CN101195842A - Primer for detecting separation purity of X, Y spermatozoon of cattle - Google Patents
Primer for detecting separation purity of X, Y spermatozoon of cattle Download PDFInfo
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- CN101195842A CN101195842A CNA2008100557081A CN200810055708A CN101195842A CN 101195842 A CN101195842 A CN 101195842A CN A2008100557081 A CNA2008100557081 A CN A2008100557081A CN 200810055708 A CN200810055708 A CN 200810055708A CN 101195842 A CN101195842 A CN 101195842A
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Abstract
The invention provides a primer for detecting the X and Y sperm separation purity. Aiming at a sex-determining gene Sry on a bull Y chromosome, the primer is designed through the PCR mismatching technology. The fragment size can be amplified by 295 bp through the primer, in order to prevent false positive from appearing, a pair of internal control primers C34 is designed through the invention according to a bull autosome 3 reported sequence, the fragment size is amplified by 208bp, dual PCR amplification is performed to single bull sperm through the two pairs of primers, and then the final evaluation is performed to the sperm separation purity according to the statistical analysis to the detection result. The invention provides the technology used for identifying the bull X and Y sperm separation purity with low cost and simple, rapid, and accurate operation, and provides reliable technical support for the popularization and the application of the subsequent sexing semen and the optimization of a sperm separation method.
Description
Technical field
The present invention relates to the amplification oligonucleotide technology, be specifically related to a kind of primer that detects ox X, y sperm separation purity, this primer can carry out purity to isolating ox X, y sperm to be identified, thereby plays sex-controlled effect in the ox reproductive process.
Background technology
Sex control has huge using value in production reality, by means of the sex control technology, can select the domestic animal of different sexes to satisfy the production needs, it is the most basic technique means of sex control that X, y sperm separate, simultaneously X, the isolating order of accuarcy of y sperm are directly connected to sex-controlled effect, thus fast and accurately appraisement system establish optimization and the raising that is beneficial to separation method.Simple and efficient in addition evaluation method is directly connected to sex-controlled credibility, and the market spreading speed is accelerated in the popularization of helping property control seminal fluid.
At present for separate back sperm purity identify main adopt flow cytometer weight analysis evaluation assessment, fluorescence in situ hybridization evaluation assessment (Fluorescent In Site Hybridization, FISH).
Flow cytometer weight analysis evaluation assessment
Flow cytometer weight analysis evaluation assessment, the sperm ultrasonic docking earlier with after separating only stays sperm head, to increase the orienting effect of sperm; Carry out chromosome dyeing with fluorescence dye Hoechst33342 then, make dyestuff quantitatively with the DNA recombine, sperm carries out weight analysis with 100～200/second speed by flow cytometer.Though flow cytometer weight analysis evaluation assessment is accurate, but be subjected to the restriction of instrumentation, when also having sacrificed the machine of a lot of preciousnesses, when X sperm and y sperm dna content difference are very little, the fluidic cell weight analysis can not guarantee accuracy, adopts same instrument to carry out the evaluation analysis of separating resulting, also may cause result's deviation, flow cytometer costs an arm and a leg simultaneously, and a lot of laboratories can not be equipped with.
Fluorescence in situ hybridization evaluation assessment (FISH evaluation assessment)
FISH is a kind of detection technique that grows up on the basis that cytogenetics, molecular genetics and immunology combine, its principle is to utilize the complementarity of base, with the karyomit(e) specific nucleic acid probe and the hybridization of the chromosome sequence in the nucleus of on-radiation fluorescent substance on the mark (as: fluorescein, DAPI etc.).The fluorescence in situ hybridization evaluation assessment can obtain high-quality qualification result owing to used karyomit(e) specific probe etc.; Probe is stable simultaneously, fluorescent reagent safety.But the FISH evaluation assessment requires to prepare a lot of samples at short notice, has increased labor capacity virtually, and finishing whole qualification process needs the long period, and fluorescent reagent costs an arm and a leg simultaneously, is unfavorable for applying of this technology.
Therefore, set up a kind of accurate, quick, economic detection X, the method for y sperm separation purity is very important.
