CN113684270A - Multiple-target capture kit for familial hypercholesterolemia and detection method thereof - Google Patents
Multiple-target capture kit for familial hypercholesterolemia and detection method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of gene detection, and discloses a multiple targeted capture kit for familial hypercholesterolemia and a detection method thereof, wherein the kit comprises primers SEQ ID NO.1-324 and primers SEQ ID NO. 347-382. The gene multi-targeting capture kit panel provided by the invention is expanded, the detection range is more comprehensive, the sequencing coverage reaches 100%, and the kit can be applied to a DNBSEQ sequencing platform.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a multiple-target capture kit for familial hypercholesterolemia and a detection method thereof.
Background
Familial Hypercholesterolaemia (FH) is an autosomal dominant hereditary disease pathologically characterized by elevated levels of total plasma cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) and early onset coronary heart disease (PCAD). Atherosclerosis (AS) is the pathological basis of coronary atherosclerotic heart disease (CAD), and FH indirectly or directly promotes AS due to abnormal blood lipid metabolism, and has close relation with the generation and development of PCAD (CAD onset age, male < 55 years, female < 65 years). The blood lipid metabolism imbalance of FH patients can accelerate the AS and PCAD processes, discovery and diagnosis of FH are carried out early, intervention is carried out early, the morbidity and mortality of coronary artery diseases can be obviously reduced, and clinical prognosis is improved.
Currently known FH-associated mutations exceed 2000, of which about 1000 have been supported by sufficient evidence to be classified into two classes, pathogenic and potentially pathogenic, these mutations are mainly distributed among three genes: LDLR (90% or more), APOB (5% to 10%), and PCSK9 (1% or less). However, with the development of gene sequencing technology and the continuous improvement of doctors and patients on FH cognition, more and more genes and mutation sites of genes are discovered, the coverage of detecting FH-related genes is still to be further improved, for example, chinese patent publication No. CN109517884B discloses a FH sequencing library, which only relates to three genes of LDLR, APOB and PCSK9, the coverage is to be further improved, and an Ion Torrent sequencing platform is adopted.
Compared with the Ion Torrent sequencing platform and the Illumina sequencing platform which are mature at present and are faced to be updated and iterated, the DNBSEQ sequencing platform is mainly positioned in the sequencing application scenes of small and medium scales at the hospital and inspection institute levels, the instrument cost is low, various performance indexes are more excellent, but relevant reports of FH gene detection libraries based on the DNBSEQ sequencing platform are not found at present.
Disclosure of Invention
The invention provides a multiple target capture kit for familial hypercholesterolemia and a detection method thereof, which at least solve one of the technical problems.
One of the objectives of the present invention is to provide:
a multiple target capture kit for familial hypercholesterolemia comprises primers SEQ ID NO. 1-382; wherein the content of the first and second substances,
the SEQ ID NO.1-14, SEQ ID NO.153-166 and SEQ ID NO.323-324 are used for detecting the LDLRAP1 gene;
the SEQ ID NO.15-32, SEQ ID NO.167-200 and SEQ ID NO.367-382 are used for detecting the PCSK9 gene;
the SEQ ID NO.33-58 and the SEQ ID NO.201-220 are used for detecting the LDLR gene;
SEQ ID NO.59-152, SEQ ID NO.221-322 and SEQ ID NO.347-366 were used for the detection of the APOB gene.
The SEQ ID NO.325-326 is used for detecting the polymorphic locus rs2479409 of the PCSK9 gene;
the SEQ ID NO.327-328 is used for detecting the polymorphic site rs629301 of the CELSR2 gene;
the SEQ ID NO.329-330 is used for detecting the ST3GAL4 gene polymorphism site rs 11220462;
the SEQ ID NO.331-332 is used for detecting NYNRIN gene polymorphism locus rs 8017377;
the SEQ ID NO.333-334 is used for detecting the LDLR gene polymorphism locus rs 6511720;
the SEQ ID NO.335-336 gene is used for detecting polymorphic sites rs429358 and rs7412 of the APOE gene;
the SEQ ID NO.337-338 is used for detecting APOB gene polymorphism site rs 1367117;
the SEQ ID NO.339-340 is used for detecting the ABCG8 gene polymorphism site rs 4299376;
SEQ ID NO.341-342 for detecting MYLIP gene polymorphic site rs 3757354;
SEQ ID NO.343-344 is used for detecting HFE gene polymorphic site rs 1800562;
the SEQ ID NO.345-346 is used for detecting the SLC22A1 gene polymorphic site rs 1564348.
Preferably, the primers SEQ ID NO.1-382 are divided into primer set 1, primer set 2 and primer set 3.
Preferably, the polypeptide also comprises an adapter sequence 1 and an adapter sequence 2, wherein the sequence of the adapter sequence 1 is SEQ ID NO.383, and the sequence of the adapter sequence 2 is SEQ ID NO. 384.
Second, the second object of the present invention is to provide:
the multiple target capture kit for familial hypercholesterolemia is used for a non-diagnosis detection method, and primers SEQ ID NO.1-324 and SEQ ID NO.347-382 are adopted to carry out 1 st round of multiple PCR reaction and 2 nd round of adaptor sequence PCR reaction on a DNA sample to obtain a gene detection library for sequencing analysis; the primers SEQ ID NO.1-324 and SEQ ID NO.347-382 were divided into primer sets 1, 2, 3.
Third, the third object of the present invention is to provide:
the multiple target capture kit for familial hypercholesterolemia is used for a non-diagnosis purpose detection method, primers SEQ ID NO.1-382 are adopted to carry out 1 st round of multiple PCR reaction and 2 nd round of joint sequence PCR reaction on a DNA sample to obtain a gene detection library, and sequencing analysis is carried out; the primers SEQ ID NO.1-382 are divided into primer groups 1, 2, 3.
Preferably, after the 1 st round of multiplex PCR reaction of the primer sets 1, 2 and 3, the multiplex PCR amplification products of the primer sets 1, 2 and 3 are combined at a volume ratio of 6-8: 5-7: 1-3.
Preferably, the reaction conditions of the 1 st round of multiplex PCR reaction of primer set 1 and primer set 2 are as follows:
preferably, the reaction conditions of the 1 st round of multiplex PCR reaction of primer set 3 are as follows:
fourthly, the fourth purpose of the invention is to provide:
a gene detection library for familial hypercholesterolemia is prepared by the detection method.
The beneficial effects of the invention are as follows:
(1) the invention designs 191 pairs of primer pairs (SEQ ID NO.1-382) applicable to a DNBSEQ sequencing platform and related to FH gene detection, wherein the primers SEQ ID NO.1-324 and the primers SEQ ID NO.347-382 are used for detecting coding exons and flanking regions (+/-10 bp) of LDLRAP1 gene, PCSK9 gene, LDLR gene and APOB gene, the mutation characteristic of monogenic inheritance is mainly considered, and the sequencing coverage can reach 100 percent.
