CN113355404A - Method for rapidly and accurately identifying mouse genetic background transformation quality - Google Patents
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Abstract
The invention belongs to the field of identification of mouse genetic background transformation quality, and particularly discloses a method for quickly and accurately identifying mouse genetic background transformation quality. The method mainly comprises the following steps: the method comprises the steps of S1 differential acquisition of different genetic backgrounds, S2 primer design, S3 extraction of mouse genome, S4 PCR amplification verification and identification of S5 born mice. The conversion quality of the mouse genetic background is judged by the biological information technology, the conventional PCR technology and the sanger sequencing technology, the defects of high technical difficulty, high workload, high cost and the like of the existing scheme are overcome, and whether the mouse genetic background is successfully converted or not can be determined quickly and accurately at relatively low cost.
Description
Technical Field
The invention belongs to the field of identification of mouse genetic background transformation quality, and particularly discloses a method for quickly and accurately identifying mouse genetic background transformation quality.
Background
The genetic background (strain) of mice can have a great influence on the experimental results, and how to quickly and accurately identify whether a conversion is successful is critical when we need to convert one strain into another.
The following methods are currently used to identify the success of mouse strain conversion:
(1) bone assay, which compares bone differences with differences in bone properties by multivariate morphological analysis. The method can accurately distinguish mice of different strains, and can reach the accuracy of 98 percent;
(2) the identification of mouse strains was carried out by constructing a whole genome library using Intragenic A-type particles (IAP) as a recognition site and obtaining the differences between mice of different strains by PCR as a genetic marker (DOI 10.1007/s 12033-010-9338-6).
Although the methods have various characteristics, the methods have the following defects:
(1) the specific properties of the mouse skeleton need to be determined, the workload is large, the accuracy is low, and the related technology is relatively complex; the bone determination needs a large amount of mouse samples, dozens of mice are generally needed, and the mice also need to be killed, so that the workload is large, and the cost is high.
(2) The differences of the Intra specific A-type particles (IAP) among different strains are inconsistent, and the IAP is interfered by false positive and false negative, the workload is high, the interference is strong, the alternative sites are few, and the like. According to the method, a large amount of PCR is carried out on a genome, then a library is constructed, finally the difference of mice with different backgrounds is determined through analysis of the library, the construction of the library needs a plurality of different PCR reactions, and the method also comprises the work with relatively high difficulty of constructing strains and the like.
Disclosure of Invention
Aiming at the problems, the invention discloses a method for rapidly and accurately identifying the transformation quality of the genetic background of a mouse.
The technical scheme of the invention is as follows:
a method for rapidly and accurately identifying the genetic background transformation quality of a mouse comprises the following steps:
acquiring genetic background difference before and after S1 transformation, designing an S2 primer, extracting a mouse genome from S3, verifying PCR amplification of S4, and identifying the transformation quality of S5 born mice. .
Further, the method for rapidly and accurately identifying the mouse genetic background transformation quality comprises the following specific steps of:
1) respectively finding the Ensembl _ gene _ id corresponding to each strain according to the Ncbi _ gene _ id of the mouse genes before and after transformation;
2) obtaining gene sequence information according to Ensembl _ gene _ id corresponding to each strain and pyensembl data packet
3) The sequences of the two lines of each gene were aligned using a Needleman-Wunsch alignment algorithm global alignment;
4) screening out the result of the difference site of 10bp or more, and sorting out the corresponding gene and position/sequence information.
Further, the method for rapidly and accurately identifying the genetic background transformation quality of the mice comprises the two lines of C57BL/6n and BALB/C.
Further, the method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse comprises the following steps of designing primers in the step S2:
1) selecting several sites on each chromosome to carry out primer design so as to cover each chromosome;
2) designing differential primers according to different sequences, wherein the designed primers have obvious size difference of amplified bands or obvious difference of specific sequences or different combination conditions of the designed primers on different templates;
3) the primer design rules are as follows: the length is 20-25bp, the Tm value is 45-60, the GC content is 40% -60%, and no obvious mismatch exists.
Further, the method for rapidly and accurately identifying the mouse genetic background transformation quality comprises the following steps of:
chromosome 1: 108900, 105244010, 21346, 17926; chromosome 2: 13067. 100503468, 100303644; chromosome 3: 102635958, 21427; chromosome 4: 100039968, 117592; chromosome 5: 100861794, 102633504, 102634580, 109202; chromosome 6: 95659, 246278, 246278; chromosome 7: 100504421, 110959; chromosome 8: 11459. 99496, 290066; chromosome 9: 235587, 108068; chromosome 10: 100504474, 13611; chromosome 11: 68097. 12261, 78889; chromosome 12: 380780, 67732; chromosome 13: 100038494, 71690, 238683; chromosome 14: 140806, 29811; chromosome 15: 17069, 110454, 110454; chromosome 16: 109857, 57808; chromosome 17: 14960. 14913, respectively; chromosome 18: 100038353, 67843; chromosome 19: 19662. 18120, 70605; chromosome X: 108160, 67564, 78248.