Summary of the invention
The object of the invention is to provide the specific amplification primer of a kind of X of detection, y sperm separation purity.
Another object of the present invention is to provide the method for a kind of X of detection, y sperm separation purity.
The present invention is directed to cattle Y-chromosome sex determining gene Sry sequence (gi:4878004, sequence report length is 2751bp) and designed sex special primer Y12, its nucleotide sequence such as sequence table SEQ ID NO.1﹠amp; Shown in 2, amplified production length is 295bp.By to the amplification of monosperm dna profiling, detect amplified production, if amplified production length is that 295bp shows that then this sperm is a y sperm.
For avoiding false-positive appearance, interior label primer can be set.The present invention is according to No. 3 euchromosome sequences of ox (gi:119890289, sequence report length is 83545bp) a pair of interior label primer C34 of design, its nucleotide sequence such as sequence table SEQ ID NO.3﹠amp; Shown in 4, this primer extension product length is 208bp.The appearance of the false negative experimental result of using two pairs of primers to increase to have avoided substance PCR method only to increase the special segment of Y chromosome and causing has improved the accuracy of detected result.
The present invention adopts two pairs of primers that single ox sperm is carried out the double PCR amplification, in isolating X or Y seminal fluid, spot-check about 100 monosperms respectively and carry out pcr amplification, the monosperm that has amplified production length and be 208bp is the X sperm, the monosperm that has amplified production length and be 295bp and 208bp is a y sperm, adds up amplification at last sperm purity is made final evaluation.
The inventive method comprises the separation of monosperm, the cracking of monosperm, and pcr amplification, electrophoresis detection and result add up five links.Specifically, the present invention adopts alkaline bleach liquor cleavage liquid (the 500mmol/L KOH of high density, 125mmol/L DTT) cracking sperm, every pipe monosperm add 1 μ l alkaline bleach liquor cleavage liquid, 65 ℃ of cracking 10-20min, neutralizer directly adds removing template total reaction system in addition simultaneously, usually adopt 25 μ L PCR reaction systems, Taq enzyme 0.7U wherein, dNTP concentration is 200 μ mol/L, primer Y12 concentration is 180 pmol/L, and interior label primer C34 concentration is 180pmol/L.Each ionic concn is respectively 96mmol/L Tris-HCl, 30mmol/L KCl and 1.5mmol/L MgCl
2Amplified production adopts GoldViewna
TMII fluorescence dye (remolding sensitivity EB is high about 10 times) dyeing is observed.Because the specific amplification segment of Y12 and C34 is at 200-300bp, the present invention passes through to change the amplification condition of conventional P CR reaction simultaneously, and realizes finishing pcr amplification about 55 minutes.In addition, utilize existing conventional instrument, use production domesticization reagent, one of ordinary skill in the art can be implemented the present invention.
Certainly, those skilled in the art are easy to primer of the present invention is become test kit with relevant reagent preparation, are suitable for convenient.
The inventive method is with low cost, simple to operate, quick, accurate, for follow-up property control seminal fluid apply and the optimization of sperm separation method provides a reliable technique support.
Description of drawings
Fig. 1 is the pcr amplification electrophorogram of bull and cow blood DNA, wherein, the 1-6 road is bull blood pcr amplification result, the 9-14 road is cow blood pcr amplification result, 7,8,15,16 negative contrasts, M is Marker I, and band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp;
Fig. 2 is the detected result of monosperm pcr amplification product, wherein the 1-6 road is that (1,2 are the X sperm to the monosperm amplification, 3～6 is y sperm), the positive contrast in 7 roads, 8, the negative contrast in 9 roads, M is Marker I, and band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp.
Embodiment
Embodiment 1: with bovine blood DNA is that template is carried out pcr amplification
The alkaline bleach liquor cleavage legal system is equipped with cow genome group DNA and needs alkaline bleach liquor cleavage liquid and two parts of neutralizer, and the composition of alkaline bleach liquor cleavage liquid comprises: KOH 500mmol/L, DTT 125mmol/L; The composition of neutralizer comprises: Tris.HCl 900mmol/L, pH=8.3.