(2) In addition to the mutation characteristics of single gene inheritance, the mutation characteristics of polygenic genetic diseases are considered, and the polymorphic site (rs629301) of CELSR2 gene, the polymorphic site (rs4299376) of ABCG8 gene, the polymorphic site (rs1564348) of SLC22A1 gene, the polymorphic site (rs1800562) of HFE gene, the polymorphic site (rs3757354) of MYLIP gene, the polymorphic site (rs11220462) of ST3GAL4 gene, the polymorphic site (rs8017377) of NYRIN gene, the polymorphic site (rs429358, rs7412) of APOE gene, the polymorphic site (rs6511720) of LDLR gene, the polymorphic site (rs1367117) of APOB gene and the polymorphic site (rs 8012479409) of PCSK9 gene are taken into the detection range, so that the coverage range of the gene detection library is further improved.
The multiple target capture kit provided by the invention can detect multiple sites of FH related gene variation, the gene panel is enlarged, the detection range is more comprehensive, the sequencing coverage can reach 100%, the probe capture is not needed, the high coverage of the region to be detected is realized by utilizing multiple PCR amplification, the multiple target capture kit has high detection coverage and high uniformity, the sample consumption is less, and the sample requirement is low.
(3) Three primer sets, mainly based on GC content, and preventing mutual pairing between the primers, wherein the GC content of the primer set 1 and the primer set 2 is approximately in the range of 40-60%, and the GC content of the primer set 3 is approximately in the range of 70-80%.
(4) The kit provided by the invention can be used for constructing libraries by adopting various samples, such as saliva gDNA, blood gDNA and the like. The invention optimizes the process steps, changes the primer digestion step after the multiple PCR is finished into the magnetic bead purification, and reduces the experimental time.
(5) Except for the primers, the detection conditions of the 1 st round of multiplex PCR reaction and the 2 nd round of adaptor sequence PCR reaction adopted by the invention ensure that the constructed gene detection library has the advantages of short construction period, high comparison rate, high capture rate, good uniformity, good repeatability, simple and convenient operation and the like.
(6) "coverage" as referred to herein refers to the percentage of sequenced bases that occupy the entire genome or target region up to a specified depth; depth refers to the average number of base sequences aligned to a known reference sequence.
Drawings
FIG. 1 is a graph showing the results of library fragment length control and concentration control of a gene assay library using an Agilent 2200 bioanalyzer.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The following examples refer to self-contained reagents and apparatus as shown in table 1 below:
TABLE 1 self-contained reagents and instruments
Example 1 construction of Gene testing library Using multiple Targeted Capture kit for familial hypercholesterolemia
A multiple target capture kit for familial hypercholesterolemia
TABLE 2 kit composition
Second, detection method of multiple target capture kit for familial hypercholesterolemia
1. Obtaining DNA samples
The collected human whole blood EDTA anticoagulation sample is subjected to whole genome DNA extraction, and the concentration and purity of the DNA are detected, wherein the concentration is required to be more than 5.6 ng/mu L, and the purity OD260/280 is 1.8-2.0.
2. Construction of Gene detection libraries
1) 1 st round of multiplex PCR reaction
The FH related genes related to this example have 4 genes and 12 polymorphic sites, 4 genes, 12 polymorphic sites and corresponding PCR amplification primer sets as follows:
a primer designed according to the LDLR (Low-density lipoprotein receptor) gene: the sequences SEQ ID NO.33-58 and the sequence SEQ ID NO. 201-220;
primers designed based on the APOB (apolipoprotein B) gene: sequence SEQ ID NO.59-152, sequence SEQ ID NO.221-322 and sequence SEQ ID NO. 347-366;
primers designed based on the PCSK9 (human proprotein convertase subtilisin/kexintype9) gene: the sequences SEQ ID NO.15-32, the sequence SEQ ID NO.167-200 and the sequence SEQ ID NO. 367-382;
primers designed according to LDLRAP1 (low density lipoprotein receptor adaptor 1) gene are SEQ ID NO.1-14, SEQ ID NO.153-166 and SEQ ID NO. 323-324;
a primer designed according to the polymorphic site (rs2479409) of the PCSK9 gene: sequence SEQ ID NO. 325-326;
a primer is designed according to a polymorphic locus (rs629301) of a CELSR2 (cadherin EGF-LAG seven-channel receptor 2 gene, cadherin EGF-LAG seven-channel-pass G-type receptor 2) gene: sequence SEQ ID NO. 327-328;
primers designed based on the polymorphic site (rs11220462) of the ST3GAL4 (sialyltransferase 4) gene: sequence SEQ ID NO. 329-330;
primers designed according to polymorphic site (rs8017377) of NYNRIN (NYN domain and retrovisual integration) gene: sequence SEQ ID NO. 331-332;
a primer designed according to the polymorphic site (rs6511720) of the LDLR gene: sequence SEQ ID NO. 333-334;
primers designed according to polymorphic sites (rs429358, rs7412) of an APOE (APOE) gene: sequence SEQ ID NO. 335-336;
a primer designed according to the polymorphic site (rs1367117) of the APOB gene: sequence SEQ ID NO. 337-338;
a primer designed according to the polymorphic site (rs4299376) of the ABCG8(ATP binding cassette B subfamily member 8 transporter gene) gene: sequence SEQ ID NO. 339-340;
primers designed based on the polymorphic site (rs3757354) of the MYLIP (myosin regulatory light chain interacting protein) gene: sequence SEQ ID NO. 341-342;
primers designed based on polymorphic site of HFE (hemochromatosis) gene (rs 1800562): sequence SEQ ID NO. 343-344;
a primer designed according to the polymorphic site (rs1564348) of the SLC22A1 (cation transporter 1) gene: the sequence SEQ ID NO. 345-346.
The primers are divided into a primer group 1, a primer group 2 and a primer group 3. The nucleotide sequences of the primers included in the primer set 1 are listed in the following table 3:
TABLE 3 nucleotide sequences of primers contained in primer set 1
The nucleotide sequences of the primers included in the primer set 2 are listed in the following table 4:
TABLE 4 nucleotide sequences of primers contained in primer set 2
The nucleotide sequences of the primers included in the primer set 3 are listed in the following table 5:
TABLE 5 nucleotide sequences of primers contained in primer set 3
The multiplex PCR reaction was divided into 3 reaction tubes, and the reagents were the same except for the primers in the primer set 1 reaction tube, the primer set 2 reaction tube, and the primer set 3 reaction tube, and the multiplex PCR reaction system (total system 30. mu.L) was as shown in Table 6 below.
