CN108588236A - The SNP rapid detection methods and SNP site and its primer of a kind of inbred strais quality of heredity monitoring - Google Patents

The SNP rapid detection methods and SNP site and its primer of a kind of inbred strais quality of heredity monitoring Download PDF

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CN108588236A
CN108588236A CN201810475211.9A CN201810475211A CN108588236A CN 108588236 A CN108588236 A CN 108588236A CN 201810475211 A CN201810475211 A CN 201810475211A CN 108588236 A CN108588236 A CN 108588236A
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CN108588236B (en
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琚存祥
赵静
马秀英
张明坤
侯欢欢
高翔
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Jiangsu Collection Pharmacy Biotechnology Co Ltd
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Abstract

The invention discloses a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring, the designs of SNP panel first：Choose inbred mouse strain, screening distinguishes site and conventional genetic quality monitoring site with other strain specificities, the former should include at least 5 pairs chromosomes that site is distinguished containing specificity as unit of chromosome, in SNP panel, and each pair of chromosome should contain 2 or more specificity and distinguish site；For the latter on the basis of specificity distinguishes site, it is 96 to supply the site of SNP panel according to 45 equidistant site principles of each pair of chromosome；Then SNP site primer is designed；Sample DNA is extracted again, carries out Genotyping with KASP methods, and compared with the SNP site information on NCBI, completes the monitoring of inbred mouse quality of heredity.The invention also discloses the SNP sites and SNP site primer of a kind of monitoring of inbred strais quality of heredity.

Description

The SNP rapid detection methods and SNP site of a kind of monitoring of inbred strais quality of heredity and its Primer
Technical field
The invention belongs to the identifications of inbred mouse strain genetic background and genetic pollution detection field, are related to a kind of inbred strais Quality of heredity monitoring SNP rapid detection methods more particularly to it is a kind of based on KASP methods carry out SNP partings detection method and SNP site and its primer.
Background technology
Quality of Experimental Animals control be experimental animal industry healthy development key problem, mouse microbial quality control and Genetic background quality control is important factor for controlling therein.Domestic quality of heredity monitoring lacks ripe professional standard, builds A kind of vertical quick and high accuracy high-throughput genotyping technique platform is most important.
Mainly there are 3 kinds for genetic background test method of quality control：Biochemical marker analytic approach, microsatellite DNA, SNP are (single The polymorphism of nucleotide) detection etc..The Genetic Detection method of world defined is biochemical marker analytic approach at present, this method is The variation of isodynamic enzyme or isomerism protein enzyme is detected to speculate corresponding gene variation；It is detected using the method, it is accurate to exist Spend the drawbacks such as low, sensitivity is low, detection site is limited, the hereditary general picture of reflection is limited.And molecular genetic marker can be dynamic to testing Object carries out finer supervision, is a kind of more perfect Quality of Experimental Animals detection means；Wherein SNP detections are used as molecular genetic A special kind of skill of label detects DNA sequence polymorphism caused by a single nucleotide variation in genomic level；SNP The polymorphism showed can monitor the variation of single base, have the characteristics that high density, representativeness, genetic stability, energy The heredity of enough comprehensively reflection genomes and variation situation.
KASP methods refer to competitive ApoE gene (Kompetitive Allele Specific PCR), with Based on highly sensitive fluoroscopic examination, accurately diallele parting is carried out to target SNPs.With traditional Taqman technologies Unlike, such method need not be closed it is not necessary that target spot, that is, specific primer/probe is marked according to each SNP site At special fluorescent primer, unique ARM PCR principles allow all site primers finally all to expand using universal fluorescent primer Increase, greatly reduces experimental cost.The PCR system of optimization can meet the needs of different loci high pass quantitative response, existing goldstandard It is accurate, and reduce use cost, there is better site adaptability than Taqman.KASP technologies are by traditional detection equipotential base 2 reactions of cause have synthesized 1 reaction, at low cost.SNPs detection not only compensate for normal PCR, cut glue, sequencing flow time it is long The shortcomings that, and the expense of sequencing is greatly saved.
Invention content
Current SNP detection panel due to being restricted by operating technology and testing cost, cause pattern detection flux compared with Low, site selection is restricted, and for disadvantage existing in the prior art, the purpose of the present invention is to provide a kind of high passes Amount, multidigit point, low cost, the SNP rapid detection methods for being rapidly used for the monitoring of inbred strais quality of heredity
The present invention also aims to provide the SNP site that one group of site is used as in the monitoring of inbred strais quality of heredity to answer With.
The present invention also aims to provide a kind of SNP site primer of inbred strais quality of heredity monitoring.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of SNP rapid detection methods of inbred strais quality of heredity monitoring, include the following steps：
(1) design of SNP panel：Determine inbred mouse strain, filter out C57BL/6 strains and other strains can Specificity distinguishes site and strain conventional genetic quality monitoring site, and it is as follows that the specificity distinguishes site：C57BL/6 strains position The base of point is different from corresponding base in 129S1/SvIm, BALB/C, A/J, CBA, DBA, FVB, NOD strain, with chromosome Should include at least 5 pairs chromosomes that site is distinguished containing specificity for unit, in SNP panel, and each pair of chromosome should contain 2 The above specificity distinguishes site；The strain conventional genetic quality monitoring site is as follows：The base in site is distinguished in the specificity On plinth, according to 4-5 site principle on each pair of chromosome, supplying for site is equally spacedly carried out, by C57BL/6 strains It is 96 that SNPpanel, which is supplied in site,；The SNP panel can be used for C57BL strains inbred mouse and corresponding mutantion line mouse Genetic monitoring and strain pollution condition detection and C57BL Strains of Mouse and other strains effective discriminating；
(2) primer for designing and synthesizing the sites above-mentioned SNP panel is not required to design probe；The sites SNP panel upstream and downstream Each 100bp sequences of sequence are pulled using programming tool in mouse genome sequences；Wherein, 5 ' end sense primer design： 5 ' end sense primers share two, include the sequence of primer precursor and one section of recognizable FAM or HEX signal；Before the primer Body is located at the upstream in the sites each SNP panel, and length 20-30bp is respectively SNP panel in the end of primer precursor Two mutating alkali yls of point；The sequence of the recognizable FAM or HEX signals is located at 5 ' ends of the primer precursor, and length is about 20bp；3 ' end downstream primers are one, length about 18-29bp；
Above-mentioned 3 primers are carried out at the same time PCR amplification, if generating one of signal in system, illustrate sample template In contain the base mutation type；To 96 sites using check clone 129S1/Svlm subbreed 129S1/SvlmJNju and C57BL/6 subbreed C57BL/6JNju carries out primer test, if success parting, then it represents that primer is successfully tested, and is otherwise lost to test Upstream and downstream of the site lost in chromosome corresponding position, which is found, replaces site；96 sites being successfully tested and PCR primer test Condition and system are determined as final detection scheme；
(3) sample to be tested DNA is extracted, when sample concentration is more than 10ng/uL, with KASP methods to 96 SNP of each sample The sites panel carry out Genotyping, analyze data and compared with the SNP site information on NCBI, complete inbred mouse heredity Quality monitoring and ore grade indexes.
Further, in the step (1), inbred mouse strain be A/J, 129S1/SvIm, BALB/C, C57BL/6, CBA、DBA、FVB、NOD；The specificity of the C57BL/6 strains and other 7 strains, which can distinguish site, 69, specifically such as Under：
Further, in the step (3), concentration necessary=5ng/uL × species to be measured genome of sample to be tested DNA Size/human genome size.
Further, the reaction system of the KASP methods is as follows：Exist including DNA (5-50ng/uL, by taking people or mouse as an example) The volume of each reacting hole is 0.8uL, and 2 × KASP Master mix are 0.778uL, KASP in the volume of each reacting hole Primer mix are 0.022uL in the volume of each reacting hole.
Further, the response procedures of the KASP methods are as follows：
Or
Or
Application of one group of 96 site in the monitoring of inbred strais quality of heredity as SNP site, 96 sites are as follows：
* it is that specificity distinguishes site.