With a bull and a cow is material, respectively gets 10 μ L anticoagulations in 0.2mL autoclaving PCR pipe, adds 90 μ l distilled waters, fully mixing.The centrifugal 2-3 of 12000rpm minute, discard supernatant liquid.Add 10 μ l alkaline bleach liquor cleavage liquid and fully inhale and beat mixing, 65 ℃ of cracking 10 minutes add 25 μ l neutralizers and fully inhale and beat mixing and be used for PCR and detect, or-20 ℃ of preservations are standby.
Design of primers
According to the Y chromosome sex determining gene Sry sequence (gi:4878004 that includes among the GenBank, sequence report length is 2751bp), design primer Y12, according to No. 3 euchromosome sequences of ox (gi:119890289, sequence report length is 83545bp), design primer C34, primer sequence and relevant information see Table 1, and all primers are synthetic by the handsome Bioisystech Co., Ltd in Shanghai.
Table 1 primer Y12, C34 sequence and relevant information
| Primer | Primer sequence | Primer length | The Tm value (℃) | GC content (%) | Product length |
| Y12 | F：CTA CTA GAC ATA CAC CGA GAC R：CCG TGC TGC CAA TGT TAC CT | 21 20 | 62 62 | 47.6 55.0 | 295bp |
| C34 | F：TTG CTG CTC TTG CCT TTG CTT R：GTC CAC CTG CCA CAA CTA AAT | 21 21 | 62 62 | 47.6 47.6 | 208bp |
The PCR reaction system
Adopt 25 μ l reaction systems, form by following components:
ddH
2O 8.2μl
dNTP(1mmol/L) 5.0μl
10×PCR buffer 1.5μl
Y12(2μmol/L) 2.25μl
C34(2μmol/L) 2.25μl
Mg
2+(25mmol/L) 0.6μl
Taq(2.5U/μL) 0.7μl
Dna profiling 4.5 μ l
The composition of 10 * PCR buffer is as follows: 500mmol/L KCl, 100mmol/L Tris.Cl, 15mmol/L MgCl
2, 0.1% gelatinum; The composition of Taq enzyme storage damping fluid is as follows: 20mmol/L Tris.Cl, 1mmol/L DTT, 0.1mmol/L EDTA, 100mmol/L KCl, 50%glycerol, 0.5%NP-40.
Each ionic concn is in the PCR reaction system: 1.5mmol/L MgCl
2, 5mmol/L DTT, 50mmol/L K
+, 96mmol/L Tris.Cl.
PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 2S, 51 ℃ of 2S, 72 ℃ of 2S, 32 circulations; 72 ℃ of 3min.
Under 120V voltage, adopt 2% agarose electrophoresis 15min, the gel imaging system check and analysis, PCR result is as shown in Figure 1.Wherein, the 1-6 road is bull blood pcr amplification result, and the 9-14 road is cow blood pcr amplification result, 7,8,15,16 negative contrasts, and M is Marker I, band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp.The result shows that the band of 295bp and 208bp has all appearred in bull blood PCR, and cow blood PCR result then only has the band of 208bp.All there is not above-mentioned band in negative control.Illustrate that the inventive method is with a high credibility, do not have non-specific amplification.
Embodiment 2: the monosperm pcr amplification
1, agarose shop sheet separates single sperm
1.1 use sucrose solution (250mM sucrose, 5mM Tris. alkali) washing sperm
A. get an autoclaving 1.5ml centrifuge tube.
B. take out a frozen semen from liquid nitrogen, putting into 40 ℃ of water moments thaws.
C. cut off tubule tampon one end with scissors, will cut off the tampon mouth of pipe and aim at the centrifugal mouth of pipe of 1.5ml, cut off the other end with scissors, sperm flows in the 1.5ml centrifuge tube along the mouth of pipe, and the residue sperm is blown in the centrifuge tube with 10 μ l pipettors.
D. the undiluted sperm of 5 μ l is added in the 495 μ l deionized waters, fully inhale with pipettor and beat mixing.
E. get about 20 μ l mixing sperms and be paved with whole cell counting count board, counting sperm number under 10 times of mirrors of ordinary optical microscope.
