CN102181521A - Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken - Google Patents
Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken Download PDFInfo
- Publication number
- CN102181521A CN102181521A CN2011100531401A CN201110053140A CN102181521A CN 102181521 A CN102181521 A CN 102181521A CN 2011100531401 A CN2011100531401 A CN 2011100531401A CN 201110053140 A CN201110053140 A CN 201110053140A CN 102181521 A CN102181521 A CN 102181521A
- Authority
- CN
- China
- Prior art keywords
- chicken
- sex
- early embryo
- pcr
- primer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a polymerase chain reaction (PCR) amplification primer used for identifying the sex of the early embryo of chicken and the nucleotide sequences of the amplification primer are shown in SEQ ID NO.1-4. The invention also discloses a method for performing PCR identification to the early embryo of chicken by using the primer. The method is as follows: the blastodermal cells of chicken are used as a template, the primer provided by the invention is utilized to amplify the target gene through the PCR technology, then the PCR amplification product is detected through agarose gel electrophoresis, the sex is judged according to the banding pattern, the female has two bands, and the male has one band. By adopting the method, the extraction step of blastodermal cell genomic DNA in the existing sex identification technology of the early embryo of chicken can be not adopted, the operation step of the sex identification can be simplified, the operation time can be shortened and the sex of the early embryo of chicken can be identified fast and effectively.
Description
Technical field
The present invention relates to bird early embryo sex authenticate technology field, be specifically related to be used to identify the pcr amplification primer and the method for chicken early embryo sex.
Background technology
Embryo Gallus domesticus is the ideal model of developmental biology research.The ovum of chicken is discharged the back from ovary and is admitted by infundibulopelvic of faliopian tube, after this with the sperm fertilization of meeting.The ovum gallinaceum after fertilization moves down in the process zygote through division in uterine tube, form transparent, the dark blastodisc on every side of central authorities.Hen is when laying eggs, and zygote was grown to embryo's the blastaea stage, contains 4～60,000 undifferentiated cells, and the blastodisc diameter is 4～5mm, the about 250 μ m of thickness.Blastoderm cells can externally operate, genetic modification and selection, with foreign gene transfection blastoderm cells, can obtain to contain the filial generation of foreign gene by blastodisc injection preparation mosaic.But, when the chimeric model of preparation, there are Gender issues for acceptor, at present the blastoderm cells mosaic still is that the cell that shifted of archeocyte mosaic all is unknown sex or mixes sex, the discordance that this just inevitably causes for the acceptor sex opposite sex occurs and transplants the preparation mosaic.Therefore, prepare same sex mosaic, study the biological mechanism or the chicken embryo sexual gland expression of gene of sex gonad development, need identify the sex of Embryo Gallus domesticus in earlier stage for purpose can be arranged.
Since after finding the site sequence of a class multiple nucleic acid restriction endonuclease Xho I and EcoR I on the female special W karyomit(e) of bird, identify the marked improvement that obtained of Embryo Gallus domesticus sex by molecular biology method.The tumor-necrosis factor glycoproteins of EcoR I family and Xho I family accounts for 70%～90% of the total DNA of whole W karyomit(e), and these big repeated fragments are made of the 21bp subunit of many placed in-line structural similitudies again.These tumor-necrosis factor glycoproteinss have constituted the chromosomal heterochromatin of W zone, are to identify the chromosomal basis of W.People such as Clinton (2001) have set up a kind of method of evaluation of Embryo Gallus domesticus sex.Design two pairs of primers 256bp fragment between the 1267th～1522nt in the Xho I site tumor-necrosis factor glycoproteins of one section 415bp on the W karyomit(e) and the 18S ribosomal gene that increases respectively; Control reaction and specific reaction are once finished in same pipe.But this method still needs by extracting genomic dna, and its step is loaded down with trivial details relatively, time-consuming.
Summary of the invention
The object of the present invention is to provide the pcr amplification primer and the method that are used to identify the chicken early embryo sex.
For achieving the above object, the invention provides the pcr amplification primer that is used to identify the chicken early embryo sex, comprise primer P1～P4 (seeing nucleotides sequence tabulation SEQ ID NO.1～4), be specially:
P1：5′-cat?gcc?ttt?cta?ccg?caa?ata?c-3′(SEQ?ID?NO.1)，
P2：5′-tta?agg?tgc?ttt?ttt?tct?ggg-3′(SEQ?ID?NO.2)；
P3：5′-tgc?agc?tct?ttc?tcg?att?ccg?tg-3′(SEQ?ID?NO.3)，
P4：5′-atg?ggg?tag?aca?caa?gct?gag?cc-3′(SEQ?ID?NO.4)；
Wherein, upstream primer P1 and the downstream primer P2 447bp fragment of highly repetitive sequence EcoR I family on the hen W karyomit(e) that is used to increase; Upstream primer P3 and the downstream primer P4 256bp fragment between cock and common 18S ribosomal gene the 1267th～1522nt that exists of hen that is used for increasing.