TABLE 6 1 round multiplex PCR reaction System
| Composition of | Addition amount (μ L) |
| ddH2O | 9-x |
| Enhancer buffer NB(1N) | 3.5 |
| Enhancer buffer M | 2.5 |
| Primer set 1/primer set 2/primer set 3 | 5 |
| gDNA | x |
| IGT-EM808polymerase mixture | 10 |
The initial amount of gDNA was 50 ng/reaction tube; the concentration of DNA was the quantitative result of the Qubit dsDNAHS Assay Kit (Thermo Fisher); the concentration of each primer in the primer set was: 10 μ M.
And shaking and uniformly mixing the reaction mixture by using a vortex oscillator, centrifuging for 5 seconds by using a mini centrifuge, and placing the mixture into a PCR instrument for reaction. The reaction conditions are shown in Table 7 (primer sets 1 and 2) and Table 8 (primer set 3).
Note that: firstly, PCR reaction is carried out according to reaction systems and reaction conditions in the following tables 7 and 8, and the quality of the library is reduced or the library construction is failed due to the change of reaction parameters; before PCR reaction, diluting all the gDNA concentrations to the same concentration, and transferring the gDNA concentrations to a PCR 8 connecting pipe, so that on one hand, the gun arrangement operation is convenient, and on the other hand, the concentration difference of the final library is reduced and the library is convenient to mix in order to add the gDNA with the same initial amount; thirdly, when multiple PCR reactions are carried out, particularly under the condition of large sample size, setting the primer group 1 as one group, setting the primer group 2 as one group and setting the primer group 3 as one group, so as to facilitate the preparation of reaction reagent mixed liquid and the gun arrangement operation; fourthly, when the multiple PCR reaction is executed, please place the unfrozen reagent on the ice box and carry out the sample adding operation on the ice box; please mark the reaction number on the upper part of the PCR tube wall or the tube cover to prevent the mark from disappearing due to high temperature or other reasons, and avoid the sample cross contamination caused by the error of the subsequent product mixing operation.
Please follow the general PCR protocol to perform the 1 st round of multiplex PCR reaction
TABLE 7 1. multiplex PCR reaction conditions (reaction tubes for primer set 1 and primer set 2)
TABLE 8 conditions for 1 st round of multiplex PCR reaction (reaction tubes for primer set 3)
2) 1 st round multiplex PCR product pooling
After the multiple PCR reaction is finished, the PCR products of 3 reaction tubes are respectively added with a linker sequence 1(SEQ ID No.383) and a linker sequence 2(SEQ ID No.384) for a connection reaction, and after the reaction is finished, the PCR products of 3 reaction tubes are combined according to the table 9, wherein the total volume is 30 mu L. The multiplex PCR products of primer sets 1, 2 and 3 were pooled at a volume ratio of 6-8: 5-7: 1-3, preferably at the volume ratio shown in Table 9.
TABLE 9 multiplex PCR product pooling
| Reaction tube numbering | T1 | T2 | T3 |
| Mixing volume | 14μL | 12μL | 4μL |
Adding 27 mu L of AMPure XP magnetic beads balanced at room temperature into 30 mu L of PCR combined products, and gently sucking and beating the mixture by using a pipette and uniformly mixing the mixture for 20 times;
after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;
thoroughly removing the supernatant, taking down the PCR tube from the magnetic frame, adding 50 μ L YF buffer B into the tube, and gently sucking and mixing the mixture for 20 times by using a pipettor;
after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;
removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;
removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;
standing at room temperature for 3min to completely volatilize residual ethanol;
taking down the PCR tube from the magnetic frame, adding 24 μ L of nucleic-free water, gently sucking the resuspension magnetic beads by a pipette to avoid generating bubbles, and standing at room temperature for 2 min;
placing the PCR tube on the magnetic frame again, and standing for 3 min;
pipette 13.5. mu.L of the supernatant into a new 200. mu.L PCR tube, the supernatant in the tube being the combined multiplex PCR product.
3) 2 nd round linker sequence PCR reaction
Using the PCR pool purified in the previous step, a linker PCR system (total 30. mu.L) was prepared as shown in Table 10:
TABLE 10 linker PCR reaction System
| Composition of | Addition amount (μ L) |
| Purified PCR pool | 13.5 |
| Enhancer buffer M | 2.5 |
| ddH2O | 2 |
| TPE1.0Index(10μM) | 1 |
| TPE2.0Index(10μM) | 1 |
| IGT-EM808polymerase mixture | 10 |
The sequence information for TPE1.0 Index is shown in Table 11 below:
TABLE 11 sequence information for TPE1.0 Index
The sequence information for the TPE2.0 Index is shown in Table 12 below:
table 12 sequence information for TPE2.0 Index
And shaking and uniformly mixing the reaction mixture by using a vortex oscillator, centrifuging for 5 seconds by using a mini centrifuge, and placing the mixture into a PCR instrument for reaction. The reaction conditions are shown in table 13:
TABLE 13 linker PCR reaction conditions
4) Round 2 magnetic bead purification
Adding 27 mu L of AMPure XP magnetic beads balanced at room temperature into the PCR reaction product obtained in the step 3), and gently sucking and beating the mixture by using a pipette and uniformly mixing the mixture for 20 times;
after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;
thoroughly removing the supernatant, taking down the PCR tube from the magnetic frame, adding 50 μ L YF buffer B into the tube, and gently sucking and mixing the mixture for 20 times by using a pipettor;
after incubation for 5min at room temperature, the PCR tube was placed on a DynaMag-96Side magnetic frame for 3 min;
removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, and standing for 30 s;
removing the supernatant, continuously placing the PCR tube on a magnetic frame, adding 180 mu L of 80% ethanol solution into the PCR tube, standing for 30s, and completely removing the supernatant;
standing at room temperature for 3min to completely volatilize residual ethanol;
taking off the PCR tube from the magnetic frame, adding 24 μ L of nucleic-free water, gently sucking by a pipette, mixing for 20 times, resuspending the magnetic beads to avoid generating bubbles, and standing at room temperature for 2 min;
placing the PCR tube on the magnetic frame again, and standing for 3 min;
pipette 20. mu.L of the supernatant, and transfer to a new PCR tube, where the supernatant is the prepared gene detection library.
3. Quality control of gene detection library
Taking 1 mu L of the gene detection library obtained in the step 4), and using an Agilent 2200 bioanalyzer to perform library fragment length quality control and concentration quality control, wherein the fragment distribution interval of the normal library is between 280 and 420bp, the main peak is about 400bp, and no obvious small fragment and large fragment impurity peak exists; otherwise, the library sample is not in accordance with the requirements, and the library is to be rebuilt.
The results of the length control and the concentration control of the gene detection library are shown in FIG. 1, and the results show that the results both meet the requirements.
2 μ L of the gene testing library was used
2.0Fluorometer (ExKubit Plus dsDNAHS assay kit) for gene detection library concentration determination, record library concentration. The concentration of the normal library ranged from 5-40 ng/. mu.l, and the library concentration was mainly related to the quality of the template.