Further, the method for rapidly and accurately identifying the transformation quality of the mouse genetic background comprises the following steps of:
chromosome 1:
108900 site: 1-3-F1 ═ SEQ ID NO: 1. 1-3-R1 ═ SEQ ID NO: 2. 1-3-F2 ═ SEQ ID NO: 3; 105244010 site: 1-6-F1 ═ SEQ ID NO: 4. 1-6-R1 ═ SEQ ID NO: 5; 21346 site: 1-8-F1 ═ SEQ ID NO: 6. 1-8-R1 ═ SEQ ID NO: 7. 1-8-F2 ═ SEQ ID NO: 8; 17926 site: 1-9-F1 ═ SEQ ID NO: 9. 1-9-R1 ═ SEQ ID NO: 10. 1-9-F2 ═ SEQ ID NO: 11;
chromosome 2:
13067 site: 2-3-F1 ═ SEQ ID NO: 12. 2-3-R1 ═ SEQ ID NO: 13; 100503468 site: 2-5-F1 ═ SEQ ID NO: 14. 2-5-R1 ═ SEQ ID NO: 15; 100303644 site: 2-8-F1 ═ SEQ ID NO: 16. 2-8-R1 ═ SEQ ID NO: 17. 2-8-R2 ═ SEQ ID NO: 18;
chromosome 3:
102635958 site: 3-3-F1 ═ SEQ ID NO: 19. 3-3-R1 ═ SEQ ID NO: 20. 3-3-R2 ═ SEQ ID NO: 21; 21427 site: 3-9-F1 ═ SEQ ID NO: 22. 3-9-R1 ═ SEQ ID NO: 23;
chromosome 4:
100039968 site: 4-1-F1 ═ SEQ ID NO: 24. 4-1-R1 ═ SEQ ID NO: 25. 4-1-F2 ═ SEQ ID NO: 26; 117592 site: 4-6-F1 ═ SEQ ID NO: 27. 4-6-R1 ═ SEQ ID NO: 28;
chromosome 5:
100861794 site: 5-3-F1 ═ SEQ ID NO: 29. 5-3-R1 ═ SEQ ID NO: 30. 5-3-R2 ═ SEQ ID NO: 31; 102633504 site: 5-4-F1 ═ SEQ ID NO: 32. 5-4-R1 ═ SEQ ID NO: 33; 102634580 site: 5-5-F1 ═ SEQ ID NO: 34. 5-5-R1 ═ SEQ ID NO: 35; 5-5-F2 ═ SEQ ID NO: 36; 109202 site: 5-6-F1 ═ SEQ ID NO: 37. 5-6-R1 ═ SEQ ID NO: 38. 5-6-R2 ═ SEQ ID NO: 39;
chromosome 6:
95659 site: 6-1-F1 ═ SEQ ID NO: 40. 6-1-R1 ═ SEQ ID NO: 41; 246278 site: 6-3-F1 ═ SEQ ID NO: 42. 6-3-R1 ═ SEQ ID NO: 43; 246278 site: 6-4-F1 ═ SEQ ID NO: 44. 6-4-R1 ═ SEQ ID NO: 45, a first step of;
chromosome 7:
100504421 site: 7-1-F1 ═ SEQ ID NO: 46. 7-1-R1 ═ SEQ ID NO: 47. 7-1-F2 ═ SEQ ID NO: 48; 110959 site: 7-5-F1 ═ SEQ ID NO: 49. 7-5-R1 ═ SEQ ID NO: 50;
chromosome 8:
position 11459: 8-1-F1 ═ SEQ ID NO: 51. 8-1-R1 ═ SEQ ID NO: 52; 99496 site: 8-2-F1 ═ SEQ ID NO 53: 8-2-R1 ═ SEQ ID NO: 54. 8-2-F2 ═ SEQ ID NO: 55; 290066 site: 8-3-F1 ═ SEQ ID NO: 56. 8-3-R1 ═ SEQ ID NO: 57. 8-3-F2 ═ SEQ ID NO: 58;
chromosome 9:
235587 site: 9-1-F1 ═ SEQ ID NO: 59. 9-1-R1 ═ SEQ ID NO: 60, adding a solvent to the mixture; 108068 site: 9-5-F1 ═ SEQ ID NO: 61. 9-5-R1 ═ SEQ ID NO: 62, a first step of mixing;
chromosome 10:
100504474 site: 10-3-F1 ═ SEQ ID NO: 63. 10-3-R1 ═ SEQ ID NO: 64. 10-3-F2 ═ SEQ ID NO: 65; 13611 site: 10-6-F1 ═ SEQ ID NO: 66. 10-6-R1 ═ SEQ ID NO: 67. 10-6-F2 ═ SEQ ID NO: 68;
chromosome 11:
68097 site: 11-2-F1 ═ SEQ ID NO: 69. 11-2-R1 ═ SEQ ID NO: 70. 11-2-R2 ═ SEQ ID NO: 71; 12261 site: 11-3-F1 ═ SEQ ID NO: 72. 11-3-R1 ═ SEQ ID NO: 73. 11-3-R2 ═ SEQ ID NO: 74; 78889 site: 11-10-F1 ═ SEQ ID NO: 75. 11-10-R1 ═ SEQ ID NO: 76. 11-10-R2 ═ SEQ ID NO: 77;
chromosome 12:
380780 site: 12-4-F1 ═ SEQ ID NO: 78. 12-4-R1 ═ SEQ ID NO: 79. 12-4-R2 ═ SEQ ID NO: 80; 67732 site: 12-6-F1 ═ SEQ ID NO: 81. 12-6-R1 ═ SEQ ID NO: 82. 12-6-F2 ═ SEQ ID NO: 83;
chromosome 13:
100038494 site: 13-1-F1 ═ SEQ ID NO: 84. 13-1-R1 ═ SEQ ID NO: 85 parts by weight; 71690 site: 13-5-F1 ═ SEQ ID NO: 86. 13-5-R1 ═ SEQ ID NO: 87. 13-5-R2 ═ SEQ ID NO: 88; 238683 site: 13-6-F1 ═ SEQ ID NO: 89. 13-6-R1 ═ SEQ ID NO: 90. 13-6-R2 ═ SEQ ID NO: 91;
chromosome 14:
140806 site: 14-4-F1 ═ SEQ ID NO: 92. 14-4-R1 ═ SEQ ID NO: 93. 14-4-R2 ═ SEQ ID NO: 94; 29811 position: 14-8-F1 ═ SEQ ID NO: 95. 14-8-R1 ═ SEQ ID NO: 96. 14-8-R2 ═ SEQ ID NO: 97, a stabilizer;
chromosome 15:
17069 site: 15-3-F1 ═ SEQ ID NO: 98. 15-3-R1 ═ SEQ ID NO: 99; 110454 site: 15-5-F1 ═ SEQ ID NO: 100. 15-5-R1 ═ SEQ ID NO: 101, a first electrode and a second electrode; 110454 site: 15-6-F1 ═ SEQ ID NO: 102. 15-6-R1 ═ SEQ ID NO: 103. 15-6-R2 ═ SEQ ID NO: 104
Chromosome 16:
109857 site: 16-5-F1 ═ SEQ ID NO: 105. 16-5-R1 ═ SEQ ID NO: 106; 57808 site: 16-6-F1 ═ SEQ ID NO: 107. 16-6-R1 ═ SEQ ID NO: 108. 16-6-R2 ═ SEQ ID NO: 109;
chromosome 17:
14960 site: 17-2-F1 ═ SEQ ID NO: 110. 17-2-R1 ═ SEQ ID NO: 111; 14913 site: 17-5-F1 ═ SEQ ID NO: 112. 17-5-R1 ═ SEQ ID NO: 113. 17-5-R2 ═ SEQ ID NO: 114, and a carrier;
chromosome 18:
100038353 site: 18-4-F1 ═ SEQ ID NO: 115. 18-4-R1 ═ SEQ ID NO: 116. 18-4-F2 ═ SEQ ID NO: 117; 67843 position: 18-8-F1 ═ SEQ ID NO: 118. 18-8-R1 ═ SEQ ID NO: 119. 18-8-F2 ═ SEQ ID NO: 120 of a solvent;
chromosome 19:
19662, site: 19-4-F1 ═ SEQ ID NO: 121. 19-4-R1 ═ SEQ ID NO: 122; 18120, site: 19-6-F1 ═ SEQ ID NO: 123. 19-6-R1 ═ SEQ ID NO: 124; 70605 site: 19-9-F1 ═ SEQ ID NO: 125. 19-9-R1 ═ SEQ ID NO: 126;
chromosome X:
108160 site: X-1-F1 ═ SEQ ID NO: 127. X-1-R1 ═ SEQ ID NO: 128; 67564 position: X-4-F1 ═ SEQ ID NO: 129. X-4-R1 ═ SEQ ID NO: 130. X-4-R2 ═ SEQ ID NO: 131; 78248 site: X-5-F1 ═ SEQ ID NO: 132. X-5-R1 ═ SEQ ID NO: 133.
further, the method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse, wherein the step S3 of extracting the genome of the mouse, comprises the following specific steps:
1) clipping 2-3mm of rat tail, and placing into an EP tube;
