CN1664110A - PCR amplification primer, kit and use thereof in identification of mammal sex - Google Patents
PCR amplification primer, kit and use thereof in identification of mammal sex Download PDFInfo
- Publication number
- CN1664110A CN1664110A CN 200410014222 CN200410014222A CN1664110A CN 1664110 A CN1664110 A CN 1664110A CN 200410014222 CN200410014222 CN 200410014222 CN 200410014222 A CN200410014222 A CN 200410014222A CN 1664110 A CN1664110 A CN 1664110A
- Authority
- CN
- China
- Prior art keywords
- embryo
- reaction
- pcr
- primer
- sample
- 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.)
- Pending
Links
- 241000124008 Mammalia Species 0.000 title claims abstract description 13
- 238000012408 PCR amplification Methods 0.000 title claims description 15
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 46
- 238000007400 DNA extraction Methods 0.000 claims abstract description 4
- 239000000872 buffer Substances 0.000 claims abstract description 4
- 239000005662 Paraffin oil Substances 0.000 claims abstract description 3
- 108010006785 Taq Polymerase Proteins 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 21
- 108700010045 sry Genes Proteins 0.000 claims description 13
- 101150059736 SRY gene Proteins 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 11
- 238000001962 electrophoresis Methods 0.000 claims description 9
- 239000012160 loading buffer Substances 0.000 claims description 9
- 239000003550 marker Substances 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims description 8
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 108010067770 Endopeptidase K Proteins 0.000 claims description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 5
- 230000004087 circulation Effects 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000001954 sterilising effect Effects 0.000 claims description 5
- 238000004659 sterilization and disinfection Methods 0.000 claims description 5
- 238000013016 damping Methods 0.000 claims description 4
- 239000000284 extract Substances 0.000 claims description 4
- 239000012530 fluid Substances 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 238000012856 packing Methods 0.000 claims description 4
- 239000011780 sodium chloride Substances 0.000 claims description 4
- 238000011144 upstream manufacturing Methods 0.000 claims description 4
- 230000001186 cumulative effect Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 229930006000 Sucrose Natural products 0.000 claims description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 claims description 2
- 239000012188 paraffin wax Substances 0.000 claims description 2
- 238000012545 processing Methods 0.000 claims description 2
- 239000011535 reaction buffer Substances 0.000 claims description 2
- 239000005720 sucrose Substances 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 3
- 239000003795 chemical substances by application Substances 0.000 abstract 3
- 230000003416 augmentation Effects 0.000 abstract 2
- 230000003190 augmentative effect Effects 0.000 abstract 2
- -1 blank control Substances 0.000 abstract 1
- 239000012295 chemical reaction liquid Substances 0.000 abstract 1
- 239000003086 colorant Substances 0.000 abstract 1
- 239000000523 sample Substances 0.000 abstract 1
- 239000012723 sample buffer Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 34
- 241000283690 Bos taurus Species 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- 241001494479 Pecora Species 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- 239000000047 product Substances 0.000 description 7
- 241000283707 Capra Species 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 102100022978 Sex-determining region Y protein Human genes 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000013461 design Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000001556 precipitation Methods 0.000 description 5
- 238000004321 preservation Methods 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 4
- 239000008267 milk Substances 0.000 description 4
- 210000004080 milk Anatomy 0.000 description 4
- 235000013336 milk Nutrition 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 239000005549 deoxyribonucleoside Substances 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000001226 triphosphate Substances 0.000 description 3
- 235000011178 triphosphate Nutrition 0.000 description 3
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 244000309464 bull Species 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000013095 identification testing Methods 0.000 description 2
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 235000019687 Lamb Nutrition 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 102100029812 Protein S100-A12 Human genes 0.000 description 1
- 101710110949 Protein S100-A12 Proteins 0.000 description 1
- 108700032475 Sex-Determining Region Y Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 210000002593 Y chromosome Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Substances OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000005547 deoxyribonucleotide Substances 0.000 description 1
- 125000002637 deoxyribonucleotide group Chemical group 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000002308 embryonic cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 244000144992 flock Species 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 210000000472 morula Anatomy 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 210000000287 oocyte Anatomy 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- LCCNCVORNKJIRZ-UHFFFAOYSA-N parathion Chemical compound CCOP(=S)(OCC)OC1=CC=C([N+]([O-])=O)C=C1 LCCNCVORNKJIRZ-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000012372 quality testing Methods 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 229960004249 sodium acetate Drugs 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a PCR augmentation primer and agent box and the application in the sex identification of mammals, which is a pair of primer augmenting the 163bp area in gene Sry, sequence of basic group is: SRY5: 5'-TGAACGCCTTCATTGTGTGGTC-3',SRY3: 5'-GCTAGTAGTCTCTGTGCCTCCT-3'. The main components of the agent box are: buffer for DNA extraction, Taq DNA polymerase, PCR reaction liquid, blank control, paraffin oil and sample buffer. The applying steps of agent box in the sex identification of mammals are: (1) sample treatment, (2) augmentation, (3) product detecting. The invention is characterized by judging the existence of Y colorant layer specific fragment by detecting the augmenting product, and the avoidance of the unnecessary pollution, and the suitness for the quick(about 1 hour) sex identifying of mammal embryo in practice, the accuracy is 100%.
