CN102206706B - Method for detecting single nucleotide polymorphism of cattle Dapper1 gene - Google Patents
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Abstract
The invention discloses a method for detecting the single nucleotide polymorphism of a cattle Dapper1 gene, comprising the following steps of: carrying out PCR (Polymerase Chain Reaction) amplification on the cattle Dapper1 gene by taking a cattle complete genome DNA, containing the Dapper1 gene, to be detected as a template and primer pairs P (P1, P2, P3, P4) as primers; respectively digesting PCR amplification products by using restriction enzymes MspI, HindII, NcoI and HhaI, then carrying out agarose gel electrophoresis on amplified fragments subjected to enzyme digestion; and identifying the single nucleotide polymorphism of a 8344th place, a 8428th place, a 10513rd place and a 10765th place of the cattle Dapper1 gene according to a result of the agarose gel electrophoresis. The detection method provided by the invention lays a foundation for the establishment of the relation between the single nucleotide polymorphism of the cattle Dapper1 gene and growth traits so as to be used for the MAS (Marker Assisted Selection) of the growth traits used for the meat of Chinese cattle, thereby fast establishing a cattle population with excellent genetic resources.
Description
Technical field
The invention belongs to the molecular genetics field, relate to the detection of gene mononucleotide polymorphism (SNP), particularly a kind of method that detects ox Dapper1 gene mononucleotide polymorphism.
Background technology
Single nucleotide polymorphism (SNP) just refers in the genomic dna sequence polymorphism that the replacement owing to single core thuja acid (A/T/C/G) causes.In any known or unknown gene or near all may find quantity not wait SNPs, SNPs (cSNPs) in the gene coding region is fewer, because the aberration rate in exon only accounts for 1/5 of sequence on every side, but it is the tool significance in the research of inherited disease and breeding, therefore receives much attention.
In recent years, people have been developed many methods be used to seeking molecular genetic marker, and modal have single-strand conformation polymorphism technology (SSCP), direct Sequencing technology and PCR-RFLP etc., but SSCP complex operation, length consuming time, the result easily causes erroneous judgement; And the direct Sequencing technical costs is higher.The PCR-RFLP method is the effective technology of a kind of SNP of detection, uses restriction enzyme to cut after finding the SNP site, then carries out the agarose gel electrophoresis analysis, just can differentiate exactly the SNP site.The PCR-RFLP method not only has the accuracy of dna sequencing method, has overcome again somewhat expensive, troublesome operation, false-positive shortcoming, and the sequence site of detecting is without the singularity requirement.
Dapper1 albumen is a kind of signals-modulating molecule.In the lower animals such as Xenopus laevis and zebra fish, Dapper1 plays an important role in a plurality of processes of fetal development in early days.At present, many on human and fish for the research of Dapper1 gene, mainly large quantity research has been done in the aspects such as the effect in the Wnt signal path and relative disease genesis mechanism.Have no the research about the variation of animal Dapper1 gene genetic both at home and abroad.The research in Chinese Cattle Dapper1 gene genetic variation field is deficient, and the functional study of this gene locus and heritable variation thereof the research related with economic characters (as: proterties such as body weight and day weight gain) is still blank.Because the Dapper1 gene function relates to the growth traitss such as body weight and day weight gain, detection method provided by the invention is that the SNP of Dapper1 gene and the foundation of growth traits relation are laid a good foundation, for use in the marker assisted selection (MAS) of Chinese Cattle growth traits, the ox population that the Rapid Establishment genetic resources is good.
Summary of the invention
The problem that the present invention solves is to utilize the PCR-RFLP method to detect ox Dapper1 gene polynorphisms, and itself and growth traits carried out association analysis, verify whether it can be used as the molecule marker of assisted Selection in the ox molecular breeding, thereby accelerate fine-variety breeding speed.
The present invention is achieved through the following technical solutions:
A kind of method that detects ox Dapper1 gene mononucleotide polymorphism, take the ox complete genome DNA to be measured that comprises the Dapper1 gene as template, take primer pair P1, P2, P3, P4 as primer, pcr amplification ox Dapper1 gene; Digest respectively after the pcr amplification product with restriction enzyme Ms mouth, HindII, NcoI, HhaI, amplified fragments after again enzyme being cut carries out agarose gel electrophoresis, identify the 8344th of ox Dapper1 gene, the 8428th, the 10513rd, the 10765th single nucleotide polymorphism according to the agarose gel electrophoresis result
Described primer pair P1 is:
Upstream primer: gttgactgtt ccccctccac accac 25bp;
Downstream primer: gaggaacatt gggctctgca cggcccc; 27bp.
