CN108588236B - SNP rapid detection method for monitoring genetic quality of inbred line, SNP locus and primer thereof - Google Patents
SNP rapid detection method for monitoring genetic quality of inbred line, SNP locus and primer thereof Download PDFInfo
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Abstract
The invention discloses a rapid SNP detection method for monitoring genetic quality of inbred lines, which comprises the following steps of firstly designing an SNP panel: selecting inbred line mouse strains, and screening specific distinguishing sites and conventional genetic quality monitoring sites of other strains, wherein the inbred line mouse strains take chromosomes as units, SNP panels comprise at least 5 pairs of chromosomes containing the specific distinguishing sites, and each pair of chromosomes contains more than 2 specific distinguishing sites; on the basis of distinguishing the sites specifically, the later complements the sites of the SNP panel to 96 sites according to the principle that each pair of chromosomes has 4-5 equidistant sites; then designing an SNP locus primer; and extracting sample DNA, genotyping by using a KASP method, and comparing with SNP locus information on NCBI to complete the monitoring of the genetic quality of the inbred line mice. The invention also discloses an SNP locus and an SNP locus primer for monitoring the genetic quality of the inbred line.
Description
Technical Field
The invention belongs to the field of inbred line mouse strain genetic background identification and genetic pollution detection, relates to an SNP rapid detection method for inbred line genetic quality monitoring, and particularly relates to a detection method for SNP typing based on a KASP method, an SNP locus and a primer thereof.
Background
The quality control of experimental animals is a core problem of healthy development of the experimental animal industry, and the control of the microbial biomass quantity and the genetic background quality of mice are important control factors. The domestic genetic quality monitoring lacks of mature industrial standards, and the establishment of a rapid and high-precision high-throughput genotyping technology platform is of great importance.
The detection method for genetic background quality control mainly comprises 3 methods: biochemical marker analysis, microsatellite DNA, SNP (single nucleotide polymorphism) detection, and the like. The current internationally prescribed genetic testing method is a biochemical marker assay, which detects changes in isoenzymes or isomerases to infer corresponding genetic changes; the method for detecting has the defects of low accuracy, low sensitivity, limited detection sites, limited reflected genetic profile and the like. The molecular genetic marker can carry out more precise supervision on the experimental animal and is a more perfect experimental animal quality detection means; wherein SNP detection is used as a technique of molecular genetic markers for detecting DNA sequence polymorphism caused by variation of single nucleotide at genome level; the polymorphism represented by SNP can monitor the variation of a single base, has the characteristics of high density, representativeness, genetic stability and the like, and can comprehensively reflect the heredity and variation conditions of a genome.
The KASP method refers to competitive Allele-Specific PCR (Kompetitive Allle Specific PCR) and is used for carrying out accurate biallelic genotyping on target SNPs based on high-sensitivity fluorescence detection. Different from the traditional Taqman technology, the method does not need to mark a target spot, namely a specific primer/probe, does not need to synthesize a specific fluorescent primer according to each SNP locus, and uses a unique ARM PCR principle to finally amplify all locus detections by using a universal fluorescent primer, thereby greatly reducing the experiment cost. The optimized PCR system can meet the requirements of high-flux reactions at different sites, has the accuracy of a gold standard, reduces the use cost and has better site adaptability than Taqman. The KASP technology synthesizes 2 reactions for traditionally detecting alleles into 1 reaction, and has low cost. The SNPs detection not only makes up the defects of long time of the traditional PCR, gel cutting and sequencing process, but also greatly saves the sequencing cost.
Disclosure of Invention
Aiming at the defects in the prior art that the current SNP detection panel is limited by operation technology and detection cost, so that the sample detection flux is low, the site selection is limited, the invention aims to provide a rapid SNP detection method which has high flux, multiple sites and low cost and is rapidly used for inbred line genetic quality monitoring
The invention also aims to provide application of the SNP loci of the group of loci in inbred line genetic quality monitoring.
The invention also aims to provide an SNP locus primer for monitoring the genetic quality of an inbred line.
