CN106498080A - Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms - Google Patents
Method and its application using PCR SSCP quick detection sheep NELF gene mononucleotide polymorphisms Download PDFInfo
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Abstract
The invention discloses a kind of method of utilization PCR SSCP quick detections sheep NELF gene mononucleotide polymorphisms and its application.With sheep complete genome DNA to be measured as template, with primer pair P as primer, PCR amplifications obtain sheep NELF genetic fragments, and PCR primer produces different swimming bands through degeneration into single stranded DNA, single stranded DNA electrophoresis in neutral polyacrylamide gel.According to the single nucleotide polymorphism that SSCP genotyping results identify sheep NELF genes the 454th.Method of the present invention can quickly detect the single nucleotide polymorphism of sheep NELF genes, SNP and the foundation of reproductive performance relation for NELF genes lays the foundation, marker assisted selection breeding for use in Chinese Sheep reproductive trait provides basic data, quickly sets up the excellent sheep population of genetic resourcess.
Description
Technical field
The invention belongs to molecular genetics field, be related to the screening of sheep single nucleotide polymorphism (SNP) molecular marker with
Detection, more particularly to using the 454th SNP of PCR-SSCP method quick detections NELF genome.Concrete next
Say, carry out polyacrylamide gel electrophoresis after exactly the product degeneration for obtaining being expanded to PCR, then determined according to electrophoresis result
Its single nucleotide polymorphism.
Background technology
Over nearly 20 years, especially over nearly 10 years, molecular genetics and molecular biotechnology have the development that advances by leaps and bounds, its
In, molecular genetic marker is at present most studied in cattle breeding content, and at no distant date most probable in cattle breeding
In the research project be used widely.Genetic marker refers to something lost that those accurately can differentiate, can reflecting individual specificity
Pass feature.Molecular genetic marker then refers to the DNA molecular marker based on individual inheritance material i.e. nucleotide sequence variation, is
The direct reflection of DNA level genetic polymorphism.Using molecular marker assisted selection breeding, it is one of modem animal molecular breeding
Main research, its are directly selected to the genotype of character on DNA level, and therefore its accuracy that chooses seeds is carried significantly
Height, overcomes the defect of conventional animal breeding method.
Single nucleotide polymorphism (SNP) is a kind of important molecular genetic marker, it be by American scholar Lander in
The third generation DNA genetic markers for proposing for 1996, are the DNA sequence polymorphism that causes of single nucleotide acid variation, bags in genome
Include the forms such as conversion, insertion and the disappearance of base.The ultimate principle of PCR-SSCP technology be PCR amplification after DNA fragmentation through become
Into single stranded DNA, single stranded DNA forms different three-dimensional conformations in neutral polyacrylamide gel to property during electrophoresis, its conformation is direct
Swimming speed, DNA its nucleotide sequence single-stranded of equal length is affected to only have the difference of single base, it is possible to produce three-dimensional structure
The difference of elephant, causes the difference of swimming speed, produces different swimming bands.Therefore, it can by PCR-SSCP technology for detection bases
Because group whether there is SNP site, and accurately differentiate the genotype of SNP site.Single strand conformation polymorphism is had been recognized that now
(SSCP) can be applied in breeding as genetic marker.The method not only has the accuracy of DNA sequencing method, overcomes expense again
Costliness, troublesome operation, false-positive shortcoming, and detection sequence site is without particular/special requirement.
Nose embryo's luteinising hormone-releasing hormo factor (Nasal embryonic LHRH factor, NELF) encodes base
Because the reproduction with animal has substantial connection.Samuel D.Quaynor etc. knock out (KO) mouse model by creating homozygosis NELF,
Prove that female mice NELF gene knockouts will be postponed vaginal orifice and be opened, do not postpone the time of first time heat, reduce son
Palace weight and reduce the neuron number of GnRH.In contrast, the adolescence of male mice is acted normally.NELF is knocked out and is led
Male and female mice is caused all to show as being damaged fertility, average nest litter size reduces.Additionally, increasing report shows, NELF
Play an important role in terms of the reproduction of mice.On No. 20 chromosome, total length 5928bp is encoded for the NELF gene mapping of sheep
Area's total length 1112bp.
At present, mice isotype animal is focused primarily upon to the research of NELF gene pleiomorphisms both at home and abroad, and for domestic animal
There is not been reported for the research of NELF gene mononucleotide polymorphisms.