Described primer pair P2 is:
Upstream primer: gaggagcggc ttggtaacca tgtca 25bp;
Downstream primer: cagcgttcac actggtcctc gg; 22bp.
Described primer pair P3 is:
Upstream primer: tgtgaagcag atacaagggg cagcc 25bp;
Downstream primer: gtggcaaagg ttttagcgaa tcc; 23bp.
Described primer pair P4 is:
Upstream primer: tacccaacat tgatgccttt ttcgc 25bp;
Downstream primer: caagacaggg tcagtggtcc aatc.24bp。
Described pcr amplification reaction program is:
94 ℃ of denaturation 5min; 94 ℃ of sex change 30s of 30～34 circulations, 65 ℃ of annealing 30s, 72 ℃ are extended 30s; 72 ℃ are extended 10min.
The mass concentration of described sepharose is 3.0%.
Describedly identify that according to the agarose gel electrophoresis result single nucleotide polymorphism of the 8344th of ox Dapper1 gene is: the TT genotype shows as the 159bp band; The CT genotype shows as 159bp, 131bp and 28bp band; The CC genotype shows as 131bp and 28bp band.The 8428th single nucleotide polymorphism is: the TT genotype shows as the 211bp band; The CT genotype shows as 211bp, 187bp and 24bp band; The CC genotype shows as 187bp and 24bp.The 10513rd single nucleotide polymorphism is: the GG genotype shows as the 206bp band; The AG genotype shows as 206bp, 183bp and 23bp band; The AA genotype shows as 183bp and 23bp.The 10765th single nucleotide polymorphism is: the CC genotype shows as the 242bp band; The CG genotype shows as 242bp, 219bp and 23bp band; The GG genotype shows as 219bp and 23bp.
The present invention is according to the primers of Dapper1 gene, take the genomic dna of 5 kinds of ox kinds as template, carries out pcr amplification, and the PCR product is checked order respectively, obtains the partial sequence of ox Dapper1 gene after the order-checking.The sequence of announcing with NCBI compares to be found to have the SNP polymorphism at the 8344th, the 8428th, the 10513rd, the 10765th.
For the above-mentioned polymorphism of SNP everywhere, the invention also discloses its examination and detection method, identify by designing increase specific digestion with restriction enzyme of specific primer PCR, can be simply, quick, cost is low, detect accurately the polymorphism of its mononucleotide.
The present invention has carried out detection and gene frequency analysis to the SNP genotype of 5 ox kinds, association analysis is carried out in above-mentioned SNP site and ox some growth proterties (body weight and day weight gain etc.), and the result shows that this site can be as the molecule marker that improves the ox growth traits.
Description of drawings
Fig. 1 is ox Dapper1 gene PCR product electrophorogram, and wherein Fig. 1 a, 1b, 1c, 1d are respectively the 8344th, the 8428th, the 10513rd, the 10765th polymorphic site PCR product electrophorogram;
Fig. 2 is that ox Dapper1 gene enzyme is cut electrophoresis result figure, and wherein Fig. 2 a, 2b, 2c, 2d are respectively the restriction enzyme digestion and electrophoresis result that ox Dapper1 gene comprises the 8344th, the 8428th, the 10513rd, the 10765th polymorphic site PCR product;
Fig. 3 is ox Dapper1 gene SNP polymorphism sequencing result figure, wherein Fig. 3 a, 3b, 3c, 3d are respectively the sequencing result figure that ox Dapper1 gene comprises the 8344th, the 8428th, the 10513rd, the 10765th polymorphic site, and the base that " square frame " of curve top comprises is mutating alkali yl;
Fig. 4 is the schematic diagram that the present invention is used for detecting SNP site mutation in the PCR design of primers of SNP polymorphism and the amplified production, it is the PCR design of primers that ox Dapper1 gene comprises the 8344th, the 8428th, the 10513rd, the 10765th polymorphic site that Fig. 4 a, 4b, 4c, 4d detect respectively, represent in the square frame that wherein mutational site, dash area are the restriction endonuclease recognition sequence, small letter is the base mismatch of introducing.
Embodiment
The present invention utilizes the PCR-RFLP method that the 8344th of ox Dapper1 gene, the 8428th, the 10513rd, the 10765th single nucleotide polymorphism are detected, below in conjunction with the present invention is described in further detail, the explanation of the invention is not limited.