Description
Technical field
The invention belongs to detection method and test kit and the primer of a kind of PCR.
Background technology
Polymerase chain reaction (Polymerarse Chain Reaction) is called for short PCR, claim the amplification in vitro technology again, it is the external enzymatic DNA amplification new technology that human inheritance portion of U.S. Cetus company, University of California and Howghes medical colleges in 1985 etc. unite establishment, has high specificity, the susceptibility height, easy and simple to handle, rapidly and efficiently wait characteristics.After round pcr comes out, excite wide spread interest immediately and pay attention to, in worldwide, be widely used and develop very soon, enter the gene diagnosis of heredopathia, detection, medical jurisprudence, archeology and the molecular biological every field of infectious pathogen rapidly.
Round pcr is actually the enzymatic building-up reactions that depends on archaeal dna polymerase under the condition of template DNA, primer and 4 kinds of deoxyribonucleotide existence.The specificity of round pcr depends on primer and template DNA bonded specificity.Reaction divided for three steps: 1. sex change (denaturation): make the hydrogen bond rupture of dna double spiral, the two strands formation single stranded DNA that dissociates by heating; 2. annealing (annealling): when temperature suddenly and its complementary template form in the part and hybridize chain, and the complementary chance is less between the template DNA two strands; 3. extend (extension): at archaeal dna polymerase and 4 kinds of deoxyribonucleoside triphosphate substrates and Mg
2+Under the condition that exists, 5 ' → 3 ' polysaccharase catalysis is the DNA chain extension reaction of starting point with the primer.More than 3 the step be a circulation, each round-robin product can be used as next round-robin template, after a few hours, the DNA fragment specific between two primers has obtained massive duplication, quantity can reach 2 * 10
6 ~ 7Copy.
The discovery of sex-determining region Y's gene (SRY) has been introduced a new stage with sex control and the embryo gender authenticate technology of animal.Because SRY is male high special sex factor, be to judge the most reliable foundation of sex, identifying for embryo gender provides technological approaches accurately, combines with the pcr amplification technology, can be applied to early embryo sex and identify.Because the Sry gene is conservative at the Mammals camber, therefore when carrying out sex identification, can differentiate different animals with identical primer with the PCR method.The pcr amplification method is the process of dna replication dna in a kind of parody, it mainly is dna sequence dna design special primer at the Sry gene, and identify embryo's sex by pcr amplification embryo SRY specific DNA sequence, this method is because of there being reliable schedule of operation, have efficiently, characteristics fast, developed rapidly in recent years.
Have the researchist to set up the technology of milk cow and goat in vitro fertilization embryo's sex identification at present: the ovocyte of milk cow and goat through maturation in vitro, in vitro fertilization and ectogenesis to morula stage, under micrurgy, draw 4 ~ 6 embryonic cells, use the Nest-PCR technology and its DNA is carried out the mensuration of sry gene to carry out the embryo gender evaluation.There are the tube cattle embryo of foreign gene and test tube sheep embryo to carry out the sry gene detection to commentaries on classics.And will go in recipient cattle and the acceptor sheep through the embryo transfer of sex identification, the lamb that gives birth to conforms to fully with the result that sex of calf and embryo gender are identified.