2) adding 98. mu.L of Triton X-100 rat tail lysate and 2. mu.L of proteinase K into the EP tube;
3) putting the sample and the EP pipe frame filled with the sample into a 56 ℃ oven, and cracking overnight;
4) the lysed sample was inactivated at 98 ℃ for 13 min.
Further, the method for rapidly and accurately identifying the transformation quality of the mouse genetic background, wherein the step S5 of identifying the transformation quality of the born mouse, comprises the following specific steps:
1) carrying out electrophoresis detection on the PCR amplification product, and judging by combining the theoretical size difference in the design of the primers;
2) and (3) sequencing and verifying the amplified band, comparing the sequencing result with a theoretical sequence, and quickly determining the genetic background of the mouse so as to quickly confirm whether the transformation is successful.
Furthermore, the method for rapidly and accurately identifying the mouse genetic background transformation quality is applied to biological experiments.
The method for rapidly and accurately identifying the genetic background transformation quality of the mouse disclosed by the invention has at least the following beneficial effects:
the improvement of the present invention over the prior art is shown in Table 1 below
TABLE 1 advancement of the invention compared to the prior art
1. Compared with the traditional method, the genetic background difference determination method has the advantages that: the difficulty of difference determination is reduced, more difference information is provided, and the risk of incapability of confirmation due to small difference is reduced due to convenience in selection.
2. Compared with the traditional method, the identification method of the invention has the advantages that: the requirement for equipment is reduced, and only sequencing and electrophoretic analysis are needed to quickly determine.
3. Compared with the traditional method, the result analysis of the invention has the advantages that: results can be analyzed quickly by relatively simple agarose gel electrophoresis and sanger sequencing.
4. Sequence alignment allows direct one-off identification of all differences, which can be used to confirm background transitions.
5. Corresponding primers are designed according to the differences, and the primers can be stored for a long time after being verified, so that the result can be quickly obtained without redesigning the primers.
Drawings
FIGS. 1A-1C are PCR bands obtained by amplification of 1-16 sets of primers in example 1;
FIG. 2 is a graph showing the results of sequencing in example 1;
FIGS. 3A-3D are PCR bands obtained by amplification of example 2 for sets 1-16 of primers;
FIG. 4 is a graph showing the sequencing results in example 2.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The chromosomal Gene loci selected and the designed primer sequences of the present invention are shown in Table 2 below
TABLE 2 chromosomal Gene loci and primer sequences designed
The experimental process comprises the following steps:
acquiring genetic background difference before and after S1 transformation, designing an S2 primer, extracting a mouse genome from S3, verifying PCR amplification of S4, and identifying the transformation quality of S5 born mice.
The experimental method comprises the following steps:
PCR amplification System and PCR reaction procedure: as shown in tables 3-4 below
TABLE 3 PCR amplification System
TABLE 4 PCR reaction procedure
2, extracting a mouse genome:
1) clipping 2-3mm of rat tail, and placing into an EP tube;
2) adding 98. mu.L of Triton X-100 rat tail lysate and 2. mu.L of proteinase K into the EP tube;
3) putting the sample and the EP pipe frame filled with the sample into a 56 ℃ oven, and cracking overnight;
4) the lysed sample was inactivated at 98 ℃ for 13 min.
3 identification of born mice
1) Carrying out electrophoresis detection on the PCR amplification product, and judging by combining the theoretical size difference in the design of the primers;
2) and (3) sequencing and verifying the amplified band, comparing the sequencing result with a theoretical sequence, and quickly determining the genetic background of the mouse so as to quickly confirm whether the conversion is successful.
Example 1
Verifying whether the background has been successfully converted
The chromosomal gene locus and the designed primer sequence as shown in the following Table 5 were selected for PCR amplification verification
TABLE 5 chromosomal Gene loci and designed primer sequences
The amplification band diagrams are shown in FIGS. 1A-1C, and the electrophoretic map shows:
1. the numbers marked on the electrophoretic chart represent the numbers of table 5 above, for example, "1" in the figure represents the previous number 1 in table 5.