The present invention also provides a kind of method of identifying the chicken early embryo sex, is template with the chicken blastoderm cell, carries out pcr amplification with above-mentioned primer, and agarose gel electrophoresis detects pcr amplification product, judges sex according to banding pattern, and female is two bands, and male is a band.
Further, the reaction system of described pcr amplification is:
Contain in the reaction system of per 25 μ l:
Described PCR reaction conditions is:
35 circulations, last 72 ℃ are extended 10min.
Further, described chicken blastoderm cell extracts from the embryo area pellucida of not hatching fertilized eggs.
Further, the instrument of described extraction chicken blastoderm cell is the glass needle at diameter 50 μ m, 30 ° of oblique angles.
Wherein, the preparation method of glass needle is as follows: set and to draw pin instrument (Narishige PN30, Japan) parameter, load onto the thin-walled glass capillary (1.0 * 100mm), fix with fixed knob, by drop-down pin instrument switch, be drawn into glass needle; Take off glass needle and be contained in and forge the holding on the punch block of pin instrument (Narishige MF-900, Japan), entry needle is moved to the glass sphere side, make its internal diameter 50 μ m places near glass sphere, point is pressed heater switch, with its severing.(Narishige EG-400, Japan) wears into 30 ° of oblique angles with the tip with grinder; In dehydrated alcohol, clean 5 times.Place super clean bench to dry, stand-by.
The present invention also provides the chicken early embryo sex that contains described primer identification kit, and described test kit also can comprise Taq archaeal dna polymerase, dNTPs, PCR damping fluid and other component that is used for pcr amplification well known in the art.
The inventive method has following advantage:
The present invention directly extracts blastoderm cells from chicken embryo area pellucida, be used for the 447bp fragment of EcoR I family on the W karyomit(e) that female specificity identifies and be used for 256bp fragment between 18S ribosomal gene the 1267th～1522nt of internal reference by PCP technology amplification, agarose gel electrophoresis detects pcr amplification product then, judge sex according to banding pattern, female is two bands, and male is a band.The inventive method has been saved blastoderm cells extracting genome DNA step of the prior art, has simplified the operation steps of early stage chicken embryo sex identification, has shortened the operating time, can identify the sex of chicken body early embryo quickly and efficiently.
Description of drawings
Fig. 1 is that chicken early embryo sex of the present invention is identified agarose gel electrophoretogram.
Embodiment
Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
12 unhatched white Leghorn kind eggs of picking will be planted egg respectively and be walked crosswise placement 6 hours, utilize kind of an egg windowing technology to window at the center line of eggshell, note trying not to destroy the eggshell inner membrance, and diameter is 8～10mm.With the blastoderm cells in microinjection pin extraction 0.1～0.3 μ l chicken embryo area pellucida, blastoderm cells is inserted in the 25 μ lPCR reaction solutions.
Two pairs of used primers of PCR reaction are respectively: the segmental upstream primer P1:5 ' of the 447bp-cat gcc ttt cta ccg caa ata c-3 ' and the downstream primer P2:5 '-tta agg tgc ttt ttt tct ggg-3 ' of amplification hen W karyomit(e) highly repetitive sequence EcoR I family; The segmental upstream primer P3:5 ' of 256bp-tgc agc tct ttc tcg att ccg tg-3 ' and downstream primer P4:5 '-atg ggg tag aca caa gct gag cc-3 ' between the 1267-1522nt in the common 18S ribosomal gene that exists of amplification cock and hen.Above-mentioned primer is synthetic by invitrogen company.The used instrument of PCR reaction is the PTC-200PCR of a MJ company amplification instrument.
The PCR reaction system is:
The PCR reaction conditions is:
35 circulations, last 72 ℃ are extended 10min.
The 447bp fragment of highly repetitive sequence EcoR I family on the amplification W karyomit(e), the 256bp fragment in the 18S ribosomal gene that increases simultaneously between the 1267th～1522nt is as the endogenous reference.Product after the amplification is that 1.2% sepharose carries out electrophoresis in concentration, after electrophoresis finishes, gel is detected, scans in monarch anticipates the east gel imaging system, and preserve image.