A, ox Dapper1 gene contain the design of the 6th exon and 3 distolateral pterion PCR primers
Take ox (NC_007308.4) sequence that NCBI was announced as reference, utilize Primer 5.0 designs to increase and comprise the PCR primer in ox Dapper1 gene the 6th exon and 3 distolateral pterions.
The primer of the 6th exon of increasing is:
Upstream primer: acaccacaat ctaattccct tgc 23bp;
Downstream primer: cttaccttga agttcggtag cag; 23bp.
The primer in 3 distolateral pterions of increasing is:
Upstream primer: ggtggtgaca gtgagtgga 19bp;
Downstream primer: ttgggctaat gtttagatgg.20bp。
With above-mentioned primer pair ox genome amplification; amplification comprises ox Dapper1 gene (NC_007308.4) the 6th exon and 3 distolateral pterion fragments; to the fragment of amplification check order identify after; there are the two SNP (NC_007308.4:8344C＞T of place at the 6th exon; 8428C＞T); simultaneously, also there are two SNP of place (NC_007308.4:10513A＞G, 10765C＞G) in 3 distolateral pterions.
By analysis, there is not natural restriction enzyme site in above SNP everywhere, therefore, and by designing primer P (P1, P2, P3, P4) respectively to SNP introducing everywhere MspI, HindII, NcoI, HhaI restriction enzyme site.
B, carry out the Dapper1 gene fragment of pcr amplification ox to be measured with primer P
1, the collection of ox sample
The present invention specifically with the population of 5 native Chinese cattle kinds as detected object, concrete collecting sample sees Table 1:
The collection of table 1 ox sample
2, the separation of blood sample genomic dna, extraction, purifying
1) freezing blood sample (being mainly hemocyte) room temperature is thawed, and transferase 45 00 μ L to 1.5mLEppendorf centrifuge tube adds equal-volume PBS liquid, abundant mixing, the centrifugal 10min of 12000r/min (4 ℃), abandoning supernatant, the repetition above-mentioned steps is transparent to supernatant liquor, precipitation is faint yellow;
2) in centrifuge tube, add DNA extraction buffer 500 μ L, shake, make the hemocyte precipitation break away from centrifuge tube tube wall, 37 ℃ of water-bath 1h;
3) add Proteinase K to 3 μ L (20mg/mL) and mixing, 55 ℃ are spent the night to clarification, and not yet the defecator can add 1 μ L Proteinase K mixing and continue digestion until clarify;
4) reaction solution is cooled to room temperature, adds the saturated phenol 500 μ L of Tris-, gentleness is shaken centrifuge tube 20min, makes its abundant mixing; 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to, repeats once;
5) add chloroform 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min,
Supernatant liquor is changed in another 1.5mL centrifuge tube;
6) add chloroform, primary isoamyl alcohol mixed solution (24: 1) 500 μ L, abundant mixing 20min, 4 ℃, the centrifugal 10min of 12000r/min changes supernatant liquor in another 1.5mL centrifuge tube over to;
7) add the NaAc damping fluid of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes, mix and rotate centrifuge tube, until the flocks of white is separated out, preserve 30～60min for-20 ℃;
8) 4 ℃, the centrifugal 10min of 12000r/min, abandoning supernatant precipitates 2 times with 70% ice-cold ethanol rinsing DNA;
9) 4 ℃, the centrifugal 10min of 12000r/min makes the ethanol volatilization clean under the abandoning supernatant, room temperature;
10) dried DNA is dissolved in the TE liquid of 80～100 μ L, preserves until DNA dissolves fully for 4 ℃, and 0.8% agarose gel electrophoresis detects its quality ,-80 ℃ of preservations.
11) adding 10%SDS in the dna solution of 500 μ L, to make its final concentration be 0.1%, adds Proteinase K to final concentration and reach 50 μ g/mL;
12) 5 ℃ are incubated about 10h;
13) equal-volume phenol, chloroform, primary isoamyl alcohol (25: 24: 1) and the extracting of chloroform difference are once;
14) the centrifugal 5min phase-splitting of 12000r/min is drawn the upper strata water to another centrifuge tube;
15) add 1/10 volume 3mol/L sodium-acetate and the 2 times of ice-cold dehydrated alcohol precipitation of volume DNA;
16) outwell liquid, dry after 70% washing with alcohol, add the dissolving of 60 μ L sterilization ultrapure water, 4 ℃ to be detected.