M stands for Marker, and 100bp Marker was used in the experiment.
3. Each serial number on the glue map corresponds to three glue holes, which respectively correspond to C57BL/6n, BALB/C and negative control (H2O). The specific information represented by '1' in the following figure is the PCR result of the first primer group for amplifying three templates (C57 BL/6n, BALB/C and negative control in turn from left to right), and the amplification result is 265bp, none and none theoretically. The actual results of the glue picture analysis are consistent with the theory.
The sequencing result is shown in FIG. 2, and the sequencing result indicates that:
(25) - (C) - (Wdr54) - (6-1-F1) represents: sequence number-line (C for C57BL/6 n; B for BALB/C) -Gene name-primer name;
it can be seen that the sequences sequenced at the same position in the mice of different strains are different, and the strain determination can be carried out by combining the above electrophoretograms.
Example 2
Verifying whether the background has been successfully converted
The chromosomal gene loci and designed primer sequences as shown in Table 6 below were selected for PCR amplification verification
TABLE 6 chromosomal Gene loci and designed primer sequences
The amplification bands are shown in FIGS. 3A to 3D, which illustrate the same example 1, the sequencing results are shown in FIG. 4, and the sequencing results are the same as in example 1.
Summary of the invention
The results of the above examples 1-2 show that the currently most used genetic background determination schemes are performed by relatively complicated biological techniques, which are time-consuming, have high thresholds and are not highly accurate.
The technology of the invention can confirm the genetic background quickly, simply and accurately by a simple technology and a clear flow.
(1) Sequence alignment allows direct one-off identification of all differences, which can be used to confirm background transitions.
(2) Corresponding primers are designed according to the differences, and the primers can be stored for a long time after being verified, so that the result can be quickly obtained without redesigning the primers.
The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.
SEQUENCE LISTING
<110> Sai industries (Suzhou) Biotechnology Ltd
<120> method for rapidly and accurately identifying mouse genetic background transformation quality
<130> 2021
<160> 133
<170> PatentIn version 3.5
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<212> DNA
<213> artificial
<400> 6
caaagaggtg ctttggagtc tca 23
<210> 7
<211> 23
<212> DNA
<213> artificial
<400> 7
agtatgatca gaggatctgc cgt 23
<210> 8
<211> 23
<212> DNA
<213> artificial
<400> 8
gtgcttttag gcagctcagg agt 23
<210> 9
<211> 21
<212> DNA
<213> artificial
<400> 9
tggtccagag cagggtccag t 21
<210> 10
<211> 23
<212> DNA
<213> artificial
<400> 10
gaggattaca ctcacactgc cag 23
<210> 11
<211> 23
<212> DNA
<213> artificial
<400> 11
ctagttcctt atgctggtcc agt 23
<210> 12
<211> 22
<212> DNA
<213> artificial
<400> 12
ttaggtggct gtggtatccg ag 22
<210> 13
<211> 24
<212> DNA
<213> artificial
<400> 13
acaccaaatc atctccttca tcag 24
<210> 14
<211> 24
<212> DNA
<213> artificial
<400> 14
tgaattacgt gagtgctgac cagt 24
<210> 15
<211> 22
<212> DNA
<213> artificial
<400> 15
agtgtttccc ctggattcca at 22
<210> 16
<211> 22
<212> DNA
<213> artificial
<400> 16
ttattcatca agtgcccttc tg 22
<210> 17
<211> 23
<212> DNA
<213> artificial
<400> 17
actcttcctt cttgtgggat tat 23
<210> 18
<211> 24
<212> DNA
<213> artificial
<400> 18
tctgggaaga taaggtagac tgat 24
<210> 19
<211> 24
<212> DNA
<213> artificial
<400> 19
taagcaggga gatgagatta tata 24
<210> 20
<211> 21
<212> DNA
<213> artificial
<400> 20
ttgccaaggt gattacattc t 21
<210> 21
<211> 25
<212> DNA
<213> artificial
<400> 21
aggtgattac attctttcta tagtt 25
<210> 22
<211> 24
<212> DNA
<213> artificial
<400> 22
atacagactc agctggcaat ggtt 24
<210> 23
<211> 24
<212> DNA
<213> artificial
<400> 23
ctctctgggc tctgtcctgt gatt 24
<210> 24
<211> 23
<212> DNA
<213> artificial
<400> 24
gccatagatg gtatacaaat gga 23
<210> 25
<211> 24
<212> DNA
<213> artificial
<400> 25
gaatccctta cttggttgca ctgg 24
<210> 26
<211> 21
<212> DNA
<213> artificial
<400> 26
gtggctcagc cgtagatgga t 21
<210> 27
<211> 22
<212> DNA
<213> artificial
<400> 27
ttaagttggg agtgcacgtc tt 22
<210> 28
<211> 23
<212> DNA
<213> artificial
<400> 28
cctgcttggt gagacagctg ctt 23
<210> 29
<211> 22
<212> DNA
<213> artificial
<400> 29
ctggagacca gagcttgcac tt 22