A kind of SNP site primer of inbred strais quality of heredity monitoring, the primer sequence such as SEQ ID NO.1 to SEQ ID Shown in NO.288.
The device have the advantages that as follows：
Detection method completes the site primer (96 sites of each sample detection) of 36 samples of 1 project, The total period needed is only 6 working days.The method of the present invention improves the detectable number of sites and high throughput of single sample Detection makes greatly shorten experimental period, ensure that the accuracy of result while improving flux in addition.
The C57BL SNP panel that the present invention uses contain 96 sites altogether, except for C57BL strains and related mutation product Outside the conventional genetic quality monitoring of system, it can be used for and the area of A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB, NOD strain Point.
The present invention, which uses, is not necessarily to that target spot, that is, specific primer is marked, and need not design probe, need not be according to every A SNP site synthesizes special fluorescent primer, and all site primers finally all use universal fluorescent primer amplification, need not pass The race glue of system PCR cuts the flows such as glue, sequencing, saves detection time, and the expense of sequencing is greatly saved；In addition KASP Method carries out diallele parting to target SNPs, and 2 reactions of traditional detection allele have been synthesized 1 reaction, cost It reduces.
Description of the drawings
Fig. 1 is the flow chart of detection method.
Specific implementation mode
The present invention is described further with reference to the accompanying drawings and examples.
The SNP rapid detection methods of the inbred strais quality of heredity monitoring of the present embodiment, flow is as shown in Figure 1, specific step Suddenly it sees below：
Embodiment 1：SNP panel designs
1, the inbred strain for including in SNP panel is determined
It chooses 13 kinds of inbred mouse strains and carries out routine SNP detections (such as table 1), 13 kinds of inbred mouse strains are by south Capital university-Nanjing biological medicine research institute (NBRI) provides (public offering), is found in screening process, C57BL/6J with C57BL/6N、C57BL/10J、C57BLKS/J、B6(Cg)-Tyrc-2JGenetic background is similar；Meanwhile DBA/1 and DBA/2 heredity Background is similar.Therefore, C57BL/6J is chosen in C57BL as representing, selection DBA/1, which is used as, in DBA represents, final to determine 8 Inbred strais detects panel (such as table 2) to establish the SNP of C57BL/6 inbred strains.The C57BL/6 strains SNP in subsequent experimental Panel can be used for conventional SNP detections, for the differentiation with A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB, NOD strain； It can be used for C57BL/6N, C57BL/10, C57BLKS, B6 (Cg)-Tyr simultaneouslyc-2JEqual C57BL strains and related mutation strain Conventional genetic quality monitoring.
1 research institute's inbreeding series of tables of table
Serial number Family name
1 C57BL/6NNju
2 C57BL/6JNju
3 CBA/CaJNju
4 DBA/2JNju
5 A/JNju
6 BALB/cJNju
7 C57BL/10JNju
8 FVB/NJNju
9 C57BLKS/JNju
10 DBA/1JNju
11 NOD/ShiLtJNju
12 B6(Cg)-Tyrc-2J/Nju
13 129S1/SvImJNju
Table 2 is used to establish the inbreeding series of tables of C57BL/6SNP detections panel
2, SNP Panel design directions
The database provided with LGC companies<mouse-marker-excel-file>It is screened.SNP panel designs Mainly carried out from two broad aspects：Filter out the site for capableing of specificity differentiation with other strains --- it is used for strain pollution detection； Screen the site of strain conventional genetic quality monitoring.
3, the site that specificity is distinguished between inbred strais and other strains is screened
A) purpose is screened：For strain pollution detection.
B) design principle：It should include at least 5 pairs as unit of chromosome, in SNP panel and distinguish site containing specificity Chromosome, and each pair of chromosome should contain 2 or more specificity and distinguish site, then it is assumed that specificity distinguishes panel can be by strain Mouse distinguishes with other inbred strais.It can specificity differentiation site：In a certain strain, site and other strain sites are not It is identical.If a SNP detection site can not be distinguished with other strains completely, cannot if being mutated after detection Determination is genetic drift or strain pollution.
The frequency of quality of heredity monitoring is 1 year/time, if polluting, the longest pollution period should be 1 year, pollute mouse Algebraically up to 4 generations.Forth generation mouse occur chromosomal contamination minimum 5 of probability (according to F1 generation and other strains with numerous, Follow-up mouse all carries out backcrossing calculating with pure background strain.Then F2 generation pollutions chromosome is that all, it is 10 that F3, which pollutes chromosome, Item, it is 5 that F4, which pollutes chromosome item number).
It is calculated with this probability, if 5 or more chromosomes contain specific position, panel can be by Strains of Mouse It is distinguished with other inbred strais.Then think that testing result has referential containing 2 or more sites on every chromosome.
C) the selection result：C57BL/6 inbred mouses filter out 69 altogether can specificity differentiation site with other strains.It should SNP detections of the panel for C57BL strains and related mutation system, can be used for A/J, 129S1/SvIm, BALB/C, CBA, The differentiation of DBA, FVB, NOD strain.
Strain specificity distinguish site screened using the mode of programming, screening principle be same strain in, screening with The site that other strains are all different.The site filtered out carries out artificial postsearch screening according to equidistant principle on chromosome (number of sites is 4-5 on every chromosome, and specific position number should be no less than 2) determines that the specificity of each strain is distinguished panel。
4, the site of strain conventional genetic quality monitoring is screened
A) purpose is screened：For strain conventional genetic quality monitoring.
B) design principle：The usual quantity in strain specificity site is very few, is fewer than conventional genetic quality monitoring.In determination On the basis of above-mentioned specificity distinguishes site panel, it is 96 that we, which supply each strain detection panel SNP quantity, Effect is as follows：Not only it can ensure SNP testing result reliabilities, but also identification is facilitated to operate.
On the basis of specific position, conventional site is carried out according to 4-5 on every chromosome equidistant site principles Supply；Meanwhile being preferentially chosen at the site of more applicabilities in a variety of strain panel.
C) the selection result：The C57BL/6J strain quality of heredity monitoring SNP detections comprising 96 sites are filtered out altogether Panel, as shown in following table (i.e. table 3).
Table 3C57BL/6 strain quality of heredity monitors SNP and detects panel
Remarks：* it is that specificity distinguishes site.
Embodiment 2：SNP site design of primers
After the completion of SNP site screening, each 100bp sequences of SNP site upstream and downstream sequence are using programming tool in murine genes It is pulled in group sequence；5 ' end design of primers：The primer of 20-30bp is separately designed in the upstream of SNP site, at the end of primer End is respectively two mutating alkali yls of SNP, and the end of primer 5 ' increases by the sequence for identifying unlike signal respectively of one section of about 20bp, such as FAM and HEX signals；3 ' design downstream primers, length about 18-29bp.
Tm (DEG C) is between 55-65 DEG C, and GC% is between 34%-60%, total design primer 288, specific primer sequence Refer to following table.The PCR amplification detection in 96 sites, 3 primers in each site is arranged each sample to be carried out at the same time PCR expansions Increase, if generating one of signal in system, illustrates to contain the base mutation type in sample template.
4 site primer information of table
Embodiment 3：KASP methods carry out Genotyping
1, the DNA of extraction detected sample is as template
The rat-tail DNA of inbred mouse is extracted using the oKtopureTM high throughput DNA extraction apparatus of LGC companies.Extraction step Rapid reference instrument explanation.The extraction flux of DNA, 3,500 sample/days can be effectively improved；Extraction rate：20-30mg originates sample This amount, 8 × 96≤1.5h, 80-100mg original samples amounts, 8 × 96≤2h.
2, DNA concentration demand
Each of most of KASP detections reaction needs 5-50ng DNA；DNA concentration demand changes according to Genome Size： Species DNA concentration demand=5ng/uL to be measured × species to be measured Genome Size/human genome size.