The technical scheme adopted by the invention for solving the technical problems is as follows:
an SNP rapid detection method for monitoring inbred line genetic quality comprises the following steps:
(1) design of SNP panel: inbred mouse strains are determined and the C57BL/6 strain is screened for the ability to specifically distinguish it from other strains and for the conventional monitoring of genetic quality of the strain as follows: the base of the C57BL/6 line locus is different from the corresponding base in 129S1/SvIm, BALB/C, A/J, CBA, DBA, FVB and NOD lines, and at least 5 pairs of chromosomes containing specific distinguishing loci are included in the SNP panel by taking a chromosome as a unit, and each pair of chromosomes contains more than 2 specific distinguishing loci; the conventional genetic quality monitoring sites of the strain are as follows: on the basis of the specific differential loci, complementing the loci at equal intervals according to the principle of 4-5 loci on each pair of chromosomes, and complementing the SNPpanel loci of the C57BL/6 strain to 96 loci; the SNP panel can be used for detecting the genetic quality and strain pollution condition of a C57BL strain inbred line mouse and a corresponding mutant line mouse and effectively identifying the C57BL strain mouse from other strains;
(2) designing and synthesizing the primer of the SNP panel locus without designing a probe; drawing 100bp sequences of the upstream and downstream sequences of the SNP panel locus in a mouse genome sequence by using a programming tool; wherein, the upstream primer design of the 5' end is as follows: the upstream primer at the 5' end comprises two primers, and each primer comprises a primer precursor and a sequence capable of recognizing FAM or HEX signals; the primer precursor is arranged at the upstream of each SNP panel locus, the length is 20-30bp, and the tail ends of the primer precursor are respectively two mutant bases of the SNP panel locus; the sequence capable of recognizing FAM or HEX signals is arranged at the 5' end of the primer precursor and is about 20bp in length; the 3 'end downstream primer is one, and the length of the 3' end downstream primer is about 18-29 bp;
carrying out PCR amplification on the 3 primers simultaneously, and if one signal is generated in the system, indicating that the sample template contains the base mutation type; carrying out primer test on 96 loci by using a control line 129S1/Svlm subline 129S1/Svlm JNju and a C57BL/6 subline C57BL/6JNju, wherein if typing is successful, the primer test is successful, and otherwise, a substitution locus is searched for at the upstream and downstream of the corresponding position of the chromosome on the locus failed in the test; determining 96 sites which are successfully tested, PCR primer test conditions and a PCR primer test system as a final detection scheme;
(3) and extracting DNA of a sample to be detected, when the sample concentration is more than 10ng/uL, carrying out genotyping on 96 SNP panel sites of each sample by using a KASP method, analyzing data and comparing with SNP site information on NCBI (N-channel polymorphism) to finish the genetic quality monitoring and strain identification of inbred line mice.
Further, in the step (1), the inbred line mouse is A/J, 129S1/SvIm, BALB/C, C57BL/6, CBA, DBA, FVB, NOD; the C57BL/6 strain has 69 specific distinguishable loci from the other 7 strains, which are as follows:
further, in the step (3), the concentration requirement of the test sample DNA is 5ng/uL × test species genome size/human genome size.
Further, the reaction system of the KASP method is as follows: including DNA (5-50ng/uL, in the case of human or mouse) at 0.8uL per reaction well, 2 XKASP Master mix at 0.778uL per reaction well, and KASP Primer mix at 0.022uL per reaction well.
Further, the reaction sequence of the KASP method is as follows:
or
Or
The application of a group of 96 sites as SNP sites in inbred genetic quality monitoring is carried out, wherein the 96 sites are as follows:
are specific differentiation sites.
An SNP locus primer for monitoring inbred line genetic quality, wherein the primer sequence is shown as SEQ ID NO. 1-SEQ ID NO. 288.
The invention has the following beneficial effects:
the detection method of the invention can complete the site detection of 36 samples of 1 project (each sample detects 96 sites), and the total period is only 6 working days. The method improves the number of detectable sites of a single sample, greatly shortens the experimental period by high-flux detection, and ensures the accuracy of the result while improving the flux.
The C57BL SNP panel used in the invention contains 96 sites in total, and can be used for distinguishing A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB and NOD strains besides being used for conventional genetic quality monitoring of the C57BL strain and related mutant strains.
