tt 22
<210>230
<211>29
<212>DNA
<213> Artificial sequence
<400>230
ttaaggacaa agttggttct tcagaatca 29
<210>231
<211>24
<212>DNA
<213> Artificial sequence
<400>231
cccagaagct gattctctgt catg 24
<210>232
<211>30
<212>DNA
<213> Artificial sequence
<400>232
tgctggcatt ttcatgatca tataaaagac 30
<210>233
<211>29
<212>DNA
<213> Artificial sequence
<400>233
ttcccatgga aaagaatcaa gatgtatgt 29
<210>234
<211>30
<212>DNA
<213> Artificial sequence
<400>234
tcctttcatt agctacttgg aagacaaaat 30
<210>235
<211>27
<212>DNA
<213> Artificial sequence
<400>235
gaacccattt tcaagaactc taccatg 27
<210>236
<211>24
<212>DNA
<213> Artificial sequence
<400>236
ctgaagctac ctccaaaact gtga 24
<210>237
<211>29
<212>DNA
<213> Artificial sequence
<400>237
agcttgtgtt gaaattgtaa ataccttgg 29
<210>238
<211>27
<212>DNA
<213> Artificial sequence
<400>238
gccttttggc taggtgttaa attatgg 27
<210>239
<211>29
<212>DNA
<213> Artificial sequence
<400>239
accagatgac tatcttaaag accacttct 29
<210>240
<211>24
<212>DNA
<213> Artificial sequence
<400>240
agctttttgc agagcttcag taga 24
<210>241
<211>30
<212>DNA
<213> Artificial sequence
<400>241
agggaaacac tcagattaaa gaagatttgt 30
<210>242
<211>30
<212>DNA
<213> Artificial sequence
<400>242
gcaattcttc tggtttctga tcaaagaaat 30
<210>243
<211>30
<212>DNA
<213> Artificial sequence
<400>243
atggacattc taagttatga ggaaacagac 30
<210>244
<211>25
<212>DNA
<213> Artificial sequence
<400>244
cactttgtcc aaagattcct ttgca 25
<210>245
<211>28
<212>DNA
<213> Artificial sequence
<400>245
cccaaagtgt aaagaaatgc agaattct 28
<210>246
<211>28
<212>DNA
<213> Artificial sequence
<400>246
ggctgaattt tcaatgactg aataaggg 28
<210>247
<211>26
<212>DNA
<213> Artificial sequence
<400>247
cagccttagc tttttacaca agttgt 26
<210>248
<211>29
<212>DNA
<213> Artificial sequence
<400>248
gaatagctgt tagacatgct actgttact 29
<210>249
<211>28
<212>DNA
<213> Artificial sequence
<400>249
tccaatgtaa aagatgcaaa tgcatacc 28
<210>250
<211>30
<212>DNA
<213> Artificial sequence
<400>250
ggtggcccta cctcaaaatt attactatta 30
<210>251
<211>30
<212>DNA
<213> Artificial sequence
<400>251
acgagaataa atcaaaaatt tgccaaacga 30
<210>252
<211>30
<212>DNA
<213> Artificial sequence
<400>252
tccaatccag acatattttg gttatgttgt 30
<210>253
<211>30
<212>DNA
<213> Artificial sequence
<400>253
atgtagtata gggaagcttc ataagtcagt 30
<210>254
<211>29
<212>DNA
<213> Artificial sequence
<400>254
agcctttttg ggatattaaa tgttctgga 29
<210>255
<211>27
<212>DNA
<213> Artificial sequence
<400>255
tcttcactat tcacctacgt ctagaca 27
<210>256
<211>28
<212>DNA
<213> Artificial sequence
<400>256
cccaaaacat gaatgttctc aacaagtg 28
<210>257
<211>29
<212>DNA