The result as shown in Figure 1,6 pieces is female Embryo Gallus domesticus, 2 bands occur; 6 pieces is male Embryo Gallus domesticus, 1 band occurs.Wherein, swimming lane 2,3,5,7,9 and 11 is female chicken embryo, swimming lane 4,6,8,10,12 and 13 male chicken embryos.Swimming lane 14 occurs 2 bands for water contrast, swimming lane 15 for the amplification of the blood sample DNA of the hen that grows up; Swimming lane 16 is the amplification of the blood sample DNA of ripe cock, 1 band occurs; Swimming lane 1 and 17 is Mark, is followed successively by from bottom to up: 100bp, 300bp, 500bp, 700bp, 900bp, 1200bp.As can be seen from Figure 1, the result of Embryo Gallus domesticus sex identification is clear, can reach the sex of Rapid identification chicken body early embryo.
In a further embodiment, extract the blastoderm cells (ddH wherein in 0.1 μ l or 0.3 μ l chicken embryo area pellucida
2It is 25 μ l that O complements to reaction system) result that detects behind the pcr amplification is too.
Though the present invention is described in detail above to have used general explanation and specific embodiment, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. be used to identify the pcr amplification primer of chicken early embryo sex, it is characterized in that, comprising:
P1：5′-cat?gcc?ttt?cta?ccg?caa?ara?c-3′，
P2：5′-tta?agg?tgc?ttt?ttt?tct?ggg-3′；
P3：5′-tgc?agc?tct?ttc?tcg?att?ccg?tg-3′，
P4：5′-atg?ggg?tag?aca?caa?gct?gag?cc-3′。
2. a method of identifying the chicken early embryo sex is characterized in that, is template with the chicken blastoderm cell, carry out pcr amplification with the described primer of claim 1, agarose gel electrophoresis detects pcr amplification product, judges sex according to banding pattern, female is two bands, and male is a band.
3. method according to claim 2 is characterized in that, the reaction system of described pcr amplification is:
Contain in the reaction system of per 25 μ l:
Described PCR reaction conditions is:
35 circulations, last 72 ℃ are extended 10min.
4. according to claim 2 or 3 described methods, it is characterized in that described chicken blastoderm cell extracts from the embryo area pellucida of not hatching fertilized eggs.
5. method according to claim 4 is characterized in that, the instrument of described extraction chicken blastoderm cell is the glass needle at diameter 50 μ m, 30 ° of oblique angles.
6. the chicken early embryo sex identification kit that comprises the described primer of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110053140 CN102181521B (en) | 2011-03-04 | 2011-03-04 | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110053140 CN102181521B (en) | 2011-03-04 | 2011-03-04 | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102181521A true CN102181521A (en) | 2011-09-14 |
| CN102181521B CN102181521B (en) | 2013-01-16 |
Family
ID=44567807
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110053140 Expired - Fee Related CN102181521B (en) | 2011-03-04 | 2011-03-04 | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102181521B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106943203A (en) * | 2017-03-17 | 2017-07-14 | 宁夏医科大学 | A kind of injection needle preparation method injected for ductuli efferentes testis |
| CN112746070A (en) * | 2021-01-28 | 2021-05-04 | 江苏省家禽科学研究所 | Primer pair and kit for early sex identification of chicken and application of primer pair and kit |
| CN114045347A (en) * | 2021-11-09 | 2022-02-15 | 江苏省家禽科学研究所 | PCR primer, kit and method for sex identification of special poultry |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3599465A1 (en) * | 2018-07-24 | 2020-01-29 | Tronico | Method for determining a specific characteristic of an embryo in an unhatched egg |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004016812A1 (en) * | 2002-08-14 | 2004-02-26 | Roslin Institute (Edinburgh) | Avian sex determination method |
| CN101130816A (en) * | 2007-07-27 | 2008-02-27 | 华中农业大学 | Method for appraising gender of chicken blastoderm by using multiple PCR |
-
2011
- 2011-03-04 CN CN 201110053140 patent/CN102181521B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004016812A1 (en) * | 2002-08-14 | 2004-02-26 | Roslin Institute (Edinburgh) | Avian sex determination method |
| CN101130816A (en) * | 2007-07-27 | 2008-02-27 | 华中农业大学 | Method for appraising gender of chicken blastoderm by using multiple PCR |