3, the preparation of PCR reaction systems
Carry out the preparation of reaction system automatically using the IntelliQube instruments of LGC companies.KASP Genotyping reactants System, as shown in the table：
Table 5KASP Genotyping reaction systems
4, PCR reacts
KASP methods are selected in PCR reactions, use the Hydrocycler2 of LGC companiesTMWater-bath PCR thermal cycler water-bath systems into Row PCR reactions.KASP Genotypings response procedures can be divided into following three kinds, any one can complete to operate, such as table 6-8 institutes Show.
Table 6KASP Genotypings response procedures (1)
Or table 7KASP Genotypings response procedures (2)
Or table 8KASP Genotypings response procedures (3)
5, high-throughput Genotyping and data analysis
It is read using IntelliQube fluoroscopic examinations, the data analysis after PCR is carried out using IntelliScore, Genotype is derived automatically to be analyzed.
6, SNP Classification Identifications are carried out to C57BL/6 subbreed C57BL/6JNju mouse
With 129S1/SvImJNju mouse, strain, the primer that 96 sites are carried out to C57BL/6JNju are tested as a contrast And the Genotyping of mouse；The result shows that C57BL/6JNju in 96 sites with the site information registered on NCBI all one It causes, refers to the following table 9.
Table 9C57BL/6JNju subbreed SNP testing results
Case study on implementation：
Detecting step and method are consistent, the difference is that sample DNA is different.10 strains are chosen altogether, separately include C57BL The mutantion line mouse of related strain and C57BL/6 carry out SNP site detection, check mouse genetic quality condition, as a result show A site rs3725940 and ncbi database are inconsistent on C57BL/10JNju Chromosome 18 in Mice, and are mutant homozygous； In C57BL/6 mutantion line mouse, the site of 9 strains is consistent with C57BL/6J, specifically see the table below.
10 10 C57BL inbred strais of table and mutantion line mouse SNP testing results
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<213>Artificial sequence
<400> 41
gaaggtcgga gtcaacggat tcagccggct ttctcatgag tg 42
<210> 42
<211> 23
<212> DNA
<213>Artificial sequence
<400> 42
ttaacatcca ggatgtgctc tgg 23
<210> 43
<211> 45
<212> DNA
<213>Artificial sequence
<400> 43
gaaggtgacc aagttcatgc tgtgcccact acaagatcag tgact 45
<210> 44
<211> 44
<212> DNA
<213>Artificial sequence
<400> 44
gaaggtcgga gtcaacggat ttgcccacta caagatcagt gacc 44
<210> 45
<211> 21
<212> DNA
<213>Artificial sequence
<400> 45
gtttggttga tgctatggcg a 21
<210> 46
<211> 46
<212> DNA
<213>Artificial sequence
<400> 46
gaaggtgacc aagttcatgc tgctgagtat gccattatca aaggaa 46
<210> 47
<211> 46
<212> DNA
<213>Artificial sequence
<400> 47
gaaggtcgga gtcaacggat tgctgagtat gccattatca aaggag 46
<210> 48
<211> 22
<212> DNA
<213>Artificial sequence
<400> 48
agaaagcaga ggatgcagaa cc 22
<210> 49
<211> 46
<212> DNA
<213>Artificial sequence
<400> 49
gaaggtgacc aagttcatgc tctccactag tccctatttt ctccca 46
<210> 50
<211> 46
<212> DNA
<213>Artificial sequence
<400> 50
gaaggtcgga gtcaacggat tctccactag tccctatttt ctcccc 46
<210> 51
<211> 23
<212> DNA
<213>Artificial sequence
<400> 51
gtgaaagggt tgtggtagga atg 23
<210> 52
<211> 46
<212> DNA
<213>Artificial sequence
<400> 52
gaaggtgacc aagttcatgc tcagttggga gtgtttaagg ttgtat 46
<210> 53
<211> 46
<212> DNA
<213>Artificial sequence
<400> 53
gaaggtcgga gtcaacggat tcagttggga gtgtttaagg ttgtag 46
<210> 54
<211> 23
<212> DNA
<213>Artificial sequence
<400> 54
tctcttatcc cagggtgggt tca 23
<210> 55
<211> 44
<212> DNA
<213>Artificial sequence
<400> 55
gaaggtgacc aagttcatgc ttgtgggact gttgggaata actg 44
<210> 56
<211> 44
<212> DNA
<213>Artificial sequence
<400> 56
gaaggtcgga gtcaacggat ttgtgggact gttgggaata actc 44
<210> 57
<211> 26
<212> DNA
<213>Artificial sequence
<400> 57
cttccacctc taccctaaga tttcta 26
<210> 58
<211> 45
<212> DNA
<213>Artificial sequence
<400> 58
gaaggtgacc aagttcatgc tttaccagtt ttggcttaat ttccg 45
<210> 59
<211> 46
<212> DNA
<213>Artificial sequence
<400> 59
gaaggtcgga gtcaacggat tcttaccagt tttggcttaa tttcca 46
<210> 60
<211> 22
<212> DNA
<213>Artificial sequence
<400> 60
aatggaatat gggagtgagg ga 22
<210> 61
<211> 43
<212> DNA
<213>Artificial sequence
<400> 61
gaaggtgacc aagttcatgc tcttgtccca ggtctagatt taa 43
<210> 62
<211> 43
<212> DNA
<213>Artificial sequence
<400> 62
gaaggtcgga gtcaacggat tcttgtccca ggtctagatt tag 43
<210> 63
<211> 21
<212> DNA
<213>Artificial sequence
<400> 63
ggcagagtag gctaatagaa t 21
<210> 64
<211> 45
<212> DNA
<213>Artificial sequence
<400> 64
gaaggtgacc aagttcatgc tcaagtctgg taagattgag gatga 45
<210> 65
<211> 45
<212> DNA
<213>Artificial sequence
<400> 65
gaaggtcgga gtcaacggat tcaagtctgg taagattgag gatgg 45
<210> 66
<211> 20
<212> DNA
<213>Artificial sequence
<400> 66
caacaatgtg gaagattgac 20
<210> 67
<211> 42
<212> DNA
<213>Artificial sequence
<400> 67
gaaggtgacc aagttcatgc tcaggaaagg gaaggctcac ca 42
<210> 68
<211> 42
<212> DNA
<213>Artificial sequence
<400> 68
gaaggtcgga gtcaacggat tcaggaaagg gaaggctcac cc 42
<210> 69
<211> 21
<212> DNA
<213>Artificial sequence
<400> 69
ccagcagtgg caaaggatgt g 21
<210> 70
<211> 44
<212> DNA
<213>Artificial sequence
<400> 70
gaaggtgacc aagttcatgc taactgctcc aagtcctgta ttga 44
<210> 71
<211> 44
<212> DNA
<213>Artificial sequence
<400> 71
gaaggtcgga gtcaacggat taactgctcc aagtcctgta ttgg 44
<210> 72
<211> 21
<212> DNA
<213>Artificial sequence
<400> 72
ttccaaaatc ccatgctttc c 21
<210> 73
<211> 46
<212> DNA
<213>Artificial sequence
<400> 73
gaaggtgacc aagttcatgc tccagcatta ccttaatcca aaggat 46
<210> 74
<211> 46
<212> DNA
<213>Artificial sequence
<400> 74
gaaggtcgga gtcaacggat tccagcatta ccttaatcca aaggac 46
<210> 75
<211> 23
<212> DNA
<213>Artificial sequence
<400> 75
ttgtcaaggt gggtttctct agc 23
<210> 76
<211> 44
<212> DNA
<213>Artificial sequence
<400> 76
gaaggtgacc aagttcatgc ttgtgatcct gatgtctgga gtcg 44