Content of the invention
It is an object of the invention to provide a kind of utilize PCR-SSCP quick detection sheep NELF Polymorphisms
Property method and its application, so as to the molecular marker assisted selection breeding for sheep provides basic data, accelerate Chinese Sheep
Germ plasm resource improvement work.
The present invention is achieved through the following technical solutions：
With the sheep complete genome DNA to be measured comprising NELF genes as template, with primer pair P as primer, PCR expands sheep
NELF genetic fragments；Degeneration is carried out to amplified fragments, polyacrylamide gel electrophoresis are then carried out；Sentenced according to Gel electrophoresis results
Determine the single nucleotide polymorphism of sheep NELF genes；
Described primer pair P is：
Forward primer F：5’-TTGCCCCTTTCGTTGCTC-3’ 18nt；
Downstream primer R：5’-TTTCTCCACTGTCACCGCC-3’ 19nt.
Described pcr amplification reaction program is：
95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 64 DEG C of annealing 50s, 72 DEG C of extension 30s, 34 circulations；72 DEG C of extensions
10min.
Sheep NELF gene accession number NC_ are identified according to polyacrylamide gel Gel electrophoresis results
019477.2：G.454 the T of position>The polymorphism of C mutation.
Identify that sheep NELF genomes the 454th have tri- kinds of bases of TT, TC, CC according to polyacrylamide gel electrophoresis result
Because of type.
Polyacrylamide gel electrophoresis of the described gel electrophoresiss for 10-12%, first at 4 DEG C, 200-250V prerunnings
20-60min, then at room temperature, 120-150V electrophoresis 3-4h.
Beneficial effects of the present invention are embodied in：
For the 454th T of sheep NELF genes>The mutation of C, the invention provides its detection method, specific by designing
Primer, PCR expands purpose fragment, and then degeneration is into single stranded DNA, then carries out polyacrylamide gel electrophoresis identification.The method
Can simply, the single nucleotide polymorphism of the high identification gene of quick, low cost, degree of accuracy.
The present invention has carried out detection and gene frequency analysis to the genotype in mutational site, and will be numerous with sheep for the site
Grow character and be associated analysis.As a result show that the site can carry out molecular marker assisted selection breeding as molecular genetic marker,
To improve Sheep Reproductive Characters.
Description of the drawings
Sequencer maps of the Fig. 1 for the 454th (site with dark-background) TT genotype individuals of sheep NELF genomes.
Sequencer maps of the Fig. 2 for the 454th CC genotype individuals of sheep NELF genomes.
Sequencer maps of the Fig. 3 for the 454th TC genotype individuals of sheep NELF genomes.
Fig. 4 be the 454th different genotype PCR primer of sheep NELF genomes through polyacrylamide gel electrophoresis result.
Specific embodiment
With reference to the accompanying drawings and examples the present invention is described in detail, described is explanation of the invention rather than limit
Fixed.
The present invention designs primer according to sheep NELF genome sequences, with the genomic DNA pond of 2 kinds of sheep varieties is respectively
Template, enters performing PCR amplification, and product sequencing obtains the partial sequence of sheep NELF genes.With the reference sequences ratio that announces on NCBI
Right, it is found that the 454th has T in sheep NELF genomes>The mutation of C, the mutational site are located at sheep NELF gene introns
Interior, produce one and be not intended to be mutated, and itself and reproductive trait are associated analysis, be defined as related to Sheep Reproductive Characters dividing
Sub- genetic marker, is detected to the mutation using PCR-SSCP methods.
First, the clone of sheep NELF Gene Partials sequence and its polymorphic detection
1. sample collection and extracting genome DNA
(1) the collection of blood sample
2 sheep varieties are present invention employs, used as detection object, the acquisition method of blood is neck to 607 individualities altogether
Venous blood collection.The collecting region of blood sample, and the data information situation of each kind is as shown in table 1：
1 laboratory animal situation of table
(2) the extraction of blood sample DNA
1. blood sample (predominantly hemocyte) thaw at RT is freezed, 500 μ L blood are drawn in 1.5mL centrifuge tubes, add etc.
The phosphate buffer (PBS) of volume is mixed, gentle shake, and 4 DEG C, 12000r/min is centrifuged 5min, and abandoning supernatant repeats above-mentioned
Step to supernatant transparent, precipitate transparent color.