3, pcr amplification
The PCR reaction system adopts mixes the application of sample method, namely according to the number of the required PCR reaction of the quantity of the required various components of each reaction system and 1 secondary response, calculate the total amount of various reactive components, join in 1 1.5mL centrifuge tube, fully instantaneous centrifugal behind the mixing, divide again to install in each 0.2mL Eppendorf PCR pipe, then add template DNA, more instantaneous centrifugal laggard performing PCR amplification;
The PCR reaction system sees Table 2:
Table 2PCR reaction system
25 μ L reaction systems comprise 0.625U Taq archaeal dna polymerase (sky, Beijing root Science and Technology Ltd.), and 2 * Buffer, 12.5 μ L (include Mg
2+, dNTPs etc.) (Mix of sky, Beijing root Science and Technology Ltd.), 50ng/ μ L contains the ox genomic dna 0.45 μ L of Dapper1 gene, each 0.5 μ L of 10pmol/ μ L upstream and downstream primer;
Genomic dna to 1185 samples of 5 ox kinds carries out pcr amplification, obtains respectively to comprise in the ox Dapper1 gene of 1185 individualities the dna fragmentation in SNP site.
C, with restriction endonuclease MspI, HindII, NcoI, HhaI respectively enzyme cut digestion pcr amplification the Dapper1 gene fragment
1, endonuclease reaction digestion system (25～30 μ L): 10～15 μ L PCR products, 10 * damping fluid (containing BSA), 2.5～3.0 μ L, restriction enzyme (10U/ μ L) is 1.0～1.5 μ L, sterilization pure water (H
2O) 11.5～16.5 μ L;
2, enzyme is cut digestion condition: digest 5～10h in 37 ℃ of constant incubators.
Agarose gel electrophoresis analysis behind d, the digestion with restriction enzyme PCR product
1) sepharose of making 3.0%, 120V voltage electrophoresis is 1 hour behind the point sample, and EB staining examine enzyme is cut the result;
2) treat that the different dna fragmentation of molecular weight separates when clear, in the imaging of BIO-RAD Gel Doc2000 gel imaging system;
3) according to agarose gel electrophoresis interpretation of result SNP polymorphism:
The single nucleotide polymorphism that the Dapper1 gene is the 8344th is: the TT genotype shows as the 159bp band; The CT genotype shows as 159bp, 131bp and 28bp band; The CC genotype shows as 131bp and 28bp band.The 8428th single nucleotide polymorphism is: the TT genotype shows as the 211bp band; The CT genotype shows as 211bp, 187bp and 24bp band; The CC genotype shows as 187bp and 24bp.The 10513rd single nucleotide polymorphism is: the GG genotype shows as the 206bp band; The AG genotype shows as 206bp, 183bp and 23bp band; The AA genotype shows as 183bp and 23bp.The 10765th single nucleotide polymorphism is: the CC genotype shows as the 242bp band; The CG genotype shows as 242bp, 219bp and 23bp band; The GG genotype shows as 219bp and 23bp.
The frequency statistics analysis of e, ox Dapper1 gene SNP site
1) gene and genotype frequency
Genotype frequency refers to that certain genotype number of individuals of a certain proterties in the colony accounts for the ratio of total individual number.P
AA=N
AA/ N, wherein P
AARepresent the AA genotype frequency in a certain site; N
AAHas the genotypic number of individuals of AA in the expression colony; N is for detecting the total quantity of colony.
Gene frequency refers to that a certain gene number is to the relative ratios of its allelotrope sum in the colony.The formula that calculates can be write as: P
A=(2N
AA+ N
Aa1+ N
Aa2+ N
Aa3+ N
Aa4+ ...+N
Aan)/2N
In the formula, P
AExpression allelotrope A frequency, N
AAHas the genotypic individual amount of AA, N in the expression colony
AaiHave Aai genotype individual amount in the expression colony, a1-an is n the mutually different multiple allelomorphos of allelotrope A.
In the SNP of different ox kind Dapper1 genes, the gene frequency rangeability is as shown in table 3.
Table 3 ox Dapper1 gene SNP Gene frequency distribution table
The association analysis of F, ox Dapper1 gene SNP site genetic effect
The genotype (CC, CG and GG) of the genotype (AA, AG and GG) of the genotype (CC, CT and TT) of the genotype (CC, CT and TT) of genotype data: MspI identification, HindII identification, NcoI identification, HhaI identification.
Production data: the body weight in Nanyang cattle June, December, 18 months and 24 months, height, body are long, chest measurement and day weight gain data.