Example 2, 30 FH patients of DNA samples were sequenced and analyzed
The multiple target capture kit for familial hypercholesterolemia provided in example 1 is used, and the gene sequencer is started by referring to the MGISEQ-2000 gene sequencer in the prior art using the instruction, so that the DNB tube, the sequencing slide and the sequencing reagent tank are loaded, and a sequencing program is started.
After sequencing was complete, data analysis was performed using bioinformatics software of the prior art, operating exactly as described. The sequencing results are shown in Table 14:
TABLE 14 sample sequencing analysis results
And (4) conclusion:
the sequencing coverage of the gene detection library constructed in the embodiment on the LDLRAP1 gene, the PCSK9 gene, the LDLR gene and the APOB gene is 99.45-100%, probe capture is not needed, high coverage of a region to be detected is realized by utilizing multiplex PCR amplification, the gene detection library has high detection coverage and high uniformity, the sample consumption is low, and the sample requirement is low.
Sequence listing
<110> Jiangsu Baishinuo medical science and technology Co Ltd
<120> multiple targeted capture kit for familial hypercholesterolemia and detection method thereof
<130> P210123CN-CN-BSN
<160> 384
<170> PatentIn version 3.5
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<400> 21
ggagcaggtc tccccaagg 19
<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 22
agcagcccca gcacctac 18
<210> 23
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 23
ctctcttggg ctcctttctc tg 22
<210> 24
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 24
agtggggtgg tgacttacca 20
<210> 25
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 25
atttctgtgg aggtcccctc a 21
<210> 26
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 26
acacacacac acacgaagga g 21
<210> 27
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 27
cctttctgtg ttttcaaagc ccat 24
<210> 28
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 28
aagcccagcc tcctacctg 19
<210> 29
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 29
agacggagca tcccagca 18
<210> 30
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 30
tcacgggcct cttcacct 18
<210> 31
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 31
gggccacgct agacatgtg 19
<210> 32
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 32
tcactggagc tcctgggag 19
<210> 33
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 33
gtttccagct aggacacagc a 21
<210> 34
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 34
tctcccaggg atggagtgat t 21
<210> 35
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 35
tctgattctg gcgttgagag ac 22
<210> 36
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 36
atggcgagac cctgtctcta tt 22
<210> 37
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 37
tggtgttggg agacttcaca c 21
<210> 38
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 38
aggtggagct gttgcactg 19
<210> 39
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 39
aagcaaactg aggctcagac ac 22
<210> 40
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 40
agcactcatg tctcagtccc tt 22
<210> 41
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 41
gtctctagcc attggggaag ag 22
<210> 42
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 42
gttcagagga tgaaactccc cc 22
<210> 43
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 43
caggtgagat gagggctcct 20
<210> 44
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 44
cgttttcctc ttcacgccct 20
<210> 45
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 45
ccagaattcg ttgcacgcat 20
<210> 46
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 46
gtctgtcctc cagcctgtg 19
<210> 47
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 47
caggcacgtg gcactca 17
<210> 48
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 48
ggacccgtct ctgggtga 18
<210> 49
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 49
ggtctcgggg gcagct 16
<210> 50
<211> 15
<212> DNA
<213> Artificial Sequence
<400> 50
agctgggagg gggcc 15
<210> 51
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 51
ttcgagttcc actgcctaag tg 22
<210> 52
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 52
ggaaatccac ttcggcacct aa 22
<210> 53
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 53
ctcatcccag tgtttaacgg gat 23
<210> 54
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 54
ttccagagtt tccacaagga gg 22
<210> 55
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 55
cctgtgtctc atcccagtgt 20
<210> 56
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 56
cagagtttcc acaaggaggt ttc 23
<210> 57
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 57
ggcctgtgtc tcatcccagt 20
<210> 58
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 58
tccagagttt ccacaaggag gtt 23
<210> 59
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 59
ctgtgctatg tgaaagttca attgga 26
<210> 60
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 60
actgcaagat ttttcagacc aactc 25
<210> 61
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 61
cttttccatc tggatcggta agga 24
<210> 62
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 62
ccatcaatac attatggccc ttcg 24
<210> 63
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 63
ctgaagattg tgttgatctc atcttgg 27
<210> 64
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 64
acacttcctt tcgagttaag gaaaca 26
<210> 65
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 65
tatatcccag gtttccccgg aa 22
<210> 66
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 66
atcgacgtga ggttccagaa ag 22
<210> 67
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 67
tgacataatc ataaaggacc cctgtg 26
<210> 68
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 68
gaagttttag tgtactatat tgtgtaattg gagt 34
<210> 69
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 69
ggtggagatg cctttcttgt ct 22
<210> 70
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 70
ttgcacaccg tgacttcagt 20
<210> 71
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 71
ctcataactc tcattgaaaa tatacagtat ctag 34
<210> 72
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 72
ctctgtacct gctggaattg tca 23
<210> 73
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 73
gatccaaagc agcaatgcca tc 22
<210> 74
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 74
gatcttcagg ttccatcgtg ca 22
<210> 75
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 75
gccattgggg tttttggtgt ac 22
<210> 76
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 76
agtccggatt cattctgggt ct 22
<210> 77
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 77
catggacctg aacaagagct ga 22
<210> 78
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 78
cttgaattcc aagagcacac gg 22
<210> 79
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 79
gtcaactgct cctttagcgg ta 22
<210> 80
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 80
gaagttagcc acagctctgt ct 22
<210> 81
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 81
cagcattggt attcagtgtg atgac 25
<210> 82
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 82
gctatgtgtt cccaaaagca gtc 23
<210> 83
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 83
gagctcgtcg tgagattttt cag 23
<210> 84
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 84
gcttgaagga attcttgaaa acgac 25
<210> 85
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 85
catttattcc tacatgggcc tcca 24
<210> 86
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 86
ctcttttctt ttcagcccag cc 22
<210> 87
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 87
cacatgctgg gaatcgactt g 21
<210> 88
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 88
ctgttgaaag ctggccacat ag 22
<210> 89
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 89
cactccatta ctaagctcca gtgt 24
<210> 90
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 90
cttccatcac tgccaaagga ga 22
<210> 91
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 91
ttgtgtgtga gatgtgggga ag 22
<210> 92
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 92
gaccagatgc tgaacagtga g 21
<210> 93
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 93
gtgtggaaaa cctggatggg at 22
<210> 94
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 94
ctgactttgc agagcaatat tctatcc 27
<210> 95
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 95
gtctcgtgta tcttctaggg tctct 25
<210> 96
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 96
gtgaggtgac tcagagactc aatg 24
<210> 97
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 97
gcatcatcaa taaatccaac caatttctc 29
<210> 98
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 98
gtcaaacact ttgttataaa tcttattggg g 31
<210> 99
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 99
ctaggtgctg gatgtctata ttctgt 26
<210> 100
<211> 34
<212> DNA
<213> Artificial Sequence
<400> 100
agctattgct aatattattg atgaaatcat tgaa 34
<210> 101
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 101
ctgtaattct atactttttt gtgagagcag t 31
<210> 102
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 102
ccattaacct cccatttttt gagacc 26
<210> 103
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 103
tgatgggctc actgagtaaa agtg 24
<210> 104