According to the invention, the target spot, namely the specific primer, is not required to be marked, a probe is not required to be designed, a specific fluorescent primer is not required to be synthesized according to each SNP locus, all locus detection is finally amplified by using a universal fluorescent primer, the processes of glue running, glue cutting, sequencing and the like of the traditional PCR are not required, the detection time is saved, and the sequencing cost is greatly saved; in addition, the KASP method carries out double allele typing on target SNPs, synthesizes 2 reactions of the traditional detection allele into 1 reaction, and reduces the cost.
Drawings
FIG. 1 is a flow chart of the detection method of the present invention.
Detailed Description
The invention is further described below with reference to the accompanying drawings and examples.
The flow of the SNP rapid detection method for monitoring inbred line genetic quality of the embodiment is shown in figure 1, and the specific steps are as follows:
example 1: SNP panel design
1. Determination of inbred lines contained in SNP panels
13 inbred line mouse strains are selected to be subjected to routine SNP detection (shown in Table 1), the 13 inbred line mouse strains are provided by Nanjing university-Nanjing Biomedical Research Institute (NBRI) (publicly sold), and C57BL/6J and C57BL/6N, C57BL/10J, C57BLKS/J, B6(Cg) -Tyr are found in the screening processc-2JThe genetic background is similar; meanwhile, DBA/1 is similar to DBA/2 genetic background. Therefore, C57BL/6J was selected as a representative in C57BL, DBA/1 was selected as a representative in DBA, and finally 8 inbred lines were determined to establish SNP detection panel of C57BL/6 inbred line (see Table 2). In subsequent experiments, the C57BL/6 strain SNP panel can be used in commonStandard SNP detection for distinguishing A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB and NOD strains; can be used for C57BL/6N, C57BL/10, C57BLKS, B6(Cg) -Tyrc-2JConventional genetic quality monitoring of the C57BL strain and related mutant strains.
TABLE 1 study inbred lines List
| Serial number | Line 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 inbred line List used to establish the C57BL/6SNP detection panel
2. SNP Panel design orientation
Screening was performed with a database < mouse-marker-excel-file > provided by LGC corporation. SNP panel design is mainly carried out from two major aspects: screening out sites which can be specifically distinguished from other strains for strain pollution detection; screening the strain for routine genetic quality monitoring sites.
3. Screening for sites of specific differentiation between inbred lines and other lines
a) Screening purposes are as follows: the method is used for strain pollution detection.
b) The design principle is as follows: in chromosome units, at least 5 pairs of chromosomes containing specific differentiation sites should be included in the SNP panel, and each pair of chromosomes should contain more than 2 specific differentiation sites, so that the specific differentiation panel is considered to be capable of differentiating the strain mice from other inbred lines. Site for specific differentiation: in a certain line, the locus is different from the loci of other lines. If one SNP detection site cannot be completely distinguished from other strains, genetic drift or strain pollution cannot be determined if mutation occurs after detection.
The frequency of genetic quality monitoring is 1 year/time, if pollution occurs, the longest pollution period is 1 year, and the generation number of the polluted mice is 4 generations. The probability of chromosome contamination of the fourth generation of mice is 5 at the lowest (according to the breeding of F1 generation and other strains, the subsequent mice are all backcrossed with pure background strains, the contamination chromosomes of F2 generation are all, the contamination chromosomes of F3 are 10, and the contamination chromosomes of F4 are 5).
Calculated with this probability, panel is able to distinguish mouse strains from other inbred lines if more than 5 chromosomes contain a specific site. The detection result is considered to have reference by containing more than 2 sites on each chromosome.
c) And (4) screening results: the C57BL/6 inbred mice were screened for 69 sites that could specifically distinguish them from other strains. The panel is used for SNP detection of a C57BL strain and related mutant lines, and can be used for distinguishing A/J, 129S1/SvIm, BALB/C, CBA, DBA, FVB and NOD strains.
The strain specificity distinguishing sites are screened in a programming mode, and the screening principle is that sites which are different from other strains are screened in the same strain. And (4) carrying out artificial secondary screening on the screened loci according to the principle of equal spacing on chromosomes (the number of the loci on each chromosome is 4-5, and the number of the specific loci is not less than 2), and determining the specific distinguishing panel of each strain.
4. Screening of loci for routine genetic quality monitoring of strains
a) Screening purposes are as follows: the method is used for routine strain genetic quality monitoring.
b) The design principle is as follows: line-specific loci are usually too few in number to be used for routine genetic quality monitoring. On the basis of the determination of the specific discrimination site panel, we complemented the number of panel SNPs detected in each line to 96, which served as follows: the reliability of the SNP detection result can be ensured, and the identification operation is convenient.