2. 500 μ L of DNA extraction buffers are added in centrifuge tube, are gently blown and beaten, make hemocyte precipitation depart from centrifugation tube wall,
37 DEG C of water-bath 1h.
3. plus E.C. 3.4.21.64 is to 3 μ L (20mg/mL), and mix, digest in 55 DEG C of water-baths overnight (16h or so) to cotton-shaped heavy
Shallow lake loses, and solution is clarified, still unclarified, can add 1 μ L E.C. 3.4.21.64s and mix and continue digestion until clarification.
4. reactant liquor is cooled to room temperature, adds 500 μ L Tris saturated phenols, gently shake 15min so as to fully mix,
4 DEG C, 12000r/min is centrifuged 10min, and upper strata aqueous phase is proceeded to another sterile centrifugation tube, repeat the above steps 1 time.
5. chloroform 500mL is added, 20min is gently shaken so as to fully mix, 4 DEG C, 12000r/min is centrifuged 15min, will
Upper strata aqueous phase proceeds to the 1.5mL centrifuge tubes of another sterilizing.
6. chloroform, isoamyl alcohol mixed liquor (24 are added:1) 500mL, is sufficiently mixed 20min, 4 DEG C, and 12000r/min is centrifuged
10min, supernatant is proceeded in another 1.5mL centrifuge tubes.
7. the NaAc buffer of 0.1 times of volume and the ice-cold dehydrated alcohol of 2 times of volumes are added, and it is straight that mixing rotates centrifuge tube
Flocculent deposit to white is separated out.
8. 4 DEG C, 12000r/min is centrifuged 10min, and abandoning supernatant is precipitated 2 times with 70% ice cold ethanol rinsing DNA.
9. 4 DEG C, 12000r/min is centrifuged 10min, and abandoning makes ethanol volatilization clean under supernatant, room temperature.
10. the ultrapure water dissolution of 80～100 μ L, 4 DEG C of preservations is added to be completely dissolved up to DNA in dried DNA solution,
Its quality, -80 DEG C of preservations are detected using UV detector.
The structure in 2.DNA ponds
With OD value of the ultraviolet light photometric determination DNA sample at 260nm, 280nm.Calculate DNA content and OD260/OD280
Ratio, DNA concentration (ng/ μ L)=50 × OD260Value × extension rate.If OD260/OD280Ratio illustrates sample less than 1.6
In containing more protein or phenol, then should carry out purification；If ratio is more than 1.8, should consider to remove RNA purification.DNA
After detection is finished, taking out a certain amount and being diluted to 50ng/ μ L, 10 concentration are then randomly choosed from 2 Sheep Populations is
5 μ L mixing constructed dnas ponds are taken in 50ng/ μ L DNA samples.
2nd, amplimer design
1. the design of sheep NELF gene PCRs primer
The sheep sequence (NC_019477.2) that is announced with NCBI, can using 5.0 software designs of Primer as reference
PCR primer of the amplification comprising the 454th site of sheep NELF genes, its primer sequence are as follows：
Forward primer F:5’-TTGCCCCTTTCGTTGCTC-3’ 18nt；
Downstream primer R:5’-TTTCTCCACTGTCACCGCC-3’ 19nt.
3rd, PCR amplifications
PCR reaction systems are as shown in table 2.
2 PCR reaction systems of table
PCR response procedures are：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 64 DEG C of annealing 50s, 72 DEG C of extension 30s, 34
Circulation；Extend 10min after 72 DEG C.
4th, PCR primer sequencing
By Hai Sheng works company limited being served with the PCR primer that DNA ponds mixed above are template amplification carry out two-way sequencing.
Sheep NELF gene purpose fragment sequencing results are compared with reference sequences, finds the 454th presence one in genome
Individual T>The mutation (referring to Fig. 1, Fig. 2 and Fig. 3) of C.
5th, PCR primer degeneration and SSCP detections
For the product degeneration first after PCR amplifications, polyacrylamide gel detection is carried out then, finally according to different
Banding pattern result judgement its SNP polymorphism.Comprise the following steps that：
(1) according to different amplified fragments, 30% acrylamide (29 is selected:1) 10%～12% polyacrylamide is made into
Amine gel (is shown in Table 3).
(2), plus after good above-mentioned solution, slowly stirred with Glass rod so as to fully mix.It is poured slowly into immediately and is ready for
Sealed bottom glass plate between, be careful not to produce bubble；After good gel, comb is plugged immediately, be sure not in comb tooth
Under leave bubble.