<210> 77
<211> 44
<212> DNA
<213>Artificial sequence
<400> 77
gaaggtcgga gtcaacggat ttgtgatcct gatgtctgga gtca 44
<210> 78
<211> 25
<212> DNA
<213>Artificial sequence
<400> 78
ccaaaccttt gttgtatcaa cattg 25
<210> 79
<211> 43
<212> DNA
<213>Artificial sequence
<400> 79
gaaggtgacc aagttcatgc taacccatgt ctgtctgcag tgc 43
<210> 80
<211> 43
<212> DNA
<213>Artificial sequence
<400> 80
gaaggtcgga gtcaacggat taacccatgt ctgtctgcag tgt 43
<210> 81
<211> 23
<212> DNA
<213>Artificial sequence
<400> 81
ggacatcgta gagcgtctca gct 23
<210> 82
<211> 42
<212> DNA
<213>Artificial sequence
<400> 82
gaaggtgacc aagttcatgc ttgtgacaga acggtcaggt cg 42
<210> 83
<211> 43
<212> DNA
<213>Artificial sequence
<400> 83
gaaggtcgga gtcaacggat tctgtgacag aacggtcagg tct 43
<210> 84
<211> 22
<212> DNA
<213>Artificial sequence
<400> 84
tgcctcacac agtcctctct cc 22
<210> 85
<211> 47
<212> DNA
<213>Artificial sequence
<400> 85
gaaggtgacc aagttcatgc taaatactct tgtaggtcag aagttga 47
<210> 86
<211> 47
<212> DNA
<213>Artificial sequence
<400> 86
gaaggtcgga gtcaacggat taaatactct tgtaggtcag aagttgg 47
<210> 87
<211> 26
<212> DNA
<213>Artificial sequence
<400> 87
agacagatta ttttcacctt agtgtc 26
<210> 88
<211> 45
<212> DNA
<213>Artificial sequence
<400> 88
gaaggtgacc aagttcatgc ttcaaagtct tccaaaagtt ttcac 45
<210> 89
<211> 44
<212> DNA
<213>Artificial sequence
<400> 89
gaaggtcgga gtcaacggat tcaaagtctt ccaaaagttt tcat 44
<210> 90
<211> 22
<212> DNA
<213>Artificial sequence
<400> 90
caaaccatcc ctttacttct gt 22
<210> 91
<211> 39
<212> DNA
<213>Artificial sequence
<400> 91
gaaggtgacc aagttcatgc tatggggctc agttgggcg 39
<210> 92
<211> 40
<212> DNA
<213>Artificial sequence
<400> 92
gaaggtcgga gtcaacggat tgatggggct cagttgggca 40
<210> 93
<211> 21
<212> DNA
<213>Artificial sequence
<400> 93
tttgtggtgc tgggaatgaa c 21
<210> 94
<211> 41
<212> DNA
<213>Artificial sequence
<400> 94
gaaggtgacc aagttcatgc taggcgatct gccagatgct g 41
<210> 95
<211> 41
<212> DNA
<213>Artificial sequence
<400> 95
gaaggtcgga gtcaacggat taggcgatct gccagatgct a 41
<210> 96
<211> 23
<212> DNA
<213>Artificial sequence
<400> 96
gctgagaggc tcttgagaca ttc 23
<210> 97
<211> 43
<212> DNA
<213>Artificial sequence
<400> 97
gaaggtgacc aagttcatgc tagagccagt cgtggtaggt cac 43
<210> 98
<211> 42
<212> DNA
<213>Artificial sequence
<400> 98
gaaggtcgga gtcaacggat tgagccagtc gtggtaggtc aa 42
<210> 99
<211> 20
<212> DNA
<213>Artificial sequence
<400> 99
gacaatgaag gaaccgaggc 20
<210> 100
<211> 41
<212> DNA
<213>Artificial sequence
<400> 100
gaaggtgacc aagttcatgc tactgtccat catcggcagc g 41
<210> 101
<211> 40
<212> DNA
<213>Artificial sequence
<400> 101
gaaggtcgga gtcaacggat tctgtccatc atcggcagca 40
<210> 102
<211> 21
<212> DNA
<213>Artificial sequence
<400> 102
aacttccctc ccctgttcac c 21
<210> 103
<211> 48
<212> DNA
<213>Artificial sequence
<400> 103
gaaggtgacc aagttcatgc tcttatatgg aggtcttcca attctgtt 48
<210> 104
<211> 47
<212> DNA
<213>Artificial sequence
<400> 104
gaaggtcgga gtcaacggat tttatatgga ggtcttccaa ttctgtc 47
<210> 105
<211> 28
<212> DNA
<213>Artificial sequence
<400> 105
ctcacccact agagtactga catacaga 28
<210> 106
<211> 44
<212> DNA
<213>Artificial sequence
<400> 106
gaaggtgacc aagttcatgc tggactctct gccttttact ggct 44
<210> 107
<211> 43
<212> DNA
<213>Artificial sequence
<400> 107
gaaggtcgga gtcaacggat tgactctctg ccttttactg gcc 43
<210> 108
<211> 25
<212> DNA
<213>Artificial sequence
<400> 108
gctgcctgtg actatttgag atgtc 25
<210> 109
<211> 44
<212> DNA
<213>Artificial sequence
<400> 109
gaaggtgacc aagttcatgc tgaggcaagt aagctctgct acat 44
<210> 110
<211> 44
<212> DNA
<213>Artificial sequence
<400> 110
gaaggtcgga gtcaacggat tgaggcaagt aagctctgct acac 44
<210> 111
<211> 20
<212> DNA
<213>Artificial sequence
<400> 111
gcagaaacag cctctaatcc 20
<210> 112
<211> 44
<212> DNA
<213>Artificial sequence
<400> 112
gaaggtgacc aagttcatgc tcagtgcaac atgggaaata tgac 44
<210> 113
<211> 44
<212> DNA
<213>Artificial sequence
<400> 113
gaaggtcgga gtcaacggat tcagtgcaac atgggaaata tgag 44
<210> 114
<211> 18
<212> DNA
<213>Artificial sequence
<400> 114
cttgtgcgac agcgtgga 18
<210> 115
<211> 41
<212> DNA
<213>Artificial sequence
<400> 115
gaaggtgacc aagttcatgc ttatttccag aacagcgggc a 41
<210> 116
<211> 41
<212> DNA
<213>Artificial sequence
<400> 116
gaaggtcgga gtcaacggat ttatttccag aacagcgggc g 41
<210> 117
<211> 24
<212> DNA
<213>Artificial sequence
<400> 117
gctggctagt ttgacatctt tgag 24
<210> 118
<211> 45
<212> DNA
<213>Artificial sequence
<400> 118
gaaggtgacc aagttcatgc tctttcaagg aaatggtagg tgtct 45
<210> 119
<211> 45
<212> DNA
<213>Artificial sequence
<400> 119
gaaggtcgga gtcaacggat tctttcaagg aaatggtagg tgtcg 45
<210> 120
<211> 20
<212> DNA
<213>Artificial sequence
<400> 120
tgaaagacgc caggaagacc 20
<210> 121
<211> 46
<212> DNA
<213>Artificial sequence
<400> 121
gaaggtgacc aagttcatgc tcctccacat tggtttctat agtggt 46
<210> 122
<211> 45
<212> DNA
<213>Artificial sequence
<400> 122
gaaggtcgga gtcaacggat tctccacatt ggtttctata gtggc 45
<210> 123
<211> 24
<212> DNA
<213>Artificial sequence
<400> 123
tctacacact gttgatggga tgct 24
<210> 124
<211> 47
<212> DNA
<213>Artificial sequence
<400> 124
gaaggtgacc aagttcatgc tctgaatcac acttctggtt gtcatta 47
<210> 125
<211> 46
<212> DNA
<213>Artificial sequence
<400> 125
gaaggtcgga gtcaacggat ttgaatcaca cttctggttg tcattg 46
<210> 126
<211> 24
<212> DNA
<213>Artificial sequence
<400> 126
agaccaagaa gtcctgtgat ttgc 24
<210> 127
<211> 48
<212> DNA
<213>Artificial sequence
<400> 127
gaaggtgacc aagttcatgc tctaggatca gtctcccata gtcttaga 48
<210> 128
<211> 47
<212> DNA
<213>Artificial sequence
<400> 128
gaaggtcgga gtcaacggat ttaggatcag tctcccatag tcttagg 47
<210> 129
<211> 26
<212> DNA
<213>Artificial sequence
<400> 129
acctgaggtg agttctattt atgtcc 26
<210> 130
<211> 42
<212> DNA
<213>Artificial sequence
<400> 130
gaaggtgacc aagttcatgc taagctgtgc cttgtgtggc ac 42
<210> 131
<211> 41
<212> DNA
<213>Artificial sequence