<210> 30
<211> 25
<212> DNA
<213> artificial
<400> 30
cacagtgtct ctaatccatt gatcc 25
<210> 31
<211> 23
<212> DNA
<213> artificial
<400> 31
ctatagactc aacccacaca gtg 23
<210> 32
<211> 22
<212> DNA
<213> artificial
<400> 32
gcagactcag agccaaatca tg 22
<210> 33
<211> 22
<212> DNA
<213> artificial
<400> 33
gcacacgcat agacctatgc ac 22
<210> 34
<211> 21
<212> DNA
<213> artificial
<400> 34
tgacacaaac taggaggcca c 21
<210> 35
<211> 24
<212> DNA
<213> artificial
<400> 35
agaaaccaca gaggagtgct gctt 24
<210> 36
<211> 22
<212> DNA
<213> artificial
<400> 36
caaactagaa gtcacctggg ag 22
<210> 37
<211> 23
<212> DNA
<213> artificial
<400> 37
ggtttccgta tacagccaga agt 23
<210> 38
<211> 24
<212> DNA
<213> artificial
<400> 38
gtacccgagt gactgcagtg ggtt 24
<210> 39
<211> 23
<212> DNA
<213> artificial
<400> 39
gatgagcctc tttgtaacac tgg 23
<210> 40
<211> 24
<212> DNA
<213> artificial
<400> 40
gaccccagca ctagacagag caaa 24
<210> 41
<211> 23
<212> DNA
<213> artificial
<400> 41
gcagacactg ggcaagaacc act 23
<210> 42
<211> 26
<212> DNA
<213> artificial
<400> 42
ccacactaag gaatttcagg tttggg 26
<210> 43
<211> 25
<212> DNA
<213> artificial
<400> 43
cactatctgc atctgaacct cagca 25
<210> 44
<211> 24
<212> DNA
<213> artificial
<400> 44
tgcctggaac tcactgagat tcac 24
<210> 45
<211> 22
<212> DNA
<213> artificial
<400> 45
agcctgcttt atggccatga gt 22
<210> 46
<211> 24
<212> DNA
<213> artificial
<400> 46
gacctcaaac tcgcagcatc caaa 24
<210> 47
<211> 24
<212> DNA
<213> artificial
<400> 47
cctagccttg atcatgggtc tagg 24
<210> 48
<211> 23
<212> DNA
<213> artificial
<400> 48
ggccacatgt gagtaaactg gaa 23
<210> 49
<211> 23
<212> DNA
<213> artificial
<400> 49
gtcagcaacc tcccaggtag ttg 23
<210> 50
<211> 26
<212> DNA
<213> artificial
<400> 50
acctatctgc ttaaagtcaa tcccag 26
<210> 51
<211> 23
<212> DNA
<213> artificial
<400> 51
ccaaagctaa ccgggagaag atg 23
<210> 52
<211> 22
<212> DNA
<213> artificial
<400> 52
ctcatagatg ggcacgttgt gg 22
<210> 53
<211> 25
<212> DNA
<213> artificial
<400> 53
gcaggtcaac ttggctgtaa gtaga 25
<210> 54
<211> 24
<212> DNA
<213> artificial
<400> 54
ggcaacagtc aagactgcaa taga 24
<210> 55
<211> 22
<212> DNA
<213> artificial
<400> 55
tgggtttgga tgggcgagtg ag 22
<210> 56
<211> 21
<212> DNA
<213> artificial
<400> 56
tcctggccac cctcagcact g 21
<210> 57
<211> 24
<212> DNA
<213> artificial
<400> 57
tttcaaaggg tacgtgggaa cagc 24
<210> 58
<211> 30
<212> DNA
<213> artificial
<400> 58
tcagtgtaac atctggctac atagtgaatt 30
<210> 59
<211> 21
<212> DNA
<213> artificial
<400> 59
agcagggctt catcacatcc a 21
<210> 60
<211> 21
<212> DNA
<213> artificial
<400> 60
gccccagcga ttccagcaga a 21
<210> 61
<211> 21
<212> DNA
<213> artificial
<400> 61
agtcctcaga ttctgctggc c 21
<210> 62
<211> 21
<212> DNA
<213> artificial
<400> 62
gggtaaatga aagcacaggc c 21
<210> 63
<211> 26
<212> DNA
<213> artificial
<400> 63
caactatctg tgaatctact tgaggc 26
<210> 64
<211> 23
<212> DNA
<213> artificial
<400> 64
ctcttgctat gggaccacag agt 23
<210> 65
<211> 26
<212> DNA
<213> artificial
<400> 65
tccagtcttc tagcagcctt cagatg 26
<210> 66
<211> 23
<212> DNA
<213> artificial
<400> 66
ctctgacccc agcacatgaa agg 23
<210> 67
<211> 25
<212> DNA
<213> artificial
<400> 67
ggagtcacta ctggacaatg ggcat 25
<210> 68
<211> 25
<212> DNA
<213> artificial
<400> 68
ggtttttcga gacagggttt ctctg 25
<210> 69
<211> 20
<212> DNA
<213> artificial
<400> 69
cactcgacct ttccttcaac 20
<210> 70
<211> 21
<212> DNA
<213> artificial
<400> 70
gctccaaaag caacactggt t 21
<210> 71
<211> 21
<212> DNA
<213> artificial
<400> 71
actacccacg gagtccgtct a 21
<210> 72
<211> 22
<212> DNA
<213> artificial
<400> 72
gcactttgta tgaacacttc ag 22
<210> 73
<211> 20
<212> DNA
<213> artificial
<400> 73
gaaagtgacc gtgatcctag 20
<210> 74
<211> 20
<212> DNA
<213> artificial
<400> 74
tctccatcct agccaatgtc 20
<210> 75
<211> 21
<212> DNA
<213> artificial
<400> 75
cttgggaaac aatgtgtgca g 21
<210> 76
<211> 20
<212> DNA
<213> artificial
<400> 76
catccaaaga ctgtgcagaa 20
<210> 77
<211> 20
<212> DNA
<213> artificial
<400> 77
gctgaccttt aagtagccca 20
<210> 78
<211> 20
<212> DNA
<213> artificial
<400> 78
agccctgaac atgacatcag 20
<210> 79
<211> 20
<212> DNA
<213> artificial
<400> 79
gtggagcgat gactagaagc 20
<210> 80
<211> 21
<212> DNA
<213> artificial
<400> 80
tagaactgaa gtacaggggt c 21
<210> 81
<211> 20
<212> DNA
<213> artificial
<400> 81
gcttggcttt ccctagtaat 20
<210> 82
<211> 21
<212> DNA
<213> artificial
<400> 82
cgttatctga acagcagaaa g 21
<210> 83
<211> 19
<212> DNA
<213> artificial
<400> 83
agatggcgca actgtcagg 19
<210> 84
<211> 21
<212> DNA
<213> artificial