(3) after gelling is solid, comb is pulled out, then glass plate is fixed to vertical by the moisture in drying loading wells gently
On straight electrophoresis tank, and 1 × tbe buffer liquid is poured into toward groove.
(4) 8 μ L PCR primers are taken, 8 μ L denaturation buffers (95% Methanamide, 10mol/L EDTA, 0.05% bromine phenol is added
Blue, 0.05% dimethylbenzene is blue or green), after brief centrifugation in PCR instrument 98 DEG C of degeneration 10min, be immediately placed on after taking-up on ice, 30min
Afterwards can loading.
(5) 4 DEG C, elder generation 250V prerunning 20min, then 120V electrophoresis 4h (according to amplified fragments size, gel strength and
The length of glass plate determines voltage swing and electrophoresis time).
(6) 1 × tbe buffer liquid in electrophoresis tank is poured out recovery, then from glass plate, takes out gel, floated with distilled water
The silver nitrate solution of appropriate 0.1% after washing 2 times, is added, shaking table lucifuge is put in and is gently shaken 15min (silver nitrate recoverables 2
～3 times).
(7) nitrite ion (+0.1% formaldehyde of 2% sodium hydroxide) submergence gel is added, is observed when shaking, shown on gel
Existing electrophoresis band.
(8) after band is clear, nitrite ion is outwelled, rapid deionized water is cleaned 2～3 times.Polyacrylamide after silver staining
Amine gel is preserved using gel imaging system imaging, is stayed and is done follow-up analytic statisticss.
The composition of 3 neutral polyacrylamide gel of table
PCR-SSCP pictures are as shown in Figure 4.Different banding patterns represents different genotype.As can be seen from the figure NELF bases
Because 454 sites have three kinds of genotype, i.e. TT, TC, CC, wherein, TT genotype and CC genotype are two bands, and TT bases
Because type is compared with CC genotype, the band away from loading end is farther apart from loading end, and TC genotype has corresponding three bands.
6th, the frequency statistics of sheep NELF gene SNP sites and its association analysiss with reproductive trait
1. gene and genotypic frequency
Genotypic frequency refers to the ratio in a colony between the various genotype of a certain character.Computing formula is as follows：
PBB=NBB/N
Wherein PBBRepresent the BB genotypic frequencies in a certain site；NBBRepresent the number of individuals in colony with BB genotype；N
For detecting the total quantity of colony.
Gene frequency refers to the relative ratios of a certain its allele of gene pairss in a colony.Computing formula can be write as：
PB=(2NBB+NBb1+NBb2+NBb3+NBb4+……+NBbn)/2N
In formula, PBRepresent allele B frequencies, NBBRepresent the individual amount in colony with BB genotype, NBbiRepresent group
There is in body BbiGenotype individuals quantity, b1～bnThe n mutually different multiple alleless for allele B.
Gene frequency in each kind is as shown in table 4 with the statistical result of genotypic frequency：
The genotype of NELF genes and gene frequency in 42 sheep varieties of table
The frequency range of allele T is 0.460～0.506 as can be seen from Table 3, the frequency of allele C
Excursion is 0.494～0.500, it can be seen that, in 2 sheep varieties, for the selection of allele C is with very big
Potentiality.
2. correlation analysiss statistical model
Dependency using SAS (9.2) software analysis gene locis and reproductive trait (litter size).First data are retouched
The property stated statistical analysiss, it is determined whether there is outlier, according to data characteristicses, using t analyses, variance analyses or multiple linear mould
Type analysis genotype effects.In data handling, different according to impact litter size factor, it is contemplated that environmental effect, age, gene
Type effect and the reciprocal effects of correlation, are analyzed using fixed model, meanwhile, accepted or rejected according to practical situation.Complete
Model is as follows：
Yijk=μ+Gj+Eijk
Wherein：YijkRecord for individual phenotype；μ is colony's average；GjGenotype effects for each site；EijkIt is with chance error
Difference.
Variance analyses between 5 NELF gene mononucleotide polymorphisms of table and Sheep Reproductive Characters
Note：Meansigma methodss shoulder is put on and represents difference not significantly (P with same letter>0.05), meansigma methodss shoulder puts on letter not
With expression significant difference (P<0.05).