On the basis of the specific locus, complementing the conventional locus according to the principle of 4-5 equidistant loci on each chromosome; at the same time, sites with multiple applicability in various lines of panel are preferably selected.
c) And (4) screening results: the C57BL/6J line containing 96 sites was co-screened for the genetic quality monitoring SNP detection panel as shown in the following table (i.e., Table 3).
TABLE 3C57BL/6 line genetic quality monitoring SNP detection panel
Remarking: are specific differentiation sites.
Example 2: SNP site primer design
After the SNP locus is screened, pulling 100bp sequences of the upstream and downstream sequences of the SNP locus in a mouse genome sequence by using a programming tool; designing a 5' end primer: respectively designing 20-30bp primers at the upstream of the SNP locus, respectively setting two mutant bases of the SNP at the tail ends of the primers, and adding a section of about 20bp sequence for respectively identifying different signals, such as FAM and HEX signals, at the 5' end of the primers; 3' designing downstream primer, length is about 18-29 bp.
The Tm (DEG C) is between 55 and 65 ℃, the GC percent is between 34 and 60 percent, 288 primers are designed, and the specific primer sequences are detailed in the following table. And (3) arranging 96 sites of PCR amplification detection on each sample, simultaneously carrying out PCR amplification on 3 primers of each site, and if one signal is generated in the system, indicating that the sample template contains the base mutation type.
TABLE 4 site primer information
Example 3: genotyping by the KASP method
1. Extracting DNA of sample to be detected as template
Rat tail DNA of inbred mice was extracted using an oKtopureTM high throughput DNA extractor from LGC. The extraction procedure was described with reference to the instrument. The extraction flux of DNA can be effectively improved, 3,500 samples per day; extraction speed: 20-30mg of initial sample size, 8 multiplied by 96 is less than or equal to 1.5h, 80-100mg of initial sample size, 8 multiplied by 96 is less than or equal to 2 h.
2. DNA concentration requirement
Most KASP assays require 5-50ng of DNA per reaction; DNA concentration requirements vary depending on genome size: the DNA concentration requirement of the species to be tested is 5ng/uL multiplied by the genome size of the species to be tested/the size of the human genome.
3. Preparation of PCR reaction System
The preparation of the reaction system was automated using an IntelliQube instrument from LGC. The KASP genotyping reaction system is shown in the following table:
TABLE 5KASP genotyping reaction System
4. PCR reaction
The KASP method was used for the PCR reaction, and Hydrocycler2 from LGC was usedTMAnd (3) carrying out PCR reaction by a water bath PCR thermal cycler water bath system. The KASP genotyping reaction program can be divided into the following three types, any of which can be performed as shown in tables 6 to 8.
TABLE 6KASP genotyping reaction sequence (1)
Or table 7KASP genotyping reaction program (2)
Or Table 8KASP genotyping reaction sequence (3)
5. High throughput genotyping and data analysis
Readings were taken using IntelliQube fluorescence detection, post-PCR data analysis was performed using IntelliScore, and genotype was automatically derived for analysis.
6. SNP typing identification of C57BL/6 subline C57BL/6JNju mice
Taking a 129S1/SvImJNju mouse as a control strain, carrying out 96-site primer test and mouse genotyping on C57BL/6 JNju; the results show that C57BL/6JNju is all consistent with the site information registered on NCBI in 96 sites, as detailed in Table 9 below.
TABLE 9C57BL/6JNju subline SNP detection results
The implementation case is as follows:
the detection steps and methods are identical, except that the sample DNA is different. Selecting 10 strains in total, carrying out SNP locus detection on mice of mutant lines respectively comprising C57BL related strains and C57BL/6, checking the genetic quality condition of the mice, and displaying that a locus rs3725940 on the chromosome 18 of the C57BL/10JNju mice is inconsistent with an NCBI database and is mutation homozygous; in the C57BL/6 mutant mice, the sites of 9 strains are identical to those of C57BL/6J, as shown in the following Table.
TABLE 1010 mouse SNP detection results of C57BL inbred lines and mutant lines
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<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 (2)
1. The application of a primer group for detecting 96 SNP sites in the genetic quality monitoring of mice of the C57BL strain, wherein the 96 SNP sites are shown as follows:
are specific differentiation sites.