<400> 84
tccattcctg actcaagagt g 21
<210> 85
<211> 22
<212> DNA
<213> artificial
<400> 85
agctggataa acctctatgt tg 22
<210> 86
<211> 20
<212> DNA
<213> artificial
<400> 86
agtaacatcg gatcctttag 20
<210> 87
<211> 21
<212> DNA
<213> artificial
<400> 87
acaacgagga gctcttccag a 21
<210> 88
<211> 22
<212> DNA
<213> artificial
<400> 88
gagtttgtgg cactcagtag tg 22
<210> 89
<211> 20
<212> DNA
<213> artificial
<400> 89
gaatgagaat cccaggctta 20
<210> 90
<211> 20
<212> DNA
<213> artificial
<400> 90
cttttggagg tgacctgatg 20
<210> 91
<211> 22
<212> DNA
<213> artificial
<400> 91
ctatacttgg agttggccag ca 22
<210> 92
<211> 20
<212> DNA
<213> artificial
<400> 92
ctatggctgg agagtagcag 20
<210> 93
<211> 21
<212> DNA
<213> artificial
<400> 93
tccctgacct cttcctggga t 21
<210> 94
<211> 20
<212> DNA
<213> artificial
<400> 94
atgctaccag aatatgtctg 20
<210> 95
<211> 20
<212> DNA
<213> artificial
<400> 95
gcatggatcg tctgatatct 20
<210> 96
<211> 22
<212> DNA
<213> artificial
<400> 96
caactcaacg accctaataa cc 22
<210> 97
<211> 22
<212> DNA
<213> artificial
<400> 97
caactcaacg accctaataa cc 22
<210> 98
<211> 21
<212> DNA
<213> artificial
<400> 98
ccaggaattc tgaggtagag a 21
<210> 99
<211> 21
<212> DNA
<213> artificial
<400> 99
tagccattaa gacaactcga g 21
<210> 100
<211> 21
<212> DNA
<213> artificial
<400> 100
acattacctc tcccatgtta g 21
<210> 101
<211> 22
<212> DNA
<213> artificial
<400> 101
ccttttatag tgggctgtta tc 22
<210> 102
<211> 20
<212> DNA
<213> artificial
<400> 102
tgggatgtga tcatcaagca 20
<210> 103
<211> 20
<212> DNA
<213> artificial
<400> 103
gaaactttgg aggatctgat 20
<210> 104
<211> 20
<212> DNA
<213> artificial
<400> 104
ttccttcagc ctaaactcca 20
<210> 105
<211> 25
<212> DNA
<213> artificial
<400> 105
ggaaattctg tcctaagctc aagcc 25
<210> 106
<211> 22
<212> DNA
<213> artificial
<400> 106
tactaaatgc cacgaagcac cg 22
<210> 107
<211> 22
<212> DNA
<213> artificial
<400> 107
ggaccgcggt ttgcctcatg ga 22
<210> 108
<211> 22
<212> DNA
<213> artificial
<400> 108
ggaccgcggt ttgcctcatg ga 22
<210> 109
<211> 21
<212> DNA
<213> artificial
<400> 109
caggaggaac gctactttct c 21
<210> 110
<211> 22
<212> DNA
<213> artificial
<400> 110
agtgaacgat gtgtacatcc ac 22
<210> 111
<211> 22
<212> DNA
<213> artificial
<400> 111
ggcaggagtc tccttttctc ac 22
<210> 112
<211> 22
<212> DNA
<213> artificial
<400> 112
tgccgaggaa ttcacagata cc 22
<210> 113
<211> 23
<212> DNA
<213> artificial
<400> 113
gacagagcca gcgctggggc agt 23
<210> 114
<211> 21
<212> DNA
<213> artificial
<400> 114
gataacggat ccagaaccca a 21
<210> 115
<211> 22
<212> DNA
<213> artificial
<400> 115
aaacacataa tgaccatgcg ga 22
<210> 116
<211> 21
<212> DNA
<213> artificial
<400> 116
actgtcagga cgaaggccca g 21
<210> 117
<211> 20
<212> DNA
<213> artificial
<400> 117
ccagggtgga ggagtctcag 20
<210> 118
<211> 20
<212> DNA
<213> artificial
<400> 118
cattaggcga agggttaccc 20
<210> 119
<211> 21
<212> DNA
<213> artificial
<400> 119
cgggcttcca gaccaactat g 21
<210> 120
<211> 22
<212> DNA
<213> artificial
<400> 120
aatccaccct taccaccacc ct 22
<210> 121
<211> 20
<212> DNA
<213> artificial
<400> 121
tgacaccatc cagagatggg 20
<210> 122
<211> 24
<212> DNA
<213> artificial
<400> 122
gtgaccatga agttcctcag taga 24
<210> 123
<211> 20
<212> DNA
<213> artificial
<400> 123
gcaggtctat gtggggacca 20
<210> 124
<211> 21
<212> DNA
<213> artificial
<400> 124
ttccacctcc aagttctgcg a 21
<210> 125
<211> 23
<212> DNA
<213> artificial
<400> 125
tcttcattgg tgttcttggg ggt 23
<210> 126
<211> 24
<212> DNA
<213> artificial
<400> 126
cccgagatgc ccctaaagca agga 24
<210> 127
<211> 22
<212> DNA
<213> artificial
<400> 127
tgccaccatg gctgtacagg tc 22
<210> 128
<211> 22
<212> DNA
<213> artificial
<400> 128
acaccaaagt cagtaccagg gc 22
<210> 129
<211> 24
<212> DNA
<213> artificial
<400> 129
gggctcaaag aggttgaagt acgg 24
<210> 130
<211> 21
<212> DNA
<213> artificial
<400> 130
gaggaagctt cctggtggtg a 21
<210> 131
<211> 21
<212> DNA
<213> artificial
<400> 131
aatactgtac ttaggatgca g 21
<210> 132
<211> 22
<212> DNA
<213> artificial
<400> 132
gaggagacca agccccaaat gt 22
<210> 133
<211> 24
<212> DNA
<213> artificial
<400> 133
acatccagct tcccgggtgc gacc 24
Claims (9)
1. A method for rapidly and accurately identifying the genetic background transformation quality of a mouse is characterized by comprising the following steps:
acquiring genetic background difference before and after S1 transformation, designing an S2 primer, extracting a mouse genome from S3, verifying PCR amplification of S4, and identifying the transformation quality of S5 born mice.
