CN104975105A - SNP (single-nucleotide polymorphism) markers and primer pairs for mouse inbred line identification, and application thereof - Google Patents
SNP (single-nucleotide polymorphism) markers and primer pairs for mouse inbred line identification, and application thereof Download PDFInfo
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses SNP (single-nucleotide polymorphism) markers and primer pairs for mouse inbred line identification, and application thereof. 7 screened SNP sites, including SNP1, SNP2, SNP3, SNP4, SNP5, SNP6 and SNP7, are utilized to design 7 primer pairs of which the sequences are disclosed as SEQ ID NO:1-SEQ ID NO:14; and each primer pair can amplify genome sequences disclosed as SEQ ID NO:15-SEQ ID NO:21 of the mouse inbred line under the same conditions. The sequences amplified by each primer pair contain one specific SNP marker. The application of the SNP marker and primer pairs can achieve the goals of quickly performing primary screening and lowering the detection cost, thereby promoting the inbred line mouse genetic quality monitoring standardization progress.
Description
Technical field
The present invention relates to and utilize full-length genome analysis of biological information technical superiority, and by pcr amplification and sanger sequence verification SNP marker, and adopt mass-spectrometric technique and SNP marker to carry out the application of Testing and appraisal to production unit mouse metallothionein-Ⅰ, particularly for SNP marker, primer pair and the application thereof of mouse metallothionein-Ⅰ qualification.
Background technology
Current domestic Bio-pharmaceutical Industry fast development, the important support condition of laboratory animal as Bio-pharmaceutical Industry and the important materials of life science, its quality is directly connected to reliability and the verity of experimental result.The quality of heredity of laboratory animal is one of important factor of Quality of Experimental Animals quality, only has the stable laboratory animal guarantee biomedical research result of application hereditary property to have the reliability of comparability and experimental result.Therefore, Quality of Experimental Animals stdn, especially the stdn of quality of heredity will directly affect the R & D Level of biological medicine.But owing to lacking unified, standardized quality of heredity control criterion, the monitoring of the quality of heredity of laboratory animal also to become in its production application comparatively distinct issues gradually.With regard to present circumstances, examination to effective genetic marker of accreditation both at home and abroad be extensively realize the technological breakthrough of laboratory animal genetic quality monitoring have one of approach.
At present, national standard (GB 14923-2010) is mainly carried out in China's laboratory animal genetic monitoring, mainly takes morphology (coat color gene method of testing), quantitative genetics (mandibular bone assay method), immune labeled DNA test (epidermization) and Biochemical markers detection method etc.These methods respectively have superiority, and vital use value is still had in the propagation production and daily monitoring management work of laboratory animal, but along with Laboratory Animal Science and molecular biological develop rapidly, traditional detection method comprises the genetic monitoring work that Biochemical markers detection method, epidermization and immunological detection that GB tries hard to recommend can not adapt to multiple strain laboratory animal completely.
Along with the discovery of a series of molecular genetic markers such as DNA fingerprint technology, random amplified polymorphism (RAPD), micro-satellite and single nucleotide polymorphism (SNP), micro-satellite and SNP show one's talent and are widely used in the Genetic Detection of laboratory animal.SNP marker is the third generation DNA molecular marker after biochemical marker and RAPD genetic marker.There is following significant advantage: 1) those some homogenic types being difficult to differentiate itself and homology strain can be differentiated relative to biochemical process and immunological method; 2) SNP detection method than biochemical process easily important advantage be do not need to put to death animal, all gene purity is produced before the next generation at it and is just guaranteed; 3) SNP detection method can reach high-throughput, low cost and automatization, can meet large-scale detection demand.Therefore, laboratory animal SNP marker has larger research potential quality and using value at home.
2002, international mouse gene order-checking alliance completed the full order-checking of classical laboratory strain C57BL/6J (B6).In the same year, Celera company also completes the complete sequence sketch of other three inbred lines.Due to SNP and micro-number of satellite huge, high-throughout gene type can reduce costs, therefore famous laboratory animal company has used SNP to carry out Genetic Detection all.For the standard that Genetic monitoring is ununified, its Sites Combination and quantity determine with the practical situation of each company as required.Well-known laboratory animal supplier in the world, Jackson Laboratory (JAX), provides 2000 SNP to assess chip, and adopts 28 SNP site to carry out Genetic Detection to tens kinds of mouse metallothionein-Ⅰ.