<400> 131
gaaggtcgga gtcaacggat tagctgtgcc ttgtgtggca t 41
<210> 132
<211> 24
<212> DNA
<213>Artificial sequence
<400> 132
gtggatatgt gggaaagtat gacg 24
<210> 133
<211> 45
<212> DNA
<213>Artificial sequence
<400> 133
gaaggtgacc aagttcatgc tacccactat ctatcagtgt gttcc 45
<210> 134
<211> 45
<212> DNA
<213>Artificial sequence
<400> 134
gaaggtcgga gtcaacggat tacccactat ctatcagtgt gttct 45
<210> 135
<211> 22
<212> DNA
<213>Artificial sequence
<400> 135
agagttcctg ttgctccatg tc 22
<210> 136
<211> 42
<212> DNA
<213>Artificial sequence
<400> 136
gaaggtgacc aagttcatgc tgatctggag tctatgccag cg 42
<210> 137
<211> 42
<212> DNA
<213>Artificial sequence
<400> 137
gaaggtcgga gtcaacggat tgatctggag tctatgccag ca 42
<210> 138
<211> 22
<212> DNA
<213>Artificial sequence
<400> 138
ggaggaaggc tatgagaatc ac 22
<210> 139
<211> 42
<212> DNA
<213>Artificial sequence
<400> 139
gaaggtgacc aagttcatgc ttgcacacag gtacacacat gg 42
<210> 140
<211> 42
<212> DNA
<213>Artificial sequence
<400> 140
gaaggtcgga gtcaacggat ttgcacacag gtacacacat ga 42
<210> 141
<211> 20
<212> DNA
<213>Artificial sequence
<400> 141
tgagtgctta tggatgggtc 20
<210> 142
<211> 44
<212> DNA
<213>Artificial sequence
<400> 142
gaaggtgacc aagttcatgc tggtctctgg ctggagtgta taat 44
<210> 143
<211> 44
<212> DNA
<213>Artificial sequence
<400> 143
gaaggtcgga gtcaacggat tggtctctgg ctggagtgta taac 44
<210> 144
<211> 21
<212> DNA
<213>Artificial sequence
<400> 144
aacccaagaa aagacaacca g 21
<210> 145
<211> 41
<212> DNA
<213>Artificial sequence
<400> 145
gaaggtgacc aagttcatgc tttgccactt gacctgagcc t 41
<210> 146
<211> 41
<212> DNA
<213>Artificial sequence
<400> 146
gaaggtcgga gtcaacggat tttgccactt gacctgagcc c 41
<210> 147
<211> 23
<212> DNA
<213>Artificial sequence
<400> 147
cctcacaaga agaaaggcaa agt 23
<210> 148
<211> 44
<212> DNA
<213>Artificial sequence
<400> 148
gaaggtgacc aagttcatgc tgcacatttg taagcagcaa tgag 44
<210> 149
<211> 45
<212> DNA
<213>Artificial sequence
<400> 149
gaaggtcgga gtcaacggat tcgcacattt gtaagcagca atgaa 45
<210> 150
<211> 24
<212> DNA
<213>Artificial sequence
<400> 150
aggagtgatg atgtctcaga tgga 24
<210> 151
<211> 45
<212> DNA
<213>Artificial sequence
<400> 151
gaaggtgacc aagttcatgc tccctgttcc ccacttataa cttaa 45
<210> 152
<211> 45
<212> DNA
<213>Artificial sequence
<400> 152
gaaggtcgga gtcaacggat tccctgttcc ccacttataa cttac 45
<210> 153
<211> 23
<212> DNA
<213>Artificial sequence
<400> 153
tctacctgcc tgttaggact gag 23
<210> 154
<211> 44
<212> DNA
<213>Artificial sequence
<400> 154
gaaggtgacc aagttcatgc tagaccaggg tctctaatga gtgg 44
<210> 155
<211> 44
<212> DNA
<213>Artificial sequence
<400> 155
gaaggtcgga gtcaacggat tagaccaggg tctctaatga gtgt 44
<210> 156
<211> 26
<212> DNA
<213>Artificial sequence
<400> 156
gtgaatgcaa acagaaattg taagtc 26
<210> 157
<211> 42
<212> DNA
<213>Artificial sequence
<400> 157
gaaggtgacc aagttcatgc tgtttgggtc cttttgtgct ca 42
<210> 158
<211> 42
<212> DNA
<213>Artificial sequence
<400> 158
gaaggtcgga gtcaacggat tgtttgggtc cttttgtgct cg 42
<210> 159
<211> 20
<212> DNA
<213>Artificial sequence
<400> 159
aggagcacgg aagcagagga 20
<210> 160
<211> 46
<212> DNA
<213>Artificial sequence
<400> 160
gaaggtgacc aagttcatgc tctcagtggc ttaccactca tagaac 46
<210> 161
<211> 46
<212> DNA
<213>Artificial sequence
<400> 161
gaaggtcgga gtcaacggat tctcagtggc ttaccactca tagaaa 46
<210> 162
<211> 25
<212> DNA
<213>Artificial sequence
<400> 162
aatgttgaat tttctagaga gcagg 25
<210> 163
<211> 40
<212> DNA
<213>Artificial sequence
<400> 163
gaaggtgacc aagttcatgc tcaggcagga aactgaggga 40
<210> 164
<211> 40
<212> DNA
<213>Artificial sequence
<400> 164
gaaggtcgga gtcaacggat tcaggcagga aactgagggg 40
<210> 165
<211> 23
<212> DNA
<213>Artificial sequence
<400> 165
gatgctctta atgtgatgtg ggg 23
<210> 166
<211> 46
<212> DNA
<213>Artificial sequence
<400> 166
gaaggtgacc aagttcatgc tcttttcttt ttgagagtcc acactt 46
<210> 167
<211> 46
<212> DNA
<213>Artificial sequence
<400> 167
gaaggtcgga gtcaacggat tcttttcttt ttgagagtcc acactg 46
<210> 168
<211> 23
<212> DNA
<213>Artificial sequence
<400> 168
gtcctaagac tcaaagccat cag 23
<210> 169
<211> 46
<212> DNA
<213>Artificial sequence
<400> 169
gaaggtgacc aagttcatgc ttctaactct ataatgtggg gaaacg 46
<210> 170
<211> 46
<212> DNA
<213>Artificial sequence
<400> 170
gaaggtcgga gtcaacggat ttctaactct ataatgtggg gaaacc 46
<210> 171
<211> 23
<212> DNA
<213>Artificial sequence
<400> 171
ttcttctctg cctcttcatc cct 23
<210> 172
<211> 43
<212> DNA
<213>Artificial sequence
<400> 172
gaaggtgacc aagttcatgc ttgaaaagag gcagtgagcc atc 43
<210> 173
<211> 43
<212> DNA
<213>Artificial sequence
<400> 173
gaaggtcgga gtcaacggat ttgaaaagag gcagtgagcc att 43
<210> 174
<211> 21
<212> DNA
<213>Artificial sequence
<400> 174
catggctcct cccagacatt c 21
<210> 175
<211> 48
<212> DNA
<213>Artificial sequence
<400> 175
gaaggtgacc aagttcatgc taaatattgt gcatagtgct agagtcag 48
<210> 176
<211> 48
<212> DNA
<213>Artificial sequence
<400> 176
gaaggtcgga gtcaacggat taaatattgt gcatagtgct agagtcaa 48
<210> 177
<211> 25
<212> DNA
<213>Artificial sequence
<400> 177
tggtggtgta gatcaaagta taggc 25
<210> 178
<211> 44
<212> DNA
<213>Artificial sequence
<400> 178
gaaggtgacc aagttcatgc tcaagaaggg atgaagtcag agga 44
<210> 179
<211> 44
<212> DNA
<213>Artificial sequence
<400> 179
gaaggtcgga gtcaacggat tcaagaaggg atgaagtcag aggg 44
<210> 180
<211> 20
<212> DNA
<213>Artificial sequence
<400> 180
aatggccgtt ccctcacttg 20
<210> 181
<211> 48
<212> DNA
<213>Artificial sequence
<400> 181
gaaggtgacc aagttcatgc ttgtcttctg tttcctgtat tatcttcg 48
<210> 182
<211> 48
<212> DNA
<213>Artificial sequence
<400> 182
gaaggtcgga gtcaacggat ttgtcttctg tttcctgtat tatcttcc 48
<210> 183
<211> 23
<212> DNA
<213>Artificial sequence
<400> 183
gaaagcaact ctgacaaaca ccc 23
<210> 184
<211> 42
<212> DNA
<213>Artificial sequence
<400> 184
gaaggtgacc aagttcatgc ttgctaaacc cagcgaatcc tc 42
<210> 185
<211> 42
<212> DNA
<213>Artificial sequence
<400> 185