<213> Artificial sequence
<400>257
tcagaaaact actttgaaac agaagcagt 29
<210>258
<211>26
<212>DNA
<213> Artificial sequence
<400>258
ggcaacacga aaggtaaaaa tgaaca 26
<210>259
<211>23
<212>DNA
<213> Artificial sequence
<400>259
gtgcctggcc tgatacaatt aac 23
<210>260
<211>30
<212>DNA
<213> Artificial sequence
<400>260
aattcctcct gaattttagt gaataaggct 30
<210>261
<211>25
<212>DNA
<213> Artificial sequence
<400>261
gtagctgtat acgtatggcg tttct 25
<210>262
<211>30
<212>DNA
<213> Artificial sequence
<400>262
ggatgaggga atacataaaa gttaacacac 30
<210>263
<211>25
<212>DNA
<213> Artificial sequence
<400>263
caagctcttt tgtctggttc aacag 25
<210>264
<211>29
<212>DNA
<213> Artificial sequence
<400>264
tccagtctta taaactggaa aggttaagc 29
<210>265
<211>27
<212>DNA
<213> Artificial sequence
<400>265
tgtttccttt tgagcaattc ttcatcc 27
<210>266
<211>27
<212>DNA
<213> Artificial sequence
<400>266
tcctgtgata gcgctaaaaa taaagca 27
<210>267
<211>32
<212>DNA
<213> Artificial sequence
<400>267
ttttaaggca gttctagaag aatgaaaact ct 32
<210>268
<211>33
<212>DNA
<213> Artificial sequence
<400>268
tcatacctgt atagggtatg ctctttgaat aat 33
<210>269
<211>22
<212>DNA
<213> Artificial sequence
<400>269
cccattgcag cacaactaag ga 22
<210>270
<211>29
<212>DNA
<213> Artificial sequence
<400>270
ctaacacact gttcaactct gtgaaaatg 29
<210>271
<211>23
<212>DNA
<213> Artificial sequence
<400>271
tctcagccca gatgacttca aag 23
<210>272
<211>30
<212>DNA
<213> Artificial sequence
<400>272
gagtcatctg aggagaattc agttcttttt 30
<210>273
<211>20
<212>DNA
<213> Artificial sequence
<400>273
ccatctcatc cctgcgtgtc 20
<210>274
<211>20
<212>DNA
<213> Artificial sequence
<400>274
atcaccgact gcccatagag 20
<210>275
<211>19
<212>DNA
<213> Artificial sequence
<400>275
ccactacgcc tccgctttc 19
<210>276
<211>11
<212>DNA
<213> Artificial sequence
<400>276
tctagctctt c 11
<210>277
<211>11
<212>DNA
<213> Artificial sequence
<400>277
tcactcggat c 11
<210>278
<211>12
<212>DNA
<213> Artificial sequence
<400>278
ttcctgcttc ac 12
<210>279
<211>11
<212>DNA
<213> Artificial sequence
<400>279
ccttagagtt c 11
<210>280
<211>12
<212>DNA
<213> Artificial sequence
<400>280
ctgagttccg ac 12
<210>281
<211>12
<212>DNA
<213> Artificial sequence
<400>281
tcctggcaca tc 12
<210>282
<211>11
<212>DNA
<213> Artificial sequence
<400>282
ccgcaatcat c 11
<210>283
<211>12
<212>DNA
<213> Artificial sequence
<400>283
ttcctaccag tc 12
<210>284
<211>11
<212>DNA
<213> Artificial sequence
<400>284
tcaagaagtt c 11
<210>285
<211>10
<212>DNA
<213> Artificial sequence
<400>285
ttcaattggc 10
<210>286
<211>11
<212>DNA
<213> Artificial sequence
<400>286
tctagctctt c 11