Relation analysis model: first data are described analysis, determine whether to exist outlier, the analysis of recycling least square is proofreaied and correct data; According to data characteristics, use in the GLM process analysis genotype of SAS (9.1) software and the bottle each behavioural effect.When being analyzed, the genotype effect adopted fixed model:
Y
ijkl＝μ+BF
i+Month
j+G
k+e
ijkl，
Wherein: Y
IjklBe the character observation value, μ is population mean, BF
iBe the fixedly effect on i kind and farm, Month
jBe the fixed effect of observation in j month, G
kBe the fixed effect of k single SNP marker genetype, e
IjklBe random error.
The result shows (seeing Table 4): at the 8344th, the genotypic whose body weight of 6 monthly age CC, height, body length, chest measurement and the weight average that increases day by day are higher than TT genotype individuality and significant difference (P＜0.05); At the 8428th, the genotypic whose body weight of 6 monthly age AA, height, body are long, chest measurement and the weight average that increases day by day are higher than GG genotype individuality and difference extremely significantly (P＜0.01).And other loci gene types and growth form concern that difference is not remarkable.The AA genotype that the 8344th CC genotype and the 8428th be described can be made the candidate molecules genetic marker that improves Weight of Yellow Cattle and day weight gain as one.
Variance analysis between table the 8344th and the 8428th polymorphic site of 4Dapper1 gene and each monthly age body weight of Nanyang cattle and the day weight gain
Annotate: have same letter and represent difference not remarkable (P＞0.05), the different expression of letter significant differences (P＜0.05).
Claims (3)
1. a method that detects ox Dapper1 gene mononucleotide polymorphism is characterized in that, take the ox complete genome DNA to be measured that comprises the Dapper1 gene as template, and take primer pair P1, P2, P3, P4 as primer, pcr amplification ox Dapper1 gene; Digest respectively after the pcr amplification product with restriction endonuclease MspI, HindII, NcoI, HhaI, amplified fragments after again enzyme being cut carries out agarose gel electrophoresis, identify the 8344th of ox Dapper1 gene, the 8428th, the 10513rd, the 10765th single nucleotide polymorphism according to the agarose gel electrophoresis result
Described primer pair P1 is:
Upstream primer: gttgactgtt ccccctccac accac
Downstream primer: gaggaacatt gggctctgca cggcccc;
Described primer pair P2 is:
Upstream primer: gaggagcggc ttggtaaccg tca
Downstream primer: cagcgttcac actggtcctc gg;
Described primer pair P3 is:
Upstream primer: tgtgaagcag atacaagggg cagcc
Downstream primer: gtggcaaagg ttttagcgaa tcc;
Described primer pair P4 is:
Upstream primer: tacccaacat tgatgcctttttcgc
Downstream primer: caagacaggg tcagtggtcc aatc;
Described single nucleotide polymorphism is:
The 8344th single nucleotide polymorphism is that the TT genotype shows as the 159bp band; The CT genotype shows as 159bp, 131bp and 28bp band; The CC genotype shows as 131bp and 28bp band;
The 8428th single nucleotide polymorphism is: the TT genotype shows as the 211bp band;
The CT genotype shows as 211bp, 187bp and 24bp band; The CC genotype shows as 187bp and 24bp;
The 10513rd single nucleotide polymorphism is: the GG genotype shows as the 206bp band; The AG genotype shows as 206bp, 183bp and 23bp band; The AA genotype shows as 183bp and 23bp;
The 10765th single nucleotide polymorphism is: the CC genotype shows as the 242bp band; The CG genotype shows as 242bp, 219bp and 23bp band; The GG genotype shows as 219bp and 23bp;
Described pcr amplification program is:
94 ℃ of denaturation 5min; 94 ℃ of sex change 30s of 30～34 circulations, 65 ℃ of annealing 30s, 72 ℃ are extended 30s; 72 ℃ are extended 10min.
2. the method for detection ox Dapper1 gene mononucleotide polymorphism as claimed in claim 1 is characterized in that, the mass concentration of described sepharose is 3.0%.
3. the application of method in the different ox of evaluation colony polymorphism of claim 1 or 2 described detection ox Dapper1 gene mononucleotide polymorphisms, identify that different ox polymorphisms are, the 8344th of ox Dapper1 gene and the 8428th 's SNP is as the molecule marker of assisted Selection in the ox molecular breeding, and the 8344th CC genotype and the 8428th 's AA genotype is for improving the candidate molecules genetic marker of Weight of Yellow Cattle and day weight gain.
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