Summary of the invention
In order to overcome the shortcoming of prior art with not enough, the object of the present invention is to provide a kind of SNP marker for mouse metallothionein-Ⅰ qualification.Cost is high at most to detect SNP site, and domestic conventional mouse species about 10 is planted, therefore, the present invention is intended to utilize existing inbred mouse snp database to sift out the SNP marker of the domestic conventional mouse metallothionein-Ⅰ of a few energy isolation identification, to reach the object of quick primary dcreening operation and reduction testing cost.
Another object of the present invention is to the primer pair being provided for mouse metallothionein-Ⅰ qualification.The primer pair provided is totally 7 right; And the sequence that the often pair of primer amplification goes out all contains a specific SNP marker.The application of primer can reach quick primary dcreening operation and reduce the object of testing cost, thus promotes inbred mouse genetic quality monitoring standardized process.
Another object of the present invention is to provide the application of described SNP marker.
Another object of the present invention is to provide the application of described primer pair.
Object of the present invention is achieved through the following technical solutions:
First the present invention carries out examination to NCBI mouse metallothionein-Ⅰ snp database, sift out 7 SNP site, and design primer, with 7 pairs of primer PCR amplifications 129 of design, the genome sequence of these 8 mouse metallothionein-Ⅰ of C3H, BALB, C57BL, CBA, DBA, FVB and ICR, then sanger order-checking, obtains sequence dna fragment, and verifies 7 SNP site, finally adopt mass spectrographic method to detect these 7 sites of domestic conventional mouse metallothionein-Ⅰ, determine the applicability of 7 SNP.
7 described SNP site are SNP1, SNP2, SNP3, SNP4, SNP5, SNP6 and SNP7.
Described SNP1 represents that this SNP site is positioned at the 91st of the nucleotide sequence shown in SEQ ID NO:15, and this position nucleotides sequence is classified as degeneracy base K (G/T);
Described SNP2 represents that this SNP site is positioned at the 196th of the nucleotide sequence shown in SEQ ID NO:16, and this position nucleotides sequence is classified as degeneracy base M (A/C);
Described SNP3 represents that this SNP site is positioned at the 112nd of the nucleotide sequence shown in SEQ ID NO:17, and this position nucleotides sequence is classified as degeneracy base R (A/G);
Described SNP4 represents that this SNP site is positioned at the 294th of the nucleotide sequence shown in SEQ ID NO:18, and this position nucleotides sequence is classified as degeneracy base Y (C/T);
Described SNP5 represents that this SNP site is positioned at the 188th of the nucleotide sequence shown in SEQ ID NO:19, and this position nucleotides sequence is classified as degeneracy base R (A/G);
Described SNP6 represents that this SNP site is positioned at the 244th of the nucleotide sequence shown in SEQ ID NO:20, and this position nucleotides sequence is classified as degeneracy base Y (C/T);
Described SNP7 represents that this SNP site is positioned at the 217th of the nucleotide sequence shown in SEQ ID NO:21, and this position nucleotides sequence is classified as degeneracy base Y (C/T);
Described primer pair is totally 7 right, and sequence is as shown in SEQ ID NO:1 ~ SEQ ID NO:14.
Described DNA fragmentation, sequence is as shown in SEQ ID NO:15 ~ SEQ ID NO:21.
7 described SNP site can be applicable to the qualification of domestic conventional mouse metallothionein-Ⅰ.
Described primer pair can be applicable to the qualification of domestic conventional mouse metallothionein-Ⅰ.
7 described SNP site are preparing the application in test kit or detection method; The purposes of described test kit or detection method is mouse metallothionein-Ⅰ qualification or species identification.
Described primer pair is preparing the application in test kit or detection method; The purposes of described test kit or detection method is mouse metallothionein-Ⅰ qualification or species identification.