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 104
tcgtgtctag gaaaagcatc agtg 24
<210> 105
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 105
agcgagtttc ccattgccat 20
<210> 106
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 106
atgccatctc ttctgctgcc 20
<210> 107
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 107
gtttcccatt gttggtgaga tcc 23
<210> 108
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 108
cttcaaggtc agtcaagaag gac 23
<210> 109
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 109
tttgcattgt gttccctgaa gc 22
<210> 110
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 110
cttcagcctg ctttctggat ca 22
<210> 111
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 11
cgtagttctc atactttagg gaagca 26
<210> 112
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 112
gttcagagtc tcttcgttct atgct 25
<210> 113
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 113
cccaggaact agatgcatcg aa 22
<210> 114
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 114
gagattgctc tggaaatgta ctgc 24
<210> 115
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 115
tgctgtagac attcgtggag ag 22
<210> 116
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 116
gatggccggg tcaaatatac ct 22
<210> 117
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 117
tctttcattt actttgcatg gttcaag 27
<210> 118
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 118
ttgctctttg aggctcatcc aa 22
<210> 119
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 119
aatcggagag atccacaggg aa 22
<210> 120
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 120
atggctccac agtttccaag ag 22
<210> 121
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 121
tctcacttct ggcttctgct tg 22
<210> 122
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 122
tctgtggctg tttctctgaa cc 22
<210> 123
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 123
caaaatccgg aatttggact tcact 25
<210> 124
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 124
accatgttag ccagaatggt ctt 23
<210> 125
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 125
catgatgtgg aaggtgagaa aatgc 25
<210> 126
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 126
ctcaggtatt tttgttcttc accagc 26
<210> 127
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 127
ctctgttctc tctttcaaac tggcta 26
<210> 128
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 128
ctgaactggt ggcaaaaccc t 21
<210> 129
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 129
gcaccctgga agaaagtaat aacc 24
<210> 130
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 130
tctcccttgt tgggtctaca gat 23
<210> 131
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 131
aaaccaacgt ctggtctcat gg 22
<210> 132
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 132
ggtaaatgga ataatgctca gtgttgag 28
<210> 133
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 133
cattgagacc caaagctttc ctt 23
<210> 134
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 134
ctgatgccat tttcttgtct gattttc 27
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 35
cccagggact ctctgtttat gatg 24
<210> 136
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 136
catggacttc agaaaattct ctcgg 25
<210> 137
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 137
ctcagggccc tcagtggta 19
<210> 138
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 138
aggaccagga ggttcttctt ca 22
<210> 139
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 139
cccctagtac cttccaaatc cttg 24
<210> 140
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 140
tctcctgcac cctgatggg 19
<210> 141
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 141
cacagaggtg caagatgttc ct 22
<210> 142
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 142
agtggctgaa acgtgtgcat 20
<210> 143
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 143
ggccaaggtt tgaaagttca gtc 23
<210> 144
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 144
atccacatca cctccaaagc ag 22
<210> 145
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 145
agccatgata ggcacatctt gag 23
<210> 146
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 146
tgcagagaca gggaaaacct tc 22
<210> 147
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 147
tgtattaata agaggatgct ccttgct 27
<210> 148
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 148
gatgttgccc gtgttgaata cat 23
<210> 149
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 149
ctcagcggac acacacaca 19
<210> 150
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 150
tcaggggcca cctctcatt 19
<210> 151
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 151
gagcaaggca caccacgat 19
<210> 152
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 152
aaggtgaaac ctgtgccgg 19
<210> 153
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 153
tgagagctgt tgctggtggt 20
<210> 154
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 154
tggtccttgg tccctggtt 19
<210> 155
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 155
gtgattgctg gggacagagt g 21
<210> 156
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 156
agtgaaaaac caagaggctc tcc 23
<210> 157
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 157
gggctccccc aaacatagtt c 21
<210> 158
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 158
aagcatttga gtggagggat gg 22
<210> 159
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 159
tctgcaactc aggggagct 19
<210> 160
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 160
aaggccactg gaccctgt 18
<210> 161
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 161
ggcttcccac atgtgctca 19
<210> 162
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 162
aaggctgcca aggccac 17
<210> 163
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 163
atgtgctcag gcccctgt 18
<210> 164
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 164
agcaaggctg ccaaggc 17
<210> 165
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 165
tgcagggctt cccacatg 18
<210> 166
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 166
agagcaaggc tgccaagg 18
<210> 167
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 167
cacggcctct aggtctcct 19
<210> 168
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 168
ggaaggtggc tgtggttcc 19
<210> 169
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 169
gtttgctggg tttcttccat gtc 23
<210> 170
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 170
ccaaaaaggg tggctcacca 20
<210> 171
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 171
gtgtttgctg ctgtccaaat gg 22
<210> 172
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 172
atggattcag ctcagatggg gt 22
<210> 173
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 173
acctgctgat ttgttatagg gtgg 24
<210> 174
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 174
gccaggtcca gatggagaga 20
<210> 175
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 175
ctttgtgtca cagagtggga ca 22
<210> 176
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 176
tcaggcctac ttcatctctg gt 22
<210> 177
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 177
gccgggccat caccat 16
<210> 178
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 178
ccaagcccca gcctgga 17
<210> 179
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 179
ctttttgcag gttggcagct 20
<210> 180
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 180
agcactgaca cacacacaca 20
<210> 181
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 181
cgtctttgac tctaaggccc aa 22
<210> 182
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 182
cagtgggtgc ataaggagaa aga 23
<210> 183
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 183
ccaggtctgg aatgcaaagt ca 22
<210> 184
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 184
agcaaaaaca gacccagcca 20
<210> 185
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 185
gacaacacgt gtgtagtcag ga 22
<210> 186
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 186
ccattttaaa gctcagcccc ag 22
<210> 187
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 187
gacgatgcct gcctctactc 20
<210> 188
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 188
cagactctgc gatgatggag g 21
<210> 189
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 189
ctactcccca gcctcagct 19
<210> 190
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 190
actctgcgat gatggaggtt tc 22
<210> 191
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 191
cctctactcc ccagcctcag 20
<210> 192
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 192
agactctgcg atgatggagg t 21
<210> 193
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 193
ggcaacttcc gggacgat 18
<210> 194
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 194
tctgacgact ccttccaaag c 21
<210> 195
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 195
ggacgatgcc tgcctctac 19
<210> 196
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 196
gactctgcga tgatggaggt tt 22
<210> 197
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 197
tacagccgcg tcctcaac 18
<210> 198
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 198
aagccagaag ggttcggc 18
<210> 199
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 199
ggcaacttcc gggacgatg 19
<210> 200
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 200