F. with centrifugal 10 minutes of undiluted seminal fluid 15000g, discard supernatant liquid.
G. add the 1ml sucrose solution and fully inhale dozen mixing, centrifugal 10 minutes of 15000g (repeating 3 times).
H. discard supernatant liquid, add 1ml ddH
2O washs sperm, centrifugal 10 minutes of 15000g.
I. discard supernatant liquid, (adjusting sperm is 1 * 10 to add an amount of sucrose solution
7/ mL) fully mixing is beaten in suction, and-20 ℃ of preservations are standby.
1.2 separate single sperm
A. get the 0.1g low melting-point agarose and be dissolved in 20ml ddH
2O makes 0.5% low melting-point agarose solution with the microwave oven heating.
B. add during 37 ℃ of left and right sides of low melting-point agarose solution fully to inhale and beat mixing through washing and resuspended sperm 1 μ l (about 1000 sperms).
The low melting-point agarose that c. will be mixed with sperm is tiled in diameter 10cm culture dish.
D. put into 37 ℃ of baking ovens after room temperature is solidified, dried 2 hours.
E. be ready to put monosperm autoclaving 0.2ml PCR pipe, the transformation of the way of 1ml syringe dig the glue spoon.
F. will complete flat board and be placed on the inverted microscope Stage microscope, under 10 times or 20 times of mirrors, seek the monosperm position.
G. use self-control to dig the glue spoon and take single sperm and put into the PCR pipe ,-20 ℃ of preservations are standby.
2, monosperm PCR
2.1 monosperm cracking
A. take out the 0.2ml PCR pipe that is placed with monosperm, 3000g is instantaneous centrifugal.
B. in PCR pipe adding 1 μ l alkaline bleach liquor cleavage liquid (500mmol/L KOH, 125mmol/LDTT),
65 ℃ of cracking 20 minutes are as the template of monosperm pcr amplification.
Attention: (900mmol/L TrisHCl pH8.3) adds the total reaction system to the sperm neutralizer.
2.2 monosperm PCR
This experiment is to utilize monosperm PCR to carry out the purity evaluation to separating seminal fluid, and as positive control, the cracked monosperm is as template with the bull blood DNA.Reaction system sees Table 2, wherein 10 * PCR buffer (500mM KCl, 100mM TrisHCl, 15mM MgCl
2, 0.1% gelatin), adopt Eppendorf Mastercycler gradient PCR instrument (Germany) to carry out the PCR reaction.
PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 2S, 51 ℃ of 2S, 72 ℃ of 2S, 35 circulations; 72 ℃ of 3min.
2.3 agarose gel electrophoresis
A. sepharose preparation: accurately take by weighing the 0.6g agarose and dissolve among 0.8 * TBE of 30mL, put to heat on the electric furnace and also shake frequently to fusing fully, room temperature is chilled to 50～60 ℃, adds 4.5 μ lGoldViewna
TMII fluorescence dye (remolding sensitivity EB is high about 10 times) shakes up immediately, pours in the electrophoresis chamber mould, places more than the 20min under the room temperature, extracts comb.
B. the preparation of sample: putting 1 μ l sample-loading buffer and 10 μ l PCR products on the cured film paper, fully the mixing point sample uses DNA Marker I as molecular weight standard.
C. electrophoresis: gel is immersed in the electrophoretic buffer, behind the point sample, connect with the mains, 120V, electrophoresis 15min.
D. observations: monosperm PCR result as shown in Figure 2, wherein the 1-6 road is that (1,2 are the X sperm to the monosperm amplification, 3～6 is y sperm), the positive contrast in 7 roads, 8, the negative contrast in 9 roads, M is Marker I, and band from bottom to up is followed successively by 100bp, 200bp, 300bp, 400bp, 500bp and 600bp.
Table 2 PCR reaction system
| Composition | Consumption | Final concentration |
| 10Xbuffer(15mM Mg 2+) MgCl 2(25mM) dNTP (1mM) primer Y12 (2 μ M) primer C34 (2 μ M) TrisHCl (900mM) ddH 2O Taq enzyme template | 1.5μl 0.6μl 5μl 2.25μl 2.25μl 2.5μl 8.2μl 0.7μl 2μl | 1×buffer 1.5mM 0.2mM 180pM 180pM 96mM - - - |
According to the method described above, the monosperm that extracts in the sample is detected, calculate X, the shared ratio of Y monosperm, thereby judge the separation purity of this sample sperm.