Differentiate that accuracy is not high but aforesaid method exists, required time is longer, is used for animal embryo sex detection method and program and is difficult to carry out in production practice.
Summary of the invention
The PCR test kit that the purpose of this invention is to provide the evaluation of quick discriminating of a kind of cattle and sheep embryo gender and embryo cloning sex, and PCR primer are used round pcr and are carried out the discriminating of mammal embryo sex quickly and accurately.
Technology contents of the present invention is as follows:
1, the design of pcr amplification primer is with synthetic:
By sequence inquiry to animal Sry genes such as mouse, pig, sheep, goat, ox among the Genbank, with Clustal W program software to the discovery that compares of these several animal Sry gene orders: mammiferous Sry gene nucleic acid sequence has very high homology and conservative property, by the rule of design of primers, design the zone of 163bp on a pair of primer amplification Sry gene.Base sequence is as follows:
5 ' end upstream primer (SRY5): 5 '-TGAACGCCTTCATTGTGTGGTC-3 '
3 ' end upstream primer (SRY3): 5 '-GCTAGTAGTCTCTGTGCCTCCT-3 '
Above primer dilutes with sterilization distilled water 1.23-1.24mL, and ultimate density is 20 μ M ,-20 ℃ of preservations.
Table 1 and table 2 have been listed the primer sequence and the homology between several Mammalss thereof of amplification Sry gene.
Table 1, the used SRY primer of sex identification and with the homology of several Mammals Sry gene nucleic acid sequences
SRY5?TGAACGCCTTCATTGTGTGGTC
Pig TGAACGCTTTCATTGTGTGGTCTCGTGATCAAAGGAGAAAAGTGGCTCTAGAGAAC
Sheep .C....................A.G...AC....G..............T
Goat .C....................A.G...AC....G..............T
Ox .C....................A.G...AC....G..............T
Mouse TGAATG.A..T..G........C....G.G.G...CAC..GT....C.AGC....T
Rabbit TGAACG.A.....G........C.AAC....G..AC.CC.G........GC....T
Pig CCTCAAATGCAAAACTCAGAGATCAGCAAGTGGCTGGGATGCAAGTGGAAAATGCT
Continuous ..CA..C.......................CA........A.G.........G...
Mountain ..AA..T.......................CA........A.G.........G...
Ox ..CA................C.........CA........ATG.........G...
Mouse ..CAGC........TA.............CA...........G........GC..
Rabbit ..CA......G.........C.........CA.......CA.C.........G...
SRY3
Pig TACAGAAGCCGAAAAGCGCCCATTCTTCGAGGAGGCACAGAGGCTACAGGCGGT
Continuous ... T..T.................T..............A....TA..TA.
Mountain ... T..T.................T..............A....TA..TA.
Ox ... T..T.................T..............A....TA..CA.
Mouse ... G...........ATTG.......................A....G...TA.
Rabbit .T.......T.....AT.G.........C.......G..A..A..G.....CA.
Table 2, the homology of various animal Sry gene 151bp
Milk cow | Sheep | Goat | Pig | Rabbit | Mouse | |
Milk cow sheep goat pig hairy rat | ??- ?97.4 ?96.7 ?84.8 ?80.8 ?71.5 | ?- ?98.7 ?86.1 ?81.5 ?72.8 | ?- ?86.1 ?80.8 ?72.2 | ?- ?78.1 ?74.2 | ?- ?76.8 | - |
The PCR test kit that contains above-mentioned primer, its main moiety are (50 reaction):
1. DNA extraction damping fluid (DNA extract buffer) 1mL, its main component is embryo's treatment solution (10mmol/LTris-HCl (pH8.3), 50mmol/L KCl, 2mmol/LMgCl
2, 0.45%NP-40,0.45%Tween-20,0.1 μ g/ μ L Proteinase K);
2. Taq archaeal dna polymerase (Taq DNA polymerase) 20 μ L are total to 100IU;
3. PCR reaction solution (PCR reaction buffer) 1500 μ L include 2.533mmol/LMgCl
2, 0.337 μ mol/L primer, 0.168mmol/L dNTPs;
4. blank (negative contrast) 250 μ L;
5. paraffin oil (paraffin liquid) 1mL;
6. sample-loading buffer (loading buffer) 100 μ L include 0.2% tetrabromophenol sulfonphthalein, 50% sucrose solution.