2. The method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse according to claim 1, wherein the step S1 of obtaining the genetic background difference before and after transformation comprises the following specific steps:
1) respectively finding the Ensembl _ gene _ id corresponding to each strain according to the Ncbi _ gene _ id of the mouse genes before and after transformation;
2) obtaining gene sequence information according to Ensembl _ gene _ id corresponding to each strain and pyensembl data packet
3) The sequences of the two lines of each gene were aligned using a Needleman-Wunsch alignment algorithm global alignment;
4) screening out the result of the difference site of 10bp or more, and sorting out the corresponding gene and position/sequence information.
3. The method of claim 2, wherein the two lines are C57BL/6n and BALB/C.
4. The method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse according to claim 1, wherein the step S2 primer design comprises the following design methods:
1) selecting several sites on each chromosome to carry out primer design so as to cover each chromosome;
2) designing differential primers according to different sequences, wherein the designed primers have obvious size difference of amplified bands or obvious difference of specific sequences or different combination conditions of the designed primers on different templates;
3) the primer design rules are as follows: the length is 20-25bp, the Tm value is 45-60, the GC content is 40% -60%, and no obvious mismatch exists.
5. The method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse as claimed in claim 4, wherein the locus is selected from one or more of the following gene IDs:
chromosome 1: 108900, 105244010, 21346, 17926
Chromosome 2: 13067. 100503468, 100303644
Chromosome 3: 102635958, 21427
Chromosome 4: 100039968, 117592
Chromosome 5: 100861794, 102633504, 102634580, 109202
Chromosome 6: 95659. 246278, 246278
Chromosome 7: 100504421, 110959
Chromosome 8: 11459. 99496, 290066
Chromosome 9: 235587, 108068
Chromosome 10: 100504474, 13611
Chromosome 11: 68097. 12261, 78889
Chromosome 12: 380780, 67732
Chromosome 13: 100038494, 71690, 238683
Chromosome 14: 140806, 29811
Chromosome 15: 17069. 110454, 110454
Chromosome 16: 109857, 57808
Chromosome 17: 14960. 14913
Chromosome 18: 100038353, 67843
Chromosome 19: 19662. 18120, 70605
Chromosome X: 108160, 67564, 78248.
6. The method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse as claimed in claim 5, wherein the primer sequence of the site is selected from the group consisting of:
chromosome 1:
108900 site: 1-3-F1= SEQ ID NO: 1. 1-3-R1= SEQ ID NO: 2. 1-3-F2= SEQ ID NO: 3;
105244010 site: 1-6-F1= SEQ ID NO: 4. 1-6-R1= SEQ ID NO: 5;
21346 site: 1-8-F1= SEQ ID NO: 6. 1-8-R1= SEQ ID NO: 7. 1-8-F2= SEQ ID NO: 8;
17926 site: 1-9-F1= SEQ ID NO: 9. 1-9-R1= SEQ ID NO: 10. 1-9-F2= SEQ ID NO: 11;
chromosome 2:
13067 site: 2-3-F1= SEQ ID NO: 12. 2-3-R1= SEQ ID NO: 13;
100503468 site: 2-5-F1= SEQ ID NO: 14. 2-5-R1= SEQ ID NO: 15;
100303644 site: 2-8-F1= SEQ ID NO: 16. 2-8-R1= SEQ ID NO: 17. 2-8-R2= SEQ ID NO: 18;
chromosome 3:
102635958 site: 3-3-F1= SEQ ID NO: 19. 3-3-R1= SEQ ID NO: 20. 3-3-R2= SEQ ID NO: 21;
21427 site: 3-9-F1= SEQ ID NO: 22. 3-9-R1= SEQ ID NO: 23;
chromosome 4:
100039968 site: 4-1-F1= SEQ ID NO: 24. 4-1-R1= SEQ ID NO: 25. 4-1-F2= SEQ ID NO: 26;
117592 site: 4-6-F1= SEQ ID NO: 27. 4-6-R1= SEQ ID NO: 28;
chromosome 5:
100861794 site: 5-3-F1= SEQ ID NO: 29. 5-3-R1= SEQ ID NO: 30. 5-3-R2= SEQ ID NO: 31;
102633504 site: 5-4-F1= SEQ ID NO: 32. 5-4-R1= SEQ ID NO: 33;
102634580 site: 5-5-F1= SEQ ID NO: 34. 5-5-R1= SEQ ID NO: 35; 5-5-F2= SEQ ID NO: 36;
109202 site: 5-6-F1= SEQ ID NO: 37. 5-6-R1= SEQ ID NO: 38. 5-6-R2= SEQ ID NO: 39;
chromosome 6:
95659 site: 6-1-F1= SEQ ID NO: 40. 6-1-R1 = SEQ ID NO: 41;
246278 site: 6-3-F1= SEQ ID NO: 42. 6-3-R1= SEQ ID NO: 43;
246278 site: 6-4-F1= SEQ ID NO: 44. 6-4-R1= SEQ ID NO: 45, a first step of;
chromosome 7:
100504421 site: 7-1-F1= SEQ ID NO: 46. 7-1-R1= SEQ ID NO: 47. 7-1-F2= SEQ ID NO: 48;
110959 site: 7-5-F1= SEQ ID NO: 49. 7-5-R1= SEQ ID NO: 50;
chromosome 8:
position 11459: 8-1-F1= SEQ ID NO: 51. 8-1-R1= SEQ ID NO: 52
99496 site: 8-2-F1= SEQ ID NO 53: 8-2-R1= SEQ ID NO: 54. 8-2-F2= SEQ ID NO: 55;
290066 site: 8-3-F1= SEQ ID NO: 56. 8-3-R1= SEQ ID NO: 57. 8-3-F2= SEQ ID NO: 58
Chromosome 9:
235587 site: 9-1-F1= SEQ ID NO: 59. 9-1-R1= SEQ ID NO: 60, adding a solvent to the mixture;