Described DNA fragmentation is in the auxiliary application carried out in mouse metallothionein-Ⅰ or species identification.
The present invention, relative to prior art, has following advantage and effect:
(1) the present invention adopts biology information technology advantage to develop a few to be used for domestic conventional inbred mouse qualification SNP marker, the application of SNP marker greatly can reduce the cost of inbred mouse qualification and simplify the operation, and promotes inbred mouse genetic quality monitoring stdn and process.
(2) 7 pairs of primer pairs provided by the invention, the sequence that the often pair of primer amplification goes out all contains a specific SNP marker; The application of primer can reach quick primary dcreening operation and reduce the object of testing cost, thus promotes inbred mouse genetic quality monitoring standardized process.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
1, laboratory animal sample
Mouse metallothionein-Ⅰ: 129, C3H, BALB, C57BL, CBA, DBA, FVB, ICR.
2, animal tissues source: Experimental Animals Supervising Station, Guangdong Prov., Ji'nan University and Nanfang Medical Univ etc.
3, the screening of target SNP
Do you utilize https: //www.ncbi.nlm.nih.gov/snp/? term=mouse database combination pertinent literature is reported, sifts out 7 SNP site.In detail in table 1.
Table 1 SNP marker and the genotype in each mouse metallothionein-Ⅰ
| SNP ID | Chr | SEQ ID | Position | 129 | C3H | BALB/cJ | C57BL/6 | C57BL/10 | CBA/C | CBA/J | DBA/1 | FVB | ICR |
| SNP1 | 7 | NO:15 | 91 | G | G | G | T | T | G | G | G | G | G |
| SNP2 | 11 | NO:16 | 196 | A | C | C | A | C | A | A | A | A | C |
| SNP3 | 15 | NO:17 | 112 | A | A | G | G | G | A | A | A | G | G |
| SNP4 | X | NO:18 | 294 | C | T | T | T | T | T | C | C | C | T |
| SNP5 | 17 | NO:19 | 188 | A | G | A | A | A | G | G | G | G | G |
| SNP6 | 3 | NO:20 | 244 | C | C | T | T | T | C | C | C | T | C |
| SNP7 | 1 | NO:21 | 217 | C | C | T | T | T | T | T | C | C | C |
4, design of primers
After target SNP has screened, intercept the fragment of each 500bp of SNP upstream and downstream, utilize primer-design software Priemer 5.0 to carry out design of primers.Ensure that primer amplification fragment contains target SNP, primer length between 18 ~ 22bp, annealing temperature 55 DEG C.Design 7 pairs of primers altogether, primer sequence is as table 2, and in sequence table, sequence numbering is SEQ ID NO:1 ~ SEQ ID NO:14.Trust Beijing Hua Da genome company synthesis after all design of primers complete.
Table 2 primer sequence information
5, target fragment pcr amplification
By 7 pairs of primer amplifications 129, C3H, BALB, C57BL, CBA, DBA, FVB and ICR gene order of design.
Extracting genome DNA operates with reference to the specification sheets of the Animal genome DNA extraction kit (centrifugal column method) that Pu Boxin bio tech ltd manufactures.The primer more than synthesized carries out of short duration centrifugal, and adding aseptic deionized water, to be fully dissolved into concentration be 10 μMs, and room temperature leaves standstill 30 minutes, 4 DEG C of preservations.
Carry out PCR reaction using the genomic dna of each animal specimen as template, PCR reaction system (20 μ L) is as table 3.
Table 3 PRC reaction system
| Title | Concentration | Volume |
| Genomic dna | 10ng/μL | 5μL |
| Upstream primer | 10μM | 0.8μL |
| Downstream primer | 10μM | 0.8μL |
| PCR kit(2×) | 10μL | |
| ddH 2O | 3.4μL |
Of short duration centrifugal after mixing, be placed in PCR instrument, arranging cycling program is:
Above-mentioned PCR reaction product carries out 1.0% agarose gel electrophoresis qualification.