gaaggtcgga gtcaacggat ttgctaaacc cagcgaatcc tt 42
<210> 186
<211> 21
<212> DNA
<213>Artificial sequence
<400> 186
ctgcccacat gctttgttct g 21
<210> 187
<211> 42
<212> DNA
<213>Artificial sequence
<400> 187
gaaggtgacc aagttcatgc tcagactcag ccttcatcca cc 42
<210> 188
<211> 43
<212> DNA
<213>Artificial sequence
<400> 188
gaaggtcgga gtcaacggat tccagactca gccttcatcc act 43
<210> 189
<211> 23
<212> DNA
<213>Artificial sequence
<400> 189
gcaccctgag tcagacttgt ggt 23
<210> 190
<211> 44
<212> DNA
<213>Artificial sequence
<400> 190
gaaggtgacc aagttcatgc tccaaactgg tcaaagccat atct 44
<210> 191
<211> 44
<212> DNA
<213>Artificial sequence
<400> 191
gaaggtcgga gtcaacggat tccaaactgg tcaaagccat atcc 44
<210> 192
<211> 20
<212> DNA
<213>Artificial sequence
<400> 192
ccattgactt gctggtggaa 20
<210> 193
<211> 46
<212> DNA
<213>Artificial sequence
<400> 193
gaaggtgacc aagttcatgc tgctttaggt ctgtctgttg gtctca 46
<210> 194
<211> 46
<212> DNA
<213>Artificial sequence
<400> 194
gaaggtcgga gtcaacggat tgctttaggt ctgtctgttg gtctcc 46
<210> 195
<211> 24
<212> DNA
<213>Artificial sequence
<400> 195
gttagttagc agcgagtgac aggt 24
<210> 196
<211> 45
<212> DNA
<213>Artificial sequence
<400> 196
gaaggtgacc aagttcatgc taccttcaga agtgagagac aagca 45
<210> 197
<211> 45
<212> DNA
<213>Artificial sequence
<400> 197
gaaggtcgga gtcaacggat taccttcaga agtgagagac aagcc 45
<210> 198
<211> 24
<212> DNA
<213>Artificial sequence
<400> 198
gcactctcag gtgatccaag aaac 24
<210> 199
<211> 43
<212> DNA
<213>Artificial sequence
<400> 199
gaaggtgacc aagttcatgc ttgaccttta gtccacgccc act 43
<210> 200
<211> 43
<212> DNA
<213>Artificial sequence
<400> 200
gaaggtcgga gtcaacggat ttgaccttta gtccacgccc acc 43
<210> 201
<211> 22
<212> DNA
<213>Artificial sequence
<400> 201
gccctcattc accactacac ca 22
<210> 202
<211> 44
<212> DNA
<213>Artificial sequence
<400> 202
gaaggtgacc aagttcatgc tggaacctta aacaaaatca tgcg 44
<210> 203
<211> 44
<212> DNA
<213>Artificial sequence
<400> 203
gaaggtcgga gtcaacggat tggaacctta aacaaaatca tgcc 44
<210> 204
<211> 21
<212> DNA
<213>Artificial sequence
<400> 204
atttgcctgg tgtgagcaac a 21
<210> 205
<211> 45
<212> DNA
<213>Artificial sequence
<400> 205
gaaggtgacc aagttcatgc taatggaggg gaaggactat agttg 45
<210> 206
<211> 45
<212> DNA
<213>Artificial sequence
<400> 206
gaaggtcgga gtcaacggat taatggaggg gaaggactat agtta 45
<210> 207
<211> 22
<212> DNA
<213>Artificial sequence
<400> 207
tgacctgatg tgttgcctga ct 22
<210> 208
<211> 46
<212> DNA
<213>Artificial sequence
<400> 208
gaaggtgacc aagttcatgc tatagggcta gtgcactgta tgaaac 46
<210> 209
<211> 46
<212> DNA
<213>Artificial sequence
<400> 209
gaaggtcgga gtcaacggat tatagggcta gtgcactgta tgaaat 46
<210> 210
<211> 25
<212> DNA
<213>Artificial sequence
<400> 210
aggggagaca ttcaactatc tccat 25
<210> 211
<211> 47
<212> DNA
<213>Artificial sequence
<400> 211
gaaggtgacc aagttcatgc tggagttttg gtagtaatag gctgatg 47
<210> 212
<211> 47
<212> DNA
<213>Artificial sequence
<400> 212
gaaggtcgga gtcaacggat tggagttttg gtagtaatag gctgatt 47
<210> 213
<211> 26
<212> DNA
<213>Artificial sequence
<400> 213
ggaactagga cagtgtatga atgtga 26
<210> 214
<211> 42
<212> DNA
<213>Artificial sequence
<400> 214
gaaggtgacc aagttcatgc tgctgttacg gagaagccga ga 42
<210> 215
<211> 42
<212> DNA
<213>Artificial sequence
<400> 215
gaaggtcgga gtcaacggat tgctgttacg gagaagccga gg 42
<210> 216
<211> 26
<212> DNA
<213>Artificial sequence
<400> 216
ggaaggtcag tcctgactct cacttt 26
<210> 217
<211> 51
<212> DNA
<213>Artificial sequence
<400> 217
gaaggtgacc aagttcatgc tatcaccaac ttaaacaatt gccattctta t 51
<210> 218
<211> 49
<212> DNA
<213>Artificial sequence
<400> 218
gaaggtcgga gtcaacggat tcaccaactt aaacaattgc cattcttac 49
<210> 219
<211> 22
<212> DNA
<213>Artificial sequence
<400> 219
gagccatcca atattgggct tt 22
<210> 220
<211> 45
<212> DNA
<213>Artificial sequence
<400> 220
gaaggtgacc aagttcatgc tggagaatgc atggcaaaca ttagg 45
<210> 221
<211> 45
<212> DNA
<213>Artificial sequence
<400> 221
gaaggtcgga gtcaacggat tggagaatgc atggcaaaca ttagt 45
<210> 222
<211> 26
<212> DNA
<213>Artificial sequence
<400> 222
cccaaggaga aagccttgag gactat 26
<210> 223
<211> 47
<212> DNA
<213>Artificial sequence
<400> 223
gaaggtgacc aagttcatgc ttaaggacct acctcaaaga gttgttc 47
<210> 224
<211> 48
<212> DNA
<213>Artificial sequence
<400> 224
gaaggtcgga gtcaacggat tgtaaggacc tacctcaaag agttgttt 48
<210> 225
<211> 24
<212> DNA
<213>Artificial sequence
<400> 225
tctgtggtga cctttgccgc aata 24
<210> 226
<211> 48
<212> DNA
<213>Artificial sequence
<400> 226
gaaggtgacc aagttcatgc tctcaactct aagaagtcca tcatatga 48
<210> 227
<211> 48
<212> DNA
<213>Artificial sequence
<400> 227
gaaggtcgga gtcaacggat tctcaactct aagaagtcca tcatatgg 48
<210> 228
<211> 25
<212> DNA
<213>Artificial sequence
<400> 228
cctgcaactc acagctcagt caata 25
<210> 229
<211> 48
<212> DNA
<213>Artificial sequence
<400> 229
gaaggtgacc aagttcatgc tacataatag gaaccagaag gaaactga 48
<210> 230
<211> 48
<212> DNA
<213>Artificial sequence
<400> 230
gaaggtcgga gtcaacggat tacataatag gaaccagaag gaaactgc 48
<210> 231
<211> 24
<212> DNA
<213>Artificial sequence
<400> 231
atgcccagcc agtggtcgtg cttt 24
<210> 232
<211> 44
<212> DNA
<213>Artificial sequence
<400> 232
gaaggtgacc aagttcatgc tatgctgacc gtccaacaca tcac 44
<210> 233
<211> 44
<212> DNA
<213>Artificial sequence
<400> 233
gaaggtcgga gtcaacggat tatgctgacc gtccaacaca tcaa 44
<210> 234
<211> 26
<212> DNA
<213>Artificial sequence
<400> 234
ctacctgtgc atccattggt gtcttt 26
<210> 235
<211> 46
<212> DNA
<213>Artificial sequence
<400> 235
gaaggtgacc aagttcatgc tcaatacagg gaaggtctct tcatga 46
<210> 236
<211> 45
<212> DNA
<213>Artificial sequence
<400> 236
gaaggtcgga gtcaacggat taatacaggg aaggtctctt catgc 45
<210> 237
<211> 24
<212> DNA
<213>Artificial sequence
<400> 237
tcagtatcca acctgggagg ctta 24
<210> 238
<211> 46
<212> DNA
<213>Artificial sequence
<400> 238
gaaggtgacc aagttcatgc tttacactgt ttggtcgttg gagacg 46