atggaggttt cgagcccct 19
<210> 201
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 201
tcagtgggtc tttcctttga gtg 23
<210> 202
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 202
gtagagacaa agtcagacca ctcc 24
<210> 203
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 203
gactgcttgg acggctcag 19
<210> 204
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 204
accatcacag cgccagc 17
<210> 205
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 205
aggccctgct tctttttctc tg 22
<210> 206
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 206
aagcagcaag gcacagagaa t 21
<210> 207
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 207
ggtggaggtt gtaatgagcc aa 22
<210> 208
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 208
catgtcagga agcgcagagg 20
<210> 209
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 209
cgacgggtcc cctctgac 18
<210> 210
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 210
tctcacctgc gggccaa 17
<210> 211
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 211
acatctactg gaccgactct gt 22
<210> 212
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 212
aaccagttcc tgaagctcct tc 22
<210> 213
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 213
gactggcatc agcacgtga 19
<210> 214
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 214
gtgtctatcc gccacctaag tg 22
<210> 215
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 215
ctggtatagc tgatgatctc gttcc 25
<210> 216
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 216
aggagggggc agttggag 18
<210> 217
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 217
tcttggtggc cttcctttag ac 22
<210> 218
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 218
ctgttccctg tccaggagaa aa 22
<210> 219
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 219
gtgtttcctg aatgctggac tg 22
<210> 220
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 220
ctgttctgcc tcccagatga at 22
<210> 221
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 221
tggacaggtc aatcaatctt ttgga 25
<210> 222
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 222
caagttgagc aatttctgca caga 24
<210> 223
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 223
tcactgtcca gccaactata cttg 24
<210> 224
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 224
acagctgaaa gagatgaaat ttacttatct t 31
<210> 225
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 225
ctttcaacag ttccctatac atcgaga 27
<210> 226
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 226
ctcactcatt gattttctga acttccc 27
<210> 227
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 227
tgtccttcca ctcttggtag gt 22
<210> 228
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 228
acctctctga aagacaacgt gc 22
<210> 229
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 229
ccaattctcc actcgctctt gg 22
<210> 230
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 230
ctcatctgtt tttctgcttt caggg 25
<210> 231
<211> 29
<212> DNA
<213> Artificial Sequence
<400> 231
ggtatggaga tgaagaaaat cacaatgag 29
<210> 232
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 232
cacaatgaaa acatcaacgt cctact 26
<210> 233
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 233
caagtggcat tatacacggg aga 23
<210> 234
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 234
gagataaccg tgcctgaatc tca 23
<210> 235
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 235
ggttgagggc aaatgatgaa gttc 24
<210> 236
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 236
gagacagcat cttcgtgttt caac 24
<210> 237
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 237
gtcaaggtgt gccttttctt gg 22
<210> 238
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 238
gagaacatac aagcaaagcc acc 23
<210> 239
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 239
caattttgga agtgccctgc a 21
<210> 240
<211> 27
<212> DNA
<213> Artificial Sequence
<400> 240
ctgtctcttc ctccatggaa tttaagt 27
<210> 241
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 241
gactaccctc cacaaatttg ttgc 24
<210> 242
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 242
tcctgccatg ggcaatatta cc 22
<210> 243
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 243
gtcagaacct aggatggaga aactag 26
<210> 244
<211> 28
<212> DNA
<213> Artificial Sequence
<400> 244
cagaaacaat gcattagatt ttgtcacc 28
<210> 245
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 245
gatttgtgat ggaatgcctg tgt 23
<210> 246
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 246
ctctgctgga aacaacgaga ac 22
<210> 247
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 247
cattgtttgt ggatgccgtg atc 23
<210> 248
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 248
ggtttatgaa tctggctccc tca 23
<210> 249
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 249
cccatttcca tgaccctttt cca 23
<210> 250
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 250
cacagtggca agtttacaca cag 23
<210> 251
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 251
aggaatgtcc tccaccttca ga 22
<210> 252
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 252
agtcccccta acagatttga gga 23
<210> 253
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 253
cagtgaaccc ttgctctacc aa 22
<210> 254
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 254
gcatctttgg ctcacatgaa gg 22
<210> 255
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 255
ttgtggtagt tccagagcct ga 22
<210> 256
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 256
gaagctaagc aatgtcctac aacaag 26
<210> 257
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 257
gctctgaagg cattgatttt ctcag 25
<210> 258
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 258
caagaaaaac tgcagcagct taaga 25
<210> 259
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 259
cggatatgat agtgctcatc aagact 26
<210> 260
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 260
gacaagtttc acatgccaag gag 23
<210> 261
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 261
ggtttctctg tacgttttcc agtac 25
<210> 262
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 262
ctctggctga cctaactcta ctaga 25
<210> 263
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 263
gggcactgac tttgtgttca ag 22
<210> 264
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 264
tgtcttccgt tctgtaatgg cc 22
<210> 265
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 265
caccctgaac cttagcaaca gt 22
<210> 266
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 266
actgttaatt tacagctaca gccct 25
<210> 267
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 267
tcagcatatg agcccatcat gtc 23
<210> 268
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 268
tggagaatga gctgaatgca gag 23
<210> 269
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 269
gtcagtgcct aagatgtcag ca 22
<210> 270
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 270
ctgatctgca aagtggcatc att 23
<210> 271
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 271
tctgacaaga caggccatat gtg 23
<210> 272
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 272
gatgggtctc tacgccacaa at 22
<210> 273
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 273
ttagagcctg ccatgaacta gc 22
<210> 274
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 274
gttgtatcaa ctgcaagtgc ctc 23
<210> 275
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 275
tgccaccaaa aggcaaagga 20
<210> 276
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 276
aggtggaggt aaaagcaaag gg 22
<210> 277
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 277
agttgaggat gtaattagca cttatatcca 30
<210> 278
<211> 30
<212> DNA
<213> Artificial Sequence
<400> 278
cagatgaaga gaagattgaa tttgaatgga 30
<210> 279
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 279
gtctcaacac ctgcattact ttgg 24
<210> 280
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 280
tggtgccaac tgattttctt tacc 24
<210> 281
<211> 17
<212> DNA
<213> Artificial Sequence
<400> 281
aggcctgcag tgcaggt 17
<210> 282
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 282
tccaggtgcg aagcagact 19
<210> 283
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 283
cctcatgaat tctgaacctg agact 25
<210> 284
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 284
ttgggaacat aatcaccatc cttga 25
<210> 285
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 285
ccgttttggt ggtagagacc aa 22
<210> 286
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 286
ggaaagaact cagccagaag ct 22
<210> 287
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 287
ccagctttta gggcaacatg ag 22
<210> 288
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 288
aggtgagaaa gggtggttga tc 22
<210> 289
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 289
tccagtttta ctccagcctt gg 22
<210> 290
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 290
ctctccaatg tatgggaggt tgt 23
<210> 291
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 291
aaaaccaagc tcctctccca ag 22
<210> 292
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 292
ctgttcctca cctggagcat tt 22
<210> 293
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 293
agccaaagtg gtccactaag ac 22
<210> 294
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 294
tggacatcaa gagtggggac ta 22
<210> 295
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 295