108068 site: 9-5-F1= SEQ ID NO: 61. 9-5-R1= SEQ ID NO: 62, a first step of mixing;
chromosome 10:
100504474 site: 10-3-F1= SEQ ID NO: 63. 10-3-R1= SEQ ID NO: 64. 10-3-F2= SEQ ID NO: 65;
13611 site: 10-6-F1= SEQ ID NO: 66. 10-6-R1= SEQ ID NO: 67. 10-6-F2= SEQ ID NO: 68;
chromosome 11:
68097 site: 11-2-F1= SEQ ID NO: 69. 11-2-R1= SEQ ID NO: 70. 11-2-R2= SEQ ID NO: 71
12261 site: 11-3-F1= SEQ ID NO: 72. 11-3-R1= SEQ ID NO: 73. 11-3-R2= SEQ ID NO: 74
78889 site: 11-10-F1= SEQ ID NO: 75. 11-10-R1= SEQ ID NO: 76. 11-10-R2= SEQ ID NO: 77
Chromosome 12:
380780 site: 12-4-F1= SEQ ID NO: 78. 12-4-R1= SEQ ID NO: 79. 12-4-R2= SEQ ID NO: 80
67732 site: 12-6-F1= SEQ ID NO: 81. 12-6-R1= SEQ ID NO: 82. 12-6-F2= SEQ ID NO: 83
Chromosome 13:
100038494 site: 13-1-F1= SEQ ID NO: 84. 13-1-R1= SEQ ID NO: 85
71690 site: 13-5-F1= SEQ ID NO: 86. 13-5-R1= SEQ ID NO: 87. 13-5-R2= SEQ ID NO: 88;
238683 site: 13-6-F1= SEQ ID NO: 89. 13-6-R1= SEQ ID NO: 90. 13-6-R2= SEQ ID NO: 91;
chromosome 14:
140806 site: 14-4-F1= SEQ ID NO: 92. 14-4-R1= SEQ ID NO: 93. 14-4-R2= SEQ ID NO: 94;
29811 position: 14-8-F1= SEQ ID NO: 95. 14-8-R1= SEQ ID NO: 96. 14-8-R2= SEQ ID NO: 97, a stabilizer;
chromosome 15:
17069 site: 15-3-F1= SEQ ID NO: 98. 15-3-R1= SEQ ID NO: 99
110454 site: 15-5-F1= SEQ ID NO: 100. 15-5-R1= SEQ ID NO: 101
110454 site: 15-6-F1= SEQ ID NO: 102. 15-6-R1= SEQ ID NO: 103. 15-6-R2= SEQ ID NO: 104
Chromosome 16:
109857 site: 16-5-F1= SEQ ID NO: 105. 16-5-R1= SEQ ID NO: 106.
57808 site: 16-6-F1= SEQ ID NO: 107. 16-6-R1= SEQ ID NO: 108. 16-6-R2= SEQ ID NO: 109
Chromosome 17:
14960 site: 17-2-F1= SEQ ID NO: 110. 17-2-R1= SEQ ID NO: 111
14913 site: 17-5-F1= SEQ ID NO: 112. 17-5-R1= SEQ ID NO: 113. 17-5-R2= SEQ ID NO: 114
Chromosome 18:
100038353 site: 18-4-F1= SEQ ID NO: 115. 18-4-R1= SEQ ID NO: 116. 18-4-F2= SEQ ID NO: 117;
67843 position: 18-8-F1= SEQ ID NO: 118. 18-8-R1= SEQ ID NO: 119. 18-8-F2= SEQ ID NO: 120 of a solvent;
chromosome 19:
19662, site: 19-4-F1= SEQ ID NO: 121. 19-4-R1= SEQ ID NO: 122;
18120, site: 19-6-F1= SEQ ID NO: 123. 19-6-R1= SEQ ID NO: 124;
70605 site: 19-9-F1= SEQ ID NO: 125. 19-9-R1= SEQ ID NO: 126;
chromosome X:
108160 site: X-1-F1= SEQ ID NO: 127. X-1-R1= SEQ ID NO: 128;
67564 position: X-4-F1= SEQ ID NO: 129. X-4-R1= SEQ ID NO: 130. X-4-R2= SEQ ID NO: 131;
78248 site: X-5-F1= SEQ ID NO: 132. X-5-R1= SEQ ID NO: 133.
7. the method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse as claimed in claim 1, wherein the step S3 of extracting the genome of the mouse comprises the following specific steps:
1) clipping 2-3mm of rat tail, and placing into an EP tube;
2) adding 98. mu.L of Triton X-100 rat tail lysate and 2. mu.L of proteinase K into the EP tube;
3) putting the sample and the EP pipe frame filled with the sample into a 56 ℃ oven, and cracking overnight;
4) the lysed sample was inactivated at 98 ℃ for 13 min.
8. The method for rapidly and accurately identifying the transformation quality of the genetic background of the mouse according to claim 1, wherein the identification of the transformation quality of the mouse born in the step S5 comprises the following specific steps:
1) carrying out electrophoresis detection on the PCR amplification product, and judging by combining the theoretical size difference in the design of the primers;
2) and (3) sequencing and verifying the amplified band, comparing the sequencing result with a theoretical sequence, and quickly determining the genetic background of the mouse so as to quickly confirm whether the transformation is successful.
9. The use of the method according to any one of claims 1 to 8 for the rapid and accurate identification of the transformation quality of the genetic background of mice in biological experiments.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109706247A (en) * | 2017-10-25 | 2019-05-03 | 上海斯莱克实验动物有限责任公司 | The method that genetic quality monitoring is carried out to inbred mouse using microsatellite technology |
| 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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| CN109706247A (en) * | 2017-10-25 | 2019-05-03 | 上海斯莱克实验动物有限责任公司 | The method that genetic quality monitoring is carried out to inbred mouse using microsatellite technology |
| 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 |
| 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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| 李生斌主编: "《中华民族基因组多态现象研究 成瘾基因组学》", 30 June 2016, 西安交通大学出版社 * |
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