Take 0.5g agarose and be added to heating for dissolving in 50mL TAE reaction solution, when temperature is down to 60 DEG C, add ethidium bromide analogue 3 μ L and mix, pour into be inserted with 2mm comb electrophoresis chamber in, wait rear use to be solidified.Every hole adds 5 μ L PCR primer, adopts 121V constant voltage, after electrophoresis 15min under ultraviolet transilluminator observations, target stripe is whole single bright.
The successful PCR primer of all amplifications, with 4 DEG C of preservations, directly carries out DNA sequencing, and whole examining order is completed by order-checking portion of Hua Da genome company, Beijing.
6, sequencing sequence analysis
6.1 sequencing sequence splicings
Because the sequence initiating terminal sequencing quality of Sanger order-checking is lower, accuracy is relatively poor, adopts forward primer and reverse primer to check order respectively, and according to order-checking peak figure, sequence is supplemented mutually, is spliced into complete sequencing sequence.The general sequence information of 8 inbred lines is shown in shown in sequence table SEQ ID NO:15 ~ SEQ ID NO:21.
6.2PCR Sequence Identification and SNP site
Sequence alignment in the sequence and protogene group that obtain checking order, by determining whether by this primer PCR sequence obtained that increases be target sequence with the Identity (homology) of original series, 7 pairs of primer PCR product sequencing results and original genomic sequence comparison homology are all more than 99%, sequence all good comparison returns corresponding genome sequence, and cover object SNP site, see sequence SEQ ID NO:15 ~ SEQID NO:21.Show that the primer designed really can amplify target area accurately and contain target SNP site.
7, mass spectrometry method is adopted to carry out somatotype to 7 of different inbred lines SNP site
Adopt mass spectroscopy MassArray to carry out the somatotype of 7 SNP site to the mouse metallothionein-Ⅰ 129 of the unit collections such as Experimental Animals Supervising Station, Guangdong Prov., Ji'nan University and Nanfang Medical Univ, C3H, BALB, C57BL, CBA, DBA, FVB, ICR, the results are shown in Table 4.PRELIMINARY RESULTS is substantially consistent with expection, but also has a few sample not to be expected results.
The genotype of table 4 SNP marker in actual mouse metallothionein-Ⅰ
| SNP ID | Chr | SEQ ID | Position | 129 | C3H | BALB | C57BL | CBA | DBA | FVB | ICR |
| SNP1 | 7 | NO:15 | 91 | G | G | G | TG | G | G | G | G |
| SNP2 | 11 | NO:16 | 196 | A | C | C | A | A | A | A | C(CA) |
| SNP3 | 15 | NO:17 | 112 | A | A | G | G | A | A | G | G |
| SNP4 | X | NO:18 | 294 | C | T | T | T | C | C | C | T |
| SNP5 | 17 | NO:19 | 188 | A | G | A | A | G | G | G | G |
| SNP6 | 3 | NO:20 | 244 | C | C | T | T | C | C | T | C |
| SNP7 | 1 | NO:21 | 217 | C | C | T | T or C | T | C | C | C or T or CT |
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1., for a SNP marker for mouse metallothionein-Ⅰ qualification, it is characterized in that comprising 7 SNP site, is SNP1, SNP2, SNP3, SNP4, SNP5, SNP6 and SNP7;
Described SNP1 represents that this SNP site is positioned at the 91st of the nucleotide sequence shown in SEQ ID NO:15, and this position nucleotides sequence is classified as degeneracy base K;
Described SNP2 represents that this SNP site is positioned at the 196th of the nucleotide sequence shown in SEQ ID NO:16, and this position nucleotides sequence is classified as degeneracy base M;
Described SNP3 represents that this SNP site is positioned at the 112nd of the nucleotide sequence shown in SEQ ID NO:17, and this position nucleotides sequence is classified as degeneracy base R;
Described SNP4 represents that this SNP site is positioned at the 294th of the nucleotide sequence shown in SEQ ID NO:18, and this position nucleotides sequence is classified as degeneracy base Y;
Described SNP5 represents that this SNP site is positioned at the 188th of the nucleotide sequence shown in SEQ ID NO:19, and this position nucleotides sequence is classified as degeneracy base R;
Described SNP6 represents that this SNP site is positioned at the 244th of the nucleotide sequence shown in SEQ ID NO:20, and this position nucleotides sequence is classified as degeneracy base Y;
Described SNP7 represents that this SNP site is positioned at the 217th of the nucleotide sequence shown in SEQ ID NO:21, and this position nucleotides sequence is classified as degeneracy base Y.