ccggagaagc atcatcaagg aa 22
<210> 296
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 296
tgctctagga aacactggca ag 22
<210> 297
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 297
gggaagtaaa aggtgtccag gaa 23
<210> 298
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 298
gacaaaccta actggggtta cca 23
<210> 299
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 299
atccctcgcc atgttgaaga tc 22
<210> 300
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 300
cagaggacac accatgagca at 22
<210> 301
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 301
cctggagagt cttcaaaaca gct 23
<210> 302
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 302
acctatggca gagccagatc ta 22
<210> 303
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 303
gagcacttga gaagtgttca gttc 24
<210> 304
<211> 26
<212> DNA
<213> Artificial Sequence
<400> 304
gcttttaaca gaagatacag ggactg 26
<210> 305
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 305
tgaagatgag tttcaagggc ca 22
<210> 306
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 306
cttccagcaa gttgtaatgg agaag 25
<210> 307
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 307
gggaaagctg tgggctctag 20
<210> 308
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 308
ctctgcccag aattggctgt 20
<210> 309
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 309
ggtttgagag ttgtgcattt gct 23
<210> 310
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 310
caagttcctg accttcacat acca 24
<210> 311
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 311
gggtttgaga gttgtgcatt tgc 23
<210> 312
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 312
caagttcctg accttcacat accag 25
<210> 313
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 313
tcttagggtt tgagagttgt gca 23
<210> 314
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 314
gttcctgacc ttcacatacc aga 23
<210> 315
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 315
gctgggtcaa gtgatggaag ag 22
<210> 316
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 316
ttggggtgca agagtcaaag g 21
<210> 317
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 317
tattttggct gaggctgggt ca 22
<210> 318
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 318
ttggggtgca agagtcaaag ga 22
<210> 319
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 319
ctgggtcaag tgatggaaga ga 22
<210> 320
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 320
cttggggtgc aagagtcaaa g 21
<210> 321
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 321
gccctccctc tggcctag 18
<210> 322
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 322
tgaggagccc gcccag 16
<210> 323
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 323
gcttcccaca tgtgctcagg 20
<210> 324
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 324
tcccacacca gacagccc 18
<210> 325
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 325
tcctgcctgg tacacaatag gt 22
<210> 326
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 326
accttaccaa ctagctgctc ct 22
<210> 327
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 327
cctcagcaat tcctgcaaag g 21
<210> 328
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 328
aggacagggt accacacaga a 21
<210> 329
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 329
gcgatgctat ccgatgaaca ga 22
<210> 330
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 330
gcttctctac ttcccagcaa ca 22
<210> 331
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 331
ttccctttgg aggattgctc ac 22
<210> 332
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 332
tccatattcg gcagactctc ca 22
<210> 333
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 333
ccggggatga tgatgattgc aa 22
<210> 334
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 334
cacaccagct ctcagaagtc t 21
<210> 335
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 335
gctgtccaag gagctgca 18
<210> 336
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 336
ggatggcgct gaggcc 16
<210> 337
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 337
aagttcatac ctcagcggac ac 22
<210> 338
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 338
tctgcagctt catcctgaag ac 22
<210> 339
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 339
caaggatcca tattcccaca gct 23
<210> 340
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 340
catcacctta ttcaatcctc actgc 25
<210> 341
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 341
caaagaactg tgtgtgggag ga 22
<210> 342
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 342
gcatttgaat gtccccgtgt atg 23
<210> 343
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 343
tactaccccc agaacatcac ca 22
<210> 344
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 344
cagatcacaa tgaggggctg at 22
<210> 345
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 345
cagtctatgg ccagagtgat catg 24
<210> 346
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 346
gaagctgcct cccaatgttt tg 22
<210> 347
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 347
ggtttgagag ttgtgcattt gct 23
<210> 348
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 348
caagttcctg accttcacat acca 24
<210> 349
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 349
gggtttgaga gttgtgcatt tgc 23
<210> 350
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 350
caagttcctg accttcacat accag 25
<210> 351
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 351
tcttagggtt tgagagttgt gca 23
<210> 352
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 352
gttcctgacc ttcacatacc aga 23
<210> 353
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 353
tcttagggtt tgagagttgt gcat 24
<210> 354
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 354
gttcctgacc ttcacatacc agaa 24
<210> 355
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 355
ggtttgagag ttgtgcattt gctt 24
<210> 356
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 356
ccttcacata ccagaattcc agct 24
<210> 357
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 357
gctgggtcaa gtgatggaag ag 22
<210> 358
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 358
ttggggtgca agagtcaaag g 21
<210> 359
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 359
tattttggct gaggctgggt ca 22
<210> 360
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 360
ttggggtgca agagtcaaag ga 22
<210> 361
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 361
ctgggtcaag tgatggaaga ga 22
<210> 362
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 362
cttggggtgc aagagtcaaa g 21
<210> 363
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 363
ggtcaagtga tggaagagaa acaga 25
<210> 364
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 364
agacccaaag acttagggtc aatg 24
<210> 365
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 365
gggtcaagtg atggaagaga aacag 25
<210> 366
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 366
cagatctaga cccaaagact taggg 25
<210> 367
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 367
cctctactcc ccagcctcag 20
<210> 368
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 368
agactctgcg atgatggagg t 21
<210> 369
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 369
gcctgcctct actcccca 18
<210> 370
<211> 24
<212> DNA
<213> Artificial Sequence
<400> 370
tgctattcag actctgcgat gatg 24
<210> 371
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 371
cctgcctcta ctccccagc 19
<210> 372
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 372
tgctattcag actctgcgat ga 22
<210> 373
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 373
gcctgcctct actccccag 19
<210> 374
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 374
tgctattcag actctgcgat gat 23
<210> 375
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 375
gacgatgcct gcctctactc 20
<210> 376
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 376
cactgctatt cagactctgc ga 22
<210> 377
<211> 18
<212> DNA
<213> Artificial Sequence
<400> 377
ggcaacttcc gggacgat 18
<210> 378
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 378
actgctattc agactctgcg atg 23
<210> 379
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 379
ggacgatgcc tgcctctac 19
<210> 380
<211> 23
<212> DNA
<213> Artificial Sequence
<400> 380
cactgctatt cagactctgc gat 23
<210> 381
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 381
gcaacttccg ggacgatgc 19
<210> 382
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 382
cactgctatt cagactctgc g 21
<210> 383
<211> 38
<212> DNA
<213> Artificial Sequence
<400> 383
ttgtcttcct aaggaacgac atggctacga tccgactt 38
<210> 384
<211> 31
<212> DNA
<213> Artificial Sequence
<400> 384
agtcggaggc caagcggtct taggaagaca a 31
Claims (10)
1. A multiple targeted capture kit for familial hypercholesterolemia, characterized in that: comprises primers SEQ ID NO.1-324 and primer SEQ ID NO. 347-382; wherein the content of the first and second substances,
the SEQ ID NO.1-14, SEQ ID NO.153-166 and SEQ ID NO.323-324 are used for detecting the LDLRAP1 gene;
the SEQ ID NO.15-32, SEQ ID NO.167-200 and SEQ ID NO.367-382 are used for detecting the PCSK9 gene;
the SEQ ID NO.33-58 and the SEQ ID NO.201-220 are used for detecting the LDLR gene;
SEQ ID NO.59-152, SEQ ID NO.221-322 and SEQ ID NO.347-366 were used for the detection of the APOB gene.