The application of described test kit on the PCR mammalian sex is identified may further comprise the steps:
1. sample preparation,
Collect 1 ~ 2 cell of mammal embryo, earlier with 0.145mol/L NaCl towards Xian 2 times, add embryo's treatment solution (10mmol/LTris-HCl (pH8.3), 50mmol/L KCl, 2mmol/L MgCl again
2, 0.45%NP-40,0.45%Tween-20,0.1 μ g/ μ L Proteinase K), making cumulative volume is 20 μ L, with embryo and treatment solution thereof at 55 ℃ of water-bath effect 60min, then with this embryo's solute directly as the template of PCR reaction.
2. amplified reaction,
Get PCR reaction solution n * 29.6 μ L, Taq archaeal dna polymerase n * 0.4 μ L and mix in a centrifuge tube by waiting to expand sample number n (the n=embryo handles sample number+2), strike with finger and severally make its mixing down, with the reaction tubes mark good after, press every pipe 30 μ L packing.Blank 50 μ L are added in the reaction tubes, get each embryo again and handle in the corresponding reaction tubes of sample (20 μ L) adding (reaction system is 50 μ L), every pipe adds 20 μ L sterilization Valelinum Liquidum, increases on the of short duration centrifugal rearmounted amplification instrument.Reaction parameter: behind 95 ℃ of pre-sex change 5min, by 95 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min programs are carried out 35 circulations, and 9min is stretched in last 72 ℃ of courts of a feudal ruler, finish reaction in 4 ℃.
3. product detects,
Get pcr amplification product 10 μ L, add 2 μ L sample-loading buffers, fully point sample behind the mixing uses PCR Marker as molecular weight standard.Finish electrophoresis with containing the painted 2% agarose gel electrophoresis 1h (3-4V/cm) of EB back.Glue is placed observation under the ultraviolet lamp, and what special band occurs is male, otherwise is female.
DNTP (deoxyribonucleoside triphosphate) is a deoxyribonucleoside triphosphate.
The designed sex identification primer of the present invention can also be used for identifying the sex of Mammals body early embryos such as pig, rabbit.The suitableeest amplification condition of amplification cow genome group DNA is adopted in the PCR reaction.
The present invention uses primer SRY5, liver and the muscle cdna group DNA sample of SRY3 amplification ox, the genome DNA sample of sheep, the genome DNA sample of rabbit, the genome DNA sample of pig, the specific amplified band of the equal clear display of electrophoretic analysis result of amplified production, size is 163bp, the electrophoretic analysis result of the amplified production of sow sample and blank does not all have corresponding specific amplified band.
The a pair of sex identification primer of the present invention's design is mixed with PCR sex identification test kit by embryo's DNA cloning condition of optimizing.The key of the PCR sex identification test kit of being prepared is Mg
2+Concentration, Mg
2+The size of concentration is very big to the experimental result influence, Mg among the present invention
2+The concentration optimum concn is 1.5mol/L, is greater than or less than this concentration and all can not successfully carries out pcr amplification.And other composition and reaction parameter and Standard PC R reaction require basically identical.Test kit of the present invention adopts round pcr that the conservative nucleotide sequence in the male specificity sry gene is increased, by the detection of amplified production being judged the Y chromosome specific fragment exists, simultaneously can avoid unnecessary pollution, be applicable to the sex of quick (about 1 hour) discriminating mammal embryo in the production practice, rate of accuracy reached to 100%.
Description of drawings
Fig. 1 is a cow genome group DNA pcr amplification experiment spectrogram.