Non-Patent Citations (5)
| Title |
|---|
| 《British Poultry Science》 20011231 M. Clinton et al. Sexing chick embryos: A rapid and simple protocol 134-138 1-6 第42卷, 第1期 * |
| 《Chromosome Research》 19980630 Irina Solovei et al. Speci(R)c chromomeres on the chicken W lampbrush chromosome contain speci(R)c repetitive DNA sequence families 323-327 1-6 第6卷, 第4期 * |
| IRINA SOLOVEI ET AL.: "Speci®c chromomeres on the chicken W lampbrush chromosome contain speci®c repetitive DNA sequence families", 《CHROMOSOME RESEARCH》 * |
| IRINA SOLOVEI ET AL.: "Speci®c chromomeres on the chicken W lampbrush chromosome contain speci®c repetitive DNA sequence families", 《CHROMOSOME RESEARCH》, vol. 6, no. 4, 30 June 1998 (1998-06-30), pages 323 - 327 * |
| M. CLINTON ET AL.: "Sexing chick embryos: A rapid and simple protocol", 《BRITISH POULTRY SCIENCE》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106943203A (en) * | 2017-03-17 | 2017-07-14 | 宁夏医科大学 | A kind of injection needle preparation method injected for ductuli efferentes testis |
| CN112746070A (en) * | 2021-01-28 | 2021-05-04 | 江苏省家禽科学研究所 | Primer pair and kit for early sex identification of chicken and application of primer pair and kit |
| CN114045347A (en) * | 2021-11-09 | 2022-02-15 | 江苏省家禽科学研究所 | PCR primer, kit and method for sex identification of special poultry |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102181521B (en) | 2013-01-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108841945B (en) | PCR amplification primer, method and kit for rapidly identifying genetic sex of Chinese softshell turtles | |
| RU2013121000A (en) | METHOD FOR IDENTIFICATION OF NATIVE PAIRS OF DNA OR RNA FRAGMENTS PRESENT IN ONE AND SAME LIVING CELLS | |
| CN102181521B (en) | Polymerase chain reaction (PCR) amplification primer and method used for identifying sex of early embryo of chicken | |
| Gòdia et al. | A systems biology framework integrating GWAS and RNA-seq to shed light on the molecular basis of sperm quality in swine | |
| CN104099330A (en) | Sheep lambing number trait related molecular marker and application thereof | |
| CN109355398B (en) | SNP (Single nucleotide polymorphism) marker primer related to number of live piglets born by Erhualian pig and application of SNP marker primer | |
| CN104498599A (en) | Microsporidium molecule universal detection primers and kit thereof | |
| CN111763723B (en) | Nondestructive sex identification method for Chinese sturgeons | |
| CN110438160A (en) | A kind of construction method of Cd2ap Gene Knock-Out Animal Model and application | |
| Francescon et al. | Assessment of homozygosity and fertility in meiotic gynogens of the European sea bass (Dicentrarchus labrax L.) | |
| CN102758016B (en) | Kit for simply, conveniently and rapidly detecting sheep multifetal gene BMPR-IB (bone morphogenetic protein receptor IB) | |
| Abdel et al. | Polymorphism in BPM-15 gene and its association with litter size in Anglo-Nubian goat | |
| CN103898231A (en) | SNP (Single Nucleotide Polymorphism) molecular marker related to pork pH characteristics and application thereof | |
| CN104651523A (en) | Applications of single-stranded nucleotide sequence of INHA gene as animal superovulation molecule marker, and detection method | |
| WO2012156563A1 (en) | Method for identifying the sex of fish | |
| CN102134600B (en) | PCR (Polymerase Chain Reaction) method for sex appraisal of Nipponia nippon | |
| Ichikawa et al. | DNA types of aspermic Fasciola species in Japan | |
| CN105779594A (en) | Pig Oct4 gene insertion/lack detection method and application thereof | |
| CN101864486B (en) | Primer for detecting egg laying performance of goose and method and application thereof | |
| Kim et al. | A novel approach for determination of chicken sexing at an early stage of development by using loop-mediated isothermal amplification method | |
| CN104195136A (en) | Method or identifying length and/or height of pig body and special primer pair therefor | |
| McKay et al. | 115 Profiling conservation of DNA methylation in cattle. | |
| CN101956009A (en) | Method for detecting number of littermate and characters of sow | |
| CN109897890B (en) | Nondestructive babuya penguin sex identification method | |
| Rao et al. | Biopsy of Indian Zebu and crossbred cattle preimplantation embryos and sexing by polymerase chain reaction |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130116 Termination date: 20140304 |