2. The multiple targeted capture kit of familial hypercholesterolemia according to claim 1, wherein: the primers SEQ ID NO.1-324 and the primers SEQ ID NO.347-382 were divided into a primer set 1, a primer set 2 and a primer set 3.
3. The multiple targeted capture kit of familial hypercholesterolemia according to claim 2, wherein: the primer set 3 further comprises a primer SEQ ID NO.325-346, wherein,
the SEQ ID NO.325-326 is used for detecting the polymorphic locus rs2479409 of the PCSK9 gene;
the SEQ ID NO.327-328 is used for detecting the polymorphic site rs629301 of the CELSR2 gene;
the SEQ ID NO.329-330 is used for detecting the ST3GAL4 gene polymorphism site rs 11220462;
the SEQ ID NO.331-332 is used for detecting NYNRIN gene polymorphism locus rs 8017377;
the SEQ ID NO.333-334 is used for detecting the LDLR gene polymorphism locus rs 6511720;
the SEQ ID NO.335-336 gene is used for detecting polymorphic sites rs429358 and rs7412 of the APOE gene;
the SEQ ID NO.337-338 is used for detecting APOB gene polymorphism site rs 1367117;
the SEQ ID NO.339-340 is used for detecting the ABCG8 gene polymorphism site rs 4299376;
SEQ ID NO.341-342 for detecting MYLIP gene polymorphic site rs 3757354;
SEQ ID NO.343-344 is used for detecting HFE gene polymorphic site rs 1800562;
the SEQ ID NO.345-346 is used for detecting the SLC22A1 gene polymorphic site rs 1564348.
4. The multiple targeted capture kit of familial hypercholesterolemia according to claim 1, wherein: the polypeptide also comprises an adapter sequence 1 and an adapter sequence 2, wherein the sequence of the adapter sequence 1 is SEQ ID NO.383, and the sequence of the adapter sequence 2 is SEQ ID NO. 384.
5. The detection method of familial hypercholesterolemia multiple-targeted capturing kit for non-diagnostic purposes as claimed in claim 1, wherein primers SEQ ID NO.1-324 and primers SEQ ID NO.347-382 are used to perform 1 st round of multiplex PCR reaction and 2 nd round of linker sequence PCR reaction on DNA sample to obtain a gene detection library for sequencing analysis; the primers SEQ ID NO.1-324 and the primers SEQ ID NO.347-382 are divided into a primer set 1, a primer set 2 and a primer set 3.
6. The method for detecting familial hypercholesterolemia multiple targeted capture kit of claim 3 for non-diagnostic purposes, characterized in that: primer SEQ ID NO.1-382 is adopted to carry out 1 st round of multiplex PCR reaction and 2 nd round of adaptor sequence PCR reaction on the DNA sample to obtain a gene detection library for sequencing analysis.
7. The method for detecting familial hypercholesterolemia multiple targeted capturing kit of claim 5 or 6 for non-diagnostic purposes, characterized in that: after the 1 st round of multiplex PCR reaction of the primer sets 1 to 3, the multiplex PCR amplification products of the primer sets 1 to 3 were pooled at a volume ratio of 6-8: 5-7: 1-3.
8. The method for detecting familial hypercholesterolemia multiple-targeted capturing kit for non-diagnostic purposes as claimed in claim 5 or 6, wherein the reaction conditions of the 1 st round of multiplex PCR reaction of the primer set 1 and the primer set 2 are as follows:
9. the method for detecting familial hypercholesterolemia multiple-targeted capturing kit for non-diagnostic purposes as claimed in claim 5 or 6, wherein the reaction conditions of the 1 st round of multiplex PCR reaction of the primer set 3 are as follows:
10. a gene testing library for familial hypercholesterolemia, comprising: the gene detection library is prepared by the detection method of any one of claims 5 to 9.
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Citations (4)
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|---|---|---|---|---|
| WO2014181107A1 (en) * | 2013-05-09 | 2014-11-13 | Medical Research Council | Genetic method of aiding the diagnosis and treatment of familial hypercholersterolaemia |
| CN109517884A (en) * | 2018-09-30 | 2019-03-26 | 北京安智因生物技术有限公司 | A kind of construction method and its kit in the genetic test library of familial hypercholesterolemia |
| CN111269995A (en) * | 2018-12-04 | 2020-06-12 | 深圳华大因源医药科技有限公司 | Primer group, kit and detection method for detecting pathogen |
| CN111808855A (en) * | 2020-07-20 | 2020-10-23 | 北京安智因生物技术有限公司 | Construction method of universal gene detection library for hereditary familial hypercholesterolemia and kit thereof |
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2021
- 2021-08-19 CN CN202110957302.8A patent/CN113684270B/en active Active
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| WO2014181107A1 (en) * | 2013-05-09 | 2014-11-13 | Medical Research Council | Genetic method of aiding the diagnosis and treatment of familial hypercholersterolaemia |
| CN109517884A (en) * | 2018-09-30 | 2019-03-26 | 北京安智因生物技术有限公司 | A kind of construction method and its kit in the genetic test library of familial hypercholesterolemia |
| CN111269995A (en) * | 2018-12-04 | 2020-06-12 | 深圳华大因源医药科技有限公司 | Primer group, kit and detection method for detecting pathogen |
| CN111808855A (en) * | 2020-07-20 | 2020-10-23 | 北京安智因生物技术有限公司 | Construction method of universal gene detection library for hereditary familial hypercholesterolemia and kit thereof |
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