Among Fig. 1, (a) 1,2,3 bulls; 4 PCR Marker; 5,6,7 cows; 8 blanks
(b) 1 PCR Marker; 2,4,6,8 bulls; 3,5,7 cows; 9 blanks
(c) 1,2,3, the 4-bull; 5 PCR Marker; 6,7,8 cows; 9 blanks
Fig. 2 is sheep and rabbit genomic dna pcr amplification experiment spectrogram.
Among Fig. 2, the 1-ram; 2,3, the 4-buck; 5-PCR Marker; 6, the 7-ewe; The female rabbit of 8-; 9-
Blank
Fig. 3 is a pig genomic dna pcr amplification experiment spectrogram.
Among Fig. 3,1 PCR Marker; 2,4,6,8 boars; 3,5,7 sows; 9 blanks
Embodiment
Example 1
Utilize animal gene group DNA detection sex
The extraction purifying and the quality examination of the genomic dna of animal tissues
1,0.5mg fresh animal muscle tissue or liver organization are cut into pieces to be placed on fills 4.5mL DNA extraction damping fluid (10 mmol/LTris-HCl, 0.1mol/LEDTA, 20 μ g/mL RNA enzymes, in homogenate pipe 0.5%SDSpH8.0), the homogenate pipe places the beaker of ice cube, adopts the about 90sec of electric driven glass refiner middling speed homogenate.Homogenate is all poured in 2 5mL centrifuge tubes, and centrifuge tube is equipped with in the beaker of ice cube, with ultrasonic cell disruptor (power 400W, ultrasonic time 3sec, off time 4sec, work number of times 15 times) pulverize.EDTA is meant ethylenediamine tetraacetic acid (EDTA).
2, after the crash cells, centrifuge tube is put 37 ℃ of incubation 1 h in the constant temperature water bath vibrator fast.
3, in centrifuge tube, add 25 μ L Proteinase Ks, to final concentration be 100 μ g/mL, leniently enzyme is sneaked in the viscid solution with a glass rod.
4, the suspension with lysing cell places 50 ℃ of constant temperature water bath vibrators water-bath, 3 h.Solution is chilled to room temperature, adds the saturated phenol of equal-volume Tris-HCl (pH7.9 ± 0.2), put upside down the about 10min of centrifuge tube lentamente back and forth, mix two-phase carefully.Centrifugal 10,000r/min, 30min.
5, with heavy caliber (3mm) pipettor head upper water is moved in the centrifuge tube of a cleaning mutually.Add isopyknic phenol: chloroform: primary isoamyl alcohol (25: 24: 1), put upside down the about 10min of centrifuge tube lentamente back and forth, mix two-phase carefully.Centrifugal 10,000r/min, 20min.
6, with heavy caliber (3mm) pipettor head upper water is moved in the centrifuge tube of a cleaning mutually.Add isopyknic chloroform: primary isoamyl alcohol (24: 1), put upside down centrifuge tube 5min lentamente back and forth, to mix two-phase carefully.Centrifugal 10,000r/min, 10min.
7, with heavy caliber (3mm) pipettor head the upper strata water is all moved in the centrifuge tube of a cleaning.Add the sodium-acetate (3mol/L pH5.2) of 0.1 times of volume and the dehydrated alcohol of 2 times of volumes in room temperature, the centrifuge tube that rolls on the table makes its abundant mixing, and DNA forms flocks immediately.With glass rod picking precipitation or the centrifugal 5000r/min of room temperature, 5min collecting precipitation.And remove liquid with the pipettor head as far as possible.
8, with 70% ethanol suspension precipitation secondary, the centrifugal 5000r/min of room temperature, 5min collects DNA.Remove 70% ethanol as far as possible.(but do not make the precipitation complete drying, otherwise be difficult for dissolving.)
9, TE (10mmol/L Tris-HCl, 1mmol/L EDTA pH 8.0) dissolving DNA precipitation is rapped it, DNA is dissolved fully after ,-20 ℃ of preservations.