<210> 239
<211> 46
<212> DNA
<213>Artificial sequence
<400> 239
gaaggtcgga gtcaacggat tttacactgt ttggtcgttg gagaca 46
<210> 240
<211> 24
<212> DNA
<213>Artificial sequence
<400> 240
tgagaaagga actcaccgct cctt 24
<210> 241
<211> 45
<212> DNA
<213>Artificial sequence
<400> 241
gaaggtgacc aagttcatgc tttctctacc ctgaagcatc aggac 45
<210> 242
<211> 45
<212> DNA
<213>Artificial sequence
<400> 242
gaaggtcgga gtcaacggat tttctctacc ctgaagcatc aggat 45
<210> 243
<211> 24
<212> DNA
<213>Artificial sequence
<400> 243
gccaagtaca taagcaacgt ggat 24
<210> 244
<211> 51
<212> DNA
<213>Artificial sequence
<400> 244
gaaggtgacc aagttcatgc ttgatgttga attaggaatt aagaaacttc g 51
<210> 245
<211> 53
<212> DNA
<213>Artificial sequence
<400> 245
gaaggtcgga gtcaacggat tgttgatgtt gaattaggaa ttaagaaact tct 53
<210> 246
<211> 28
<212> DNA
<213>Artificial sequence
<400> 246
gataagtcaa gtctagcctt gccacaat 28
<210> 247
<211> 44
<212> DNA
<213>Artificial sequence
<400> 247
gaaggtgacc aagttcatgc tcaggaagca gagtctgtga aacg 44
<210> 248
<211> 45
<212> DNA
<213>Artificial sequence
<400> 248
gaaggtcgga gtcaacggat tgcaggaagc agagtctgtg aaaca 45
<210> 249
<211> 22
<212> DNA
<213>Artificial sequence
<400> 249
ccaacatgtc cctattaccc aa 22
<210> 250
<211> 44
<212> DNA
<213>Artificial sequence
<400> 250
gaaggtgacc aagttcatgc tatctgtatt gaacaggccc aggc 44
<210> 251
<211> 44
<212> DNA
<213>Artificial sequence
<400> 251
gaaggtcgga gtcaacggat tatctgtatt gaacaggccc aggg 44
<210> 252
<211> 27
<212> DNA
<213>Artificial sequence
<400> 252
cagcccatag acattgtatc acctcta 27
<210> 253
<211> 48
<212> DNA
<213>Artificial sequence
<400> 253
gaaggtgacc aagttcatgc ttacaaaatc aggccaattg agttcagt 48
<210> 254
<211> 44
<212> DNA
<213>Artificial sequence
<400> 254
gaaggtcgga gtcaacggat taaatcaggc caattgagtt cagc 44
<210> 255
<211> 24
<212> DNA
<213>Artificial sequence
<400> 255
tctaagccaa ccaggaacgg tcaa 24
<210> 256
<211> 50
<212> DNA
<213>Artificial sequence
<400> 256
gaaggtgacc aagttcatgc ttaacctaag aaatgcacaa tgaacattca 50
<210> 257
<211> 48
<212> DNA
<213>Artificial sequence
<400> 257
gaaggtcgga gtcaacggat tacctaagaa atgcacaatg aacattcg 48
<210> 258
<211> 25
<212> DNA
<213>Artificial sequence
<400> 258
cttctcccag agtctctgtt tccaa 25
<210> 259
<211> 44
<212> DNA
<213>Artificial sequence
<400> 259
gaaggtgacc aagttcatgc tgaggaaagt gaagcagaag aggg 44
<210> 260
<211> 45
<212> DNA
<213>Artificial sequence
<400> 260
gaaggtcgga gtcaacggat tggaggaaag tgaagcagaa gagga 45
<210> 261
<211> 25
<212> DNA
<213>Artificial sequence
<400> 261
cagccacaac tcctaccgta atgta 25
<210> 262
<211> 51
<212> DNA
<213>Artificial sequence
<400> 262
gaaggtgacc aagttcatgc tcgttctaat tctcagaaca ttaagcacaa c 51
<210> 263
<211> 51
<212> DNA
<213>Artificial sequence
<400> 263
gaaggtcgga gtcaacggat tcgttctaat tctcagaaca ttaagcacaa t 51
<210> 264
<211> 21
<212> DNA
<213>Artificial sequence
<400> 264
ctctcctcca tccacttcca t 21
<210> 265
<211> 45
<212> DNA
<213>Artificial sequence
<400> 265
gaaggtgacc aagttcatgc tcccagcaac caagtataca acctg 45
<210> 266
<211> 45
<212> DNA
<213>Artificial sequence
<400> 266
gaaggtcgga gtcaacggat tcccagcaac caagtataca accta 45
<210> 267
<211> 25
<212> DNA
<213>Artificial sequence
<400> 267
aagctaatag aaccggttgc cctaa 25
<210> 268
<211> 45
<212> DNA
<213>Artificial sequence
<400> 268
gaaggtgacc aagttcatgc tggatactga gaaaggccca gtgta 45
<210> 269
<211> 44
<212> DNA
<213>Artificial sequence
<400> 269
gaaggtcgga gtcaacggat tgatactgag aaaggcccag tgtg 44
<210> 270
<211> 22
<212> DNA
<213>Artificial sequence
<400> 270
ggagcccact cattcctttg tt 22
<210> 271
<211> 41
<212> DNA
<213>Artificial sequence
<400> 271
gaaggtgacc aagttcatgc tgcggagtac ctgggtcctt c 41
<210> 272
<211> 41
<212> DNA
<213>Artificial sequence
<400> 272
gaaggtcgga gtcaacggat tgcggagtac ctgggtcctt t 41
<210> 273
<211> 24
<212> DNA
<213>Artificial sequence
<400> 273
tgggagatcc cttcctgttg acta 24
<210> 274
<211> 43
<212> DNA
<213>Artificial sequence
<400> 274
gaaggtgacc aagttcatgc tcactgtgcc tagtgactag acg 43
<210> 275
<211> 43
<212> DNA
<213>Artificial sequence
<400> 275
gaaggtcgga gtcaacggat tcactgtgcc tagtgactag acc 43
<210> 276
<211> 25
<212> DNA
<213>Artificial sequence
<400> 276
tatgctggga gcatgagaag gcttt 25
<210> 277
<211> 45
<212> DNA
<213>Artificial sequence
<400> 277
gaaggtgacc aagttcatgc ttccccgttt tcagagaaca gcaca 45
<210> 278
<211> 43
<212> DNA
<213>Artificial sequence
<400> 278
gaaggtcgga gtcaacggat tcccgttttc agagaacagc acg 43
<210> 279
<211> 25
<212> DNA
<213>Artificial sequence
<400> 279
accacatgag ccagctagag ccatt 25
<210> 280
<211> 45
<212> DNA
<213>Artificial sequence
<400> 280
gaaggtgacc aagttcatgc tttgctacct tccagttgat ggaga 45
<210> 281
<211> 45
<212> DNA
<213>Artificial sequence
<400> 281
gaaggtcgga gtcaacggat tttgctacct tccagttgat ggagg 45
<210> 282
<211> 24
<212> DNA
<213>Artificial sequence
<400> 282
ggtgctgcca tcctaaagag gaaa 24
<210> 283
<211> 45
<212> DNA
<213>Artificial sequence
<400> 283
gaaggtgacc aagttcatgc tctatctccc tattgacctc cttca 45
<210> 284
<211> 45
<212> DNA
<213>Artificial sequence
<400> 284
gaaggtcgga gtcaacggat tctatctccc tattgacctc cttcg 45
<210> 285
<211> 27
<212> DNA
<213>Artificial sequence
<400> 285
cacctacttt caacacttgc agcacaa 27
<210> 286
<211> 48
<212> DNA
<213>Artificial sequence
<400> 286
gaaggtgacc aagttcatgc tcctgcacat gtaactacaa tgcaatca 48
<210> 287
<211> 47
<212> DNA
<213>Artificial sequence
<400> 287
gaaggtcgga gtcaacggat tctgcacatg taactacaat gcaatcg 47
<210> 288
<211> 29
<212> DNA
<213>Artificial sequence
<400> 288
cctgtgaaga tttaccacac tgcttgtta 29

Claims (7)

1. a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring, which is characterized in that include the following steps：