2. An SNP locus primer group for monitoring inbred line genetic quality is characterized in that the sequences of primers in the locus primer group are shown as SEQ ID NO.1 to SEQ ID NO. 288.
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| CN201810475211.9A CN108588236B (en) | 2018-05-17 | 2018-05-17 | SNP rapid detection method for monitoring genetic quality of inbred line, SNP locus and primer thereof |
| PCT/CN2019/075790 WO2019218743A1 (en) | 2018-05-17 | 2019-02-22 | Snp rapid detection method and snp locus for monitoring genetic quality of inbred strains and primer for snp locus |
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| CN108588236B (en) * | 2018-05-17 | 2021-02-26 | 江苏集萃药康生物科技股份有限公司 | SNP rapid detection method for monitoring genetic quality of inbred line, SNP locus and primer thereof |
| CN109609659B (en) * | 2018-12-21 | 2021-09-28 | 江苏集萃药康生物科技股份有限公司 | SNP loci for monitoring genetic quality of CBA/CaJ inbred line mice, primer combination and application thereof |
| CN109694914B (en) * | 2018-12-28 | 2022-06-03 | 江苏集萃药康生物科技股份有限公司 | Method for monitoring genetic quality of DBA inbred line mice, primer group and application thereof |
| CN110452969B (en) * | 2019-07-18 | 2020-09-04 | 苏州西山生物技术有限公司 | Rat genetic quality monitoring SNP marker typing method and kit based on KASP |
| CN110484627B (en) * | 2019-07-30 | 2023-05-02 | 江苏集萃药康生物科技股份有限公司 | Method for monitoring genetic quality of A/J inbred line mice, primer set and application thereof |
| CN110484608B (en) * | 2019-07-30 | 2021-02-05 | 江苏集萃药康生物科技股份有限公司 | Method for monitoring genetic quality of 129S1/SvImJ inbred line mouse, primer set and application thereof |
| CN110305974B (en) * | 2019-08-08 | 2023-06-23 | 广东省实验动物监测所 | PCR analysis primer for distinguishing common mouse inbred lines based on detection of five SNP loci and analysis method thereof |
| CN110423826B (en) * | 2019-08-30 | 2023-10-31 | 苏州西山生物技术有限公司 | C57BL/6 subline mouse KASP genetic detection kit and primer |
| CN110358847B (en) * | 2019-09-03 | 2021-03-05 | 江苏集萃药康生物科技股份有限公司 | SNP loci for monitoring genetic quality of BALB/cJ inbred line mice, primer combination and application thereof |
| CN116004842B (en) * | 2022-07-21 | 2023-08-18 | 斯贝福（北京）生物技术有限公司 | Application of SNP marker in inbred line rat genetic quality detection and/or inbred line rat strain identification, and primer sequence |
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| CN108034728A (en) * | 2018-02-07 | 2018-05-15 | 北京诺赛基因组研究中心有限公司 | SNP marker for detecting lung cancer susceptibility combines, primer combines and kit |
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| CN104975105B (en) * | 2015-07-31 | 2018-04-13 | 华南理工大学 | SNP marker, primer pair and its application for mouse metallothionein-Ⅰ identification |
| CN108588236B (en) * | 2018-05-17 | 2021-02-26 | 江苏集萃药康生物科技股份有限公司 | SNP rapid detection method for monitoring genetic quality of inbred line, SNP locus and primer thereof |
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| CN108034728A (en) * | 2018-02-07 | 2018-05-15 | 北京诺赛基因组研究中心有限公司 | SNP marker for detecting lung cancer susceptibility combines, primer combines and kit |
Non-Patent Citations (2)
| Title |
|---|
| 2018年实验动物中心繁殖C57BL/6J遗传质量SNP检测结果;清华大学实验动物中心;《http://www.larc.tsinghua.edu.cn/post/499》;20180306;第2-3页 * |
| A sequence-based variation map of 8.27 million SNPs in inbred mouse strains;Kelly A. Frazer et al.;《NATURE》;20070830;第448卷;第1052页右栏倒数第3段、附件表8 * |
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| WO2019218743A1 (en) | 2019-11-21 |
| CN108588236A (en) | 2018-09-28 |
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