10, ultraviolet spectrophotometry detects purity and the content of DNA
In quartz colorimetric utensil, add 15 μ L DNA samples, add water to 3mL, measure the ultraviolet absorption value of DNA sample behind the mixing respectively, and calculate A at 260nm and 280nm place
260/ A
280Value.A
260/ A
280Should be between 1.75-1.8, be lower than 1.75 and show and retain remarkable protein in the prepared product, should add SDS this moment and be 0.5% and repeat the above-mentioned 2-10 step to final concentration; Show in the DNA sample that greater than 1.8 RNA is arranged, should add the RNA enzyme this moment, and repeat the above-mentioned 2-10 step.Calculate DNA concentration: 10 * A
260(μ g/ μ L)
11, agarose gel electrophoresis detects DNA purity.
1. the preparation of 2% gel
Accurately take by weighing the 0.18g agarose and dissolve in 1 * TAE (0.04mol/L Tris-HCl of 18mL, 0.02mol/LNaAC, 2 mmol/LEDTA pH8.0) in, put to heat on the electric furnace and also shake frequently to fusing fully, room temperature is chilled to 50-60 ℃, and the 10mg/mL EB that adds 1 μ L shakes up immediately, pours into rapidly and seals in the electrophoresis chamber of mouth with the adhesive waterproof tape of the bacterium of going out, place more than the 30min under the room temperature, extract comb.
2. the preparation of sample
Putting 2 μ L sample-loading buffers and 10 μ L DNA samples on the cured film paper, abundant mixing, and with PCRMarker as molecular weight standard.
3. electrophoresis
Gel is immersed in the electrophoretic buffer, behind the point sample, connect with the mains, 3 V/cm are more than the electrophoresis 1h.
4. observations
Use the hand-held ultraviolet lamp observations, and the preservation of taking pictures.
Example 2
The extraction purifying and the quality examination of the genomic dna of animal serum
1, collects about 6mL fresh blood, add in the centrifuge tube that the acid acid citrate dextrose solution B of 1mL (glucose 1.47g adds water to 100mL for ACD solution-citric acid 0.48g, Trisodium Citrate 1.32g) is housed.Blood can be in 0 ℃ of preservation a couple of days or in-70 ℃ of prolonged preservation before preparation DNA.
A, the centrifugal 4500r/min of fresh blood (2mL), 15min abandons upper plasma, carefully yellowish layer moved in the new centrifuge tube with suction pipe, and recentrifuge 4500r/min, 15min.After centrifugal yellowish layer is resuspended in 1.5mLDNA and extracts in the damping fluid, in 37 ℃ of incubation 1h, later step is with the 3-11 step of example 1.
B, refrigeration blood (2mL), room temperature is melted, and moves in the new centrifuge tube, with equal-volume phosphate buffered saline(PBS) PBS (8g NaCl, 0.2g KCl, 1.44g Na
2HPO
4, 0.24g KH
2PO
4, add water to 1 L adjust pH 7.4) and dilution, subsequently in the centrifugal 7000r/min of room temperature, 15min.And supernatant discarded, with the resuspended throw out of 1.5mLDNA extraction buffer, in 37 ℃ of incubation 1 h, later step is with the 3-11 step of example 1.
2, amplified reaction
Get PCR reaction solution n * 29.6 μ L, Taq archaeal dna polymerase n * 0.4 μ L and mix in a centrifuge tube by waiting to expand sample number n (the n=embryo handles sample number+2), strike with finger and severally make its mixing down, with the reaction tubes mark good after, press every pipe 30 μ L packing.Blank 50 μ are added in the reaction tubes, get each embryo again and handle in the corresponding reaction tubes of sample (20 μ L) adding (reaction system is 50 μ L), every pipe adds 20 μ L sterilization Valelinum Liquidum, increases on the of short duration centrifugal rearmounted amplification instrument.Reaction parameter: behind 95 ℃ of pre-sex change 5min, by 95 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min programs are carried out 35 circulations, and 9min is stretched in last 72 ℃ of courts of a feudal ruler, finish reaction in 4 ℃.
3, product detects
Get pcr amplification product 10 μ L, add 2 μ L sample-loading buffers, fully point sample behind the mixing uses PCR Marker as molecular weight standard.Finish electrophoresis with containing EB painted 2% agarose gel electrophoresis 1 h (3-4V/cm) back.
Glue is placed observation under the ultraviolet lamp, and what special band occurs is