(1) design of SNP panel：Determine inbred mouse strain, filter out C57BL/6 strains and other strains can be special Property distinguish site and strain conventional genetic quality monitoring site, it is as follows that the specificity distinguishes site：C57BL/6 strains site Base is different from corresponding base in 129S1/SvIm, BALB/C, A/J, CBA, DBA, FVB, NOD strain, is single with chromosome , should include at least 5 pairs chromosomes that site is distinguished containing specificity in SNP panel, and each pair of chromosome should contain 2 or more Specificity distinguishes site；The strain conventional genetic quality monitoring site is as follows：On the basis of the specificity distinguishes site, According to 4-5 site principle on each pair of chromosome, supplying for site is equally spacedly carried out, by the SNP panel of C57BL/6 strains It is 96 that site, which is supplied,；The SNP panel can be used for the hereditary matter of C57BL strains inbred mouse and corresponding mutantion line mouse Amount detection and the detection of strain pollution condition and effective discriminating of C57BL Strains of Mouse and other strains；
(2) primer for designing and synthesizing the sites above-mentioned SNP panel is not required to design probe；The sites SNP panel upstream and downstream sequence It is pulled in mouse genome sequences using programming tool；Wherein, 5 ' end sense primer design：5 ' end sense primers are shared Two, include the sequence of primer precursor and one section of recognizable FAM or HEX signal；The primer precursor is located at each SNP The upstream in the sites panel, length 20-30bp are respectively two mutation alkali in the sites SNP panel in the end of primer precursor Base；The sequence of the recognizable FAM or HEX signals is located at 5 ' ends of the primer precursor, and length is about 20bp；Draw at 3 ' ends downstream Object is one, length about 18-29bp；
Above-mentioned 3 primers are carried out at the same time PCR amplification, if generating one of signal in system, illustrate to contain in sample template There is the base mutation type；Primer test is carried out using check clone to 96 sites, if success parting, then it represents that primer is tested Success, otherwise the upstream and downstream to the site of test crash in chromosome corresponding position, which is found, replaces site；96 be successfully tested Site and PCR primer test condition and system are determined as final detection scheme；
(3) sample to be tested DNA is extracted, when sample concentration is more than 10ng/uL, with KASP methods to 96 SNP of each sample The sites panel carry out Genotyping, analyze data and compared with the SNP site information on NCBI, complete inbred mouse heredity Quality monitoring and ore grade indexes.
2. a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring as described in claim 1, which is characterized in that institute It states in step (1), inbred mouse strain is A/J, 129S1/SvIm, BALB/C, C57BL/6, CBA, DBA, FVB, NOD；Institute The specificity of the subbreed C57BL/6J strains and other 7 strains of stating C57BL/6, which can distinguish site, 69, specific as follows：
3. a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring as claimed in claim 1 or 2, which is characterized in that In the step (3), concentration necessary=5ng/uL × species to be measured Genome Size/human genome of sample to be tested DNA is big It is small.
4. a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring as claimed in claim 1 or 2, which is characterized in that The reaction system of the KASP methods is as follows：DNA including a concentration of 5-50ng/uL each reacting hole volume be 0.8uL, 2 × KASP Master mix are 0.778uL, bodies of the KASP Primer mix in each reacting hole in the volume of each reacting hole Product is 0.022uL.
5. a kind of SNP rapid detection methods of inbred strais quality of heredity monitoring as claimed in claim 1 or 2, which is characterized in that The response procedures of the KASP methods are as follows：
Or
Or
6. application of one group of site in the monitoring of inbred strais quality of heredity as SNP site, which is characterized in that one group of site There are 96, it is specific as follows
* it is that specificity distinguishes site.
7. a kind of SNP site primer of inbred strais quality of heredity monitoring, which is characterized in that the site primer such as SEQ ID Shown in NO.1 to SEQ ID NO.288.
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CN109609659A (en) * 2018-12-21 2019-04-12 江苏集萃药康生物科技有限公司 One group for the SNP site of CBA/CaJ inbred mouse quality of heredity monitoring and its primer combination and application
CN109694914A (en) * 2018-12-28 2019-04-30 江苏集萃药康生物科技有限公司 For monitoring the method, primer sets and its application of DBA inbred mouse quality of heredity
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CN110358847A (en) * 2019-09-03 2019-10-22 江苏集萃药康生物科技有限公司 One group for the SNP site of BALB/cJ inbred mouse quality of heredity monitoring and its primer combination and application
CN110423826A (en) * 2019-08-30 2019-11-08 苏州西山生物技术有限公司 A kind of C57BL/6 subbreed mouse KASP genetic detection kit and primer
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WO2019218743A1 (en) * 2018-05-17 2019-11-21 江苏集萃药康生物科技有限公司 Snp rapid detection method and snp locus for monitoring genetic quality of inbred strains and primer for snp locus
CN110484627A (en) * 2019-07-30 2019-11-22 江苏集萃药康生物科技有限公司 For monitoring the method, primer sets and its application of A/J inbred mouse quality of heredity
CN110484608A (en) * 2019-07-30 2019-11-22 江苏集萃药康生物科技有限公司 For monitoring the method, primer sets and its application of 129S1/SvImJ inbred mouse quality of heredity

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CN110358847A (en) * 2019-09-03 2019-10-22 江苏集萃药康生物科技有限公司 One group for the SNP site of BALB/cJ inbred mouse quality of heredity monitoring and its primer combination and application

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