2. the SNP marker for mouse metallothionein-Ⅰ qualification according to claim 1, is characterized in that: described mouse metallothionein-Ⅰ is 129, at least one in C3H, BALB, C57BL, CBA, DBA, FVB and ICR.
3., for a primer pair for mouse metallothionein-Ⅰ qualification, it is characterized in that: comprise at least one pair of in primer pair SNP1F/R, primer pair SNP2F/R, primer pair SNP3F/R, primer pair SNP4F/R, primer pair SNP5F/R, primer pair SNP6F/R and primer pair SNP7F/R;
The sequence of primer pair SNP1F/R is as shown in SEQ ID NO:1 and SEQ ID NO:2;
The sequence of primer pair SNP2F/R is as shown in SEQ ID NO:3 and SEQ ID NO:4;
The sequence of primer pair SNP3F/R is as shown in SEQ ID NO:5 and SEQ ID NO:6;
The sequence of primer pair SNP4F/R is as shown in SEQ ID NO:7 and SEQ ID NO:8;
The sequence of primer pair SNP5F/R is as shown in SEQ ID NO:9 and SEQ ID NO:10;
The sequence of primer pair SNP6F/R is as shown in SEQ ID NO:11 and SEQ ID NO:12;
The sequence of primer pair SNP7F/R is as shown in SEQ ID NO:13 and SEQ ID NO:14.
4. the primer pair for mouse metallothionein-Ⅰ qualification according to claim 3, is characterized in that: described mouse metallothionein-Ⅰ is 129, at least one in C3H, BALB, C57BL, CBA, DBA, FVB and ICR.
5. the DNA sequence dna utilizing the primer pair amplifies described in claim 3 or 4 to obtain, is characterized in that: described DNA sequence dna is as shown in SEQ ID NO:15 ~ SEQ ID NO:21.
6. the application of the SNP marker described in claim 1 or 2 in the qualification of mouse metallothionein-Ⅰ.
7. the SNP marker described in claim 1 or 2 is preparing the application in test kit or detection method, it is characterized in that: described test kit or the purposes of detection method are mouse metallothionein-Ⅰ qualification or species identification.
8. the application of the primer pair described in claim 3 or 4 in the qualification of mouse metallothionein-Ⅰ.
9. the primer pair described in claim 3 or 4 is preparing the application in test kit or detection method, it is characterized in that: described test kit or the purposes of detection method are mouse metallothionein-Ⅰ qualification or species identification.
10. DNA sequence dna according to claim 5 is in the auxiliary application carried out in mouse metallothionein-Ⅰ or species identification.
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| CN105671040A (en) * | 2016-03-18 | 2016-06-15 | 中国人民解放军第二军医大学 | Naked mole rat MHC gene SNP locus and application thereof |
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| CN108588236A (en) * | 2018-05-17 | 2018-09-28 | 江苏集萃药康生物科技有限公司 | The SNP rapid detection methods and SNP site and its primer of a kind of inbred strais quality of heredity monitoring |
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| CN110452969A (en) * | 2019-07-18 | 2019-11-15 | 苏州西山生物技术有限公司 | A kind of rat quality of heredity monitoring SNP marker classifying method and kit based on KASP |
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| 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 |
| 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 |
| CN114317767A (en) * | 2021-12-21 | 2022-04-12 | 斯贝福（北京）生物技术有限公司 | Primer, kit and method for distinguishing C57BL/6 subline mouse |
| CN114507744A (en) * | 2022-04-19 | 2022-05-17 | 斯贝福（北京）生物技术有限公司 | Application of SNP (Single nucleotide polymorphism) marker in inbred line mouse strain identification and primer sequence |
| CN116004842A (en) * | 2022-07-21 | 2023-04-25 | 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 |
| 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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