Claims (10)

  1. The BRCA1/2 gene variation combined detection kit is characterized by comprising the following reagents: BRCA1 gene primer group, BRCA2 gene primer group, polymerase, PCR reaction liquid, digestive enzyme, ligase buffer solution, a connecting joint, a fluorescent probe, Taq enzyme for qPCR reaction, qPCR primer and qPCR reaction liquid;
    the BRCA1 gene primer group comprises 52 pairs of primers, and the nucleic acid sequence of the BRCA1 gene primer is shown as Seq ID No 1-Seq ID No 104;
    the BRCA2 gene primer group comprises 84 pairs of primers; the nucleic acid sequences of the primers for BRCA2 gene are shown in Seq ID No105 to Seq ID No 272.
  2. 2. The kit of claim 1, wherein the nucleotide sequence of the qPCR primer is shown as Seq ID No273 to Seq ID No 274.
  3. 3. The kit of claim 1, wherein the fluorescent probe has the nucleotide sequence shown as Seq ID No 275.
  4. 4. The kit according to claim 1, wherein the nucleotide sequence of the linker is shown as Seq ID No276 to Seq ID No 286.
  5. 5. The kit of claim 1, wherein the digestive enzyme is a phosphodiesterase; the enzyme activity of the phosphodiesterase is 2U/mu L-8U/mu L.
  6. 6. The kit of claim 1, wherein the ligase is T4 ligase; the enzyme activity of the T4 ligase is 0.5U/mu L-2U/mu L.
  7. 7. The kit according to claim 1, wherein the PCR reaction solution and the qPCR reaction solution comprise the following components in content: a 5 Xamplification enzyme reaction solution containing a dNTP mixture at a molar concentration of 10 mmol/L; the 5 Xamplification enzyme reaction solution contains 25 mmol/L-75 mmol/L LTris-HCl and 25mmol/L～75mmol/LKCl、5mmol/L～10mmol/LMg2+And glycerol with the mass concentration of 25-70 percent.
  8. 8. The kit according to claim 1, wherein the enzymatic activity of the polymerase is 2.5U/μ L to 7U/μ L.
  9. 9. The kit of claim 1, wherein the ligase buffer comprises the following components in amounts: a 5 Xamplification enzyme reaction solution containing a dNTP mixture at a molar concentration of 10 mmol/L; the 5 Xamplification enzyme reaction solution contains 25 mmol/L-75 mmol/L LTris-HCl, 25 mmol/L-75 mmol/LMg2+2.5 mmol/L-7.5 mmol/LATP, 2.5 mmol/L-7.5 mmol/LDTT and polyethylene glycol with the mass concentration of 15-35 percent.
  10. 10. Use of the kit of any one of claims 1 to 9 in the preparation of a reagent for detecting mutations in BRCA1 and/or BRCA2 genes.
CN201710447510.7A 2017-06-14 2017-06-14 BRCA1/2 gene variation combined detection kit and application thereof Active CN107083442B (en)

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CN108085387B (en) * 2017-11-27 2020-03-27 天津诺禾致源生物信息科技有限公司 Specific capture probe, kit and sequencing library for detecting human BRCA1/2 gene mutation and construction method thereof
CN108624686B (en) * 2018-03-30 2019-06-04 南京世和基因生物技术有限公司 A kind of probe library, detection method and the kit of detection BRCA1/2 mutation
CN110129414A (en) * 2019-04-28 2019-08-16 安徽鼎晶生物科技有限公司 A kind of BRCA high-throughput sequencing library and its construction method and application

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CN101748200B (en) * 2008-12-16 2012-02-01 上海基康生物技术有限公司 Six pair of primers for breast cancer-associated locus detection
CN104531862B (en) * 2014-12-19 2017-12-29 钱学庆 Detect the method and primer in the full exon sequence mutational site of mankind's BRCA1 and BRCA2 gene
CN105586427B (en) * 2016-03-10 2020-06-19 厦门艾德生物医药科技股份有限公司 Primers, kit and method for detecting human BRCA1 and BRCA2 gene mutation
CN106636442B (en) * 2017-02-23 2020-12-01 上海鼎晶生物医药科技股份有限公司 Human tumor gene variation joint detection kit

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