Variance analyses between 6 NELF gene mononucleotide polymorphisms of table and sheep reproductive trait
Variance analyses between 7 NELF gene mononucleotide polymorphisms of table and Small-fat-tail sheep reproductive trait
Referring to table 5,6,7, as a result show, on the 454th mononucleotide polymorphic site of sheep NELF gene orders
Different genotype shows that with sheep litter size association analysiss the litter size of the individuality of TC genes is significantly higher than two kinds of genes of TT and CC
The individuality of type.If from the point of view of graded kind, for sheep, the litter size of the individuality of TC genotype is significantly higher than TT and CC genes
Type is individual.But for Small-fat-tail sheep, the litter size of the individuality of TC genotype is not notable with TT and CC genotype individuals differences.
From the point of view of colony, TC genotype with TT and CC genotypic differences significantly, but only detects diversity on sheep in kind,
And be not detected by Small-fat-tail sheep, this is probably that varietY specificity is caused.Therefore speculate that TC genotype can be used as sheep morning
The molecular breeding genetic marker that phase selects.
In a word, method of the present invention can quickly detect the single nucleotide polymorphism of sheep NELF genes, be
The SNP of NELF genes is laid the foundation with the foundation of reproductive performance relation, and the labelling for use in Chinese Sheep reproductive trait is auxiliary
Help selection (MAS) breeding to provide basic data, quickly set up the excellent sheep population of genetic resourcess.
Nucleotides sequence list
<110>Lanzhou University
<120>Method and its application using PCR-SSCP quick detection sheep NELF gene mononucleotide polymorphisms
<160> 2
<210> 1
<211> 18
<212> DNA
<213>Synthetic
<400> 1
ttgccccttt cgttgctc 18
<210> 2
<211> 19
<212> DNA
<213>Synthetic
<400> 2
tttctccact gtcaccgcc 19
Claims (9)
1. using the method for PCR-SSCP quick detection sheep NELF gene mononucleotide polymorphisms, it is characterised in that：With to be measured
Sheep complete genome DNA is template, and with primer pair P as primer, PCR expands sheep NELF genetic fragments, the fragment that PCR is expanded
Then degeneration is detected by polyacrylamide gel electrophoresis into single stranded DNA, according to Gel electrophoresis results judgement sheep NELF
The genotype of mononucleotide polymorphism site on gene；
Described primer pair P is：
Forward primer：5’-TTGCCCCTTTCGTTGCTC-3’
Downstream primer：5’-TTTCTCCACTGTCACCGCC-3’.
2. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1,
It is characterized in that：The pcr amplification reaction program is：95 DEG C of denaturations 5min；95 DEG C of degeneration 30s, 64 DEG C of annealing 50s, 72 DEG C
Extend 30s, 34 circulations；72 DEG C of extension 10min.
3. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1,
It is characterized in that：The single nucleotide polymorphism of the sheep NELF genes is the 454th T of sheep NELF genes>The monokaryon of C mutation
Nucleotide polymorphism.
4. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1,
It is characterized in that：The concentration of the polyacrylamide gel is 10-12%.
5. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1,
It is characterized in that：The condition of the polyacrylamide gel electrophoresis includes：Prior to 200-250V prerunning 20-60min, then at
120-150V electrophoresis 3-4h.
6. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1,
It is characterized in that：The single nucleotide polymorphism of the sheep NELF genes shows as tri- kinds of genotype of TT, TC and CC, and electrophoresis is tied
Fruit is respectively：There are TC genotype three bands, TT and CC genotype there are two bands.
7. the method for utilization PCR-SSCP quick detections sheep NELF gene mononucleotide polymorphisms as claimed in claim 1 exists
Application in sheep molecular breeding.
8. application as claimed in claim 7, it is characterised in that：The Sheep Populations that genotype is TC are set up, the product of sheep is improved
Lamb number.
9. application as claimed in claim 8, it is characterised in that：The sheep is selected from sheep.
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| CN109576376A (en) * | 2018-11-20 | 2019-04-05 | 甘肃农业大学 | Genetic marker relevant to tibetan sheep high altitude hypoxia adaptation and its application |
| CN111118148A (en) * | 2020-03-02 | 2020-05-08 | 天津奥群牧业有限公司 | Detection primer pair, kit, method and application of sheep CSF1R gene insertion/deletion polymorphism |
| CN112342301A (en) * | 2020-11-12 | 2021-02-09 | 扬州大学 | Method for detecting Hu sheep NSMF gene CNV marker and application thereof |
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| CN112342301B (en) * | 2020-11-12 | 2023-11-24 | 扬州大学 | Method for detecting CNV (CNV) mark of NSMF (NSMF) gene of Hu sheep and application of CNV mark |
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