Sequence table
＜110〉the Chinese Academy of Agricultural Sciences's animal and veterinary institute
＜120〉a kind of primer that detects ox X, y sperm separation purity
<130>
<160>4
<170>PatentIn version 3.3
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<211>21
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＜213〉artificial sequence
<400>1
ctactagaca tacaccgaga c 21
<210>2
<211>20
<212>DNA
＜213〉artificial sequence
<400>2
ccgtgctgcc aatgttacct 20
<210>3
<211>21
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＜213〉artificial sequence
<400>3
ttgctgctct tgcctttgct t 21
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
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gtccacctgc cacaactaaa t 21
Claims (8)
1. one kind is detected the primer that separates X, y sperm purity, and it comprises the nucleotide sequence shown in SEQ ID NO.1 and 2.
2. primer as claimed in claim 1, it also comprises interior label primer.
3. primer as claimed in claim 2, wherein said interior label primer has the nucleotide sequence shown in SEQ IDNO.3 and 4.
4. each described primer of claim 1～3 is detecting the application that separates in X, the y sperm purity.
5. one kind is detected the method for separating back X, y sperm purity, it adopts each described primer of claim 1～3 that the single sperm DNA of ox is carried out pcr amplification, and detect its amplified production, and detected result is carried out statistical study, judge and separate back X, y sperm purity.
6. method as claimed in claim 5 is characterized in that, when adopting the described primer of claim 3 to increase, the monosperm that has amplified production length and be 208bp is the X sperm, and the monosperm that has amplified production length and be 295bp and 208bp is a y sperm.
7. as claim 5 or 6 described methods, it is characterized in that the PCR reaction conditions is: 94 ℃ of 4min; 94 ℃ of 2S, 51 ℃ of 2S, 72 ℃ of 2S, 35 circulations; 72 ℃ of 3min.
8. the test kit that contains each described primer of claim 1～3.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810055708 CN101195842B (en) | 2008-01-07 | 2008-01-07 | Primer for detecting separation purity of X, Y spermatozoon of cattle |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810055708 CN101195842B (en) | 2008-01-07 | 2008-01-07 | Primer for detecting separation purity of X, Y spermatozoon of cattle |
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| Publication Number | Publication Date |
|---|---|
| CN101195842A true CN101195842A (en) | 2008-06-11 |
| CN101195842B CN101195842B (en) | 2010-07-28 |
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|---|---|---|---|
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935698A (en) * | 2010-05-19 | 2011-01-05 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting separation purity of bull sperms X and Y |
| CN101962680A (en) * | 2010-10-13 | 2011-02-02 | 南方医科大学 | Double PCR molecular diagnosis kit for detecting inactivation of X chromosome |
| CN106544411A (en) * | 2016-08-27 | 2017-03-29 | 华中农业大学 | A kind of method for identifying mammal y sperm typing using mark analysis of protein |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
| CN110669847A (en) * | 2019-11-07 | 2020-01-10 | 河北农业大学 | Detection method and kit for animal sexual control sperm purity and application |
-
2008
- 2008-01-07 CN CN 200810055708 patent/CN101195842B/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935698A (en) * | 2010-05-19 | 2011-01-05 | 中国农业科学院北京畜牧兽医研究所 | Method for detecting separation purity of bull sperms X and Y |
| CN101962680A (en) * | 2010-10-13 | 2011-02-02 | 南方医科大学 | Double PCR molecular diagnosis kit for detecting inactivation of X chromosome |
| CN106544411A (en) * | 2016-08-27 | 2017-03-29 | 华中农业大学 | A kind of method for identifying mammal y sperm typing using mark analysis of protein |
| CN108642001A (en) * | 2018-05-08 | 2018-10-12 | 中国农业科学院北京畜牧兽医研究所 | A method of it improving obstinacy control and freezes essence ability in vitro fertilization |
| CN110669847A (en) * | 2019-11-07 | 2020-01-10 | 河北农业大学 | Detection method and kit for animal sexual control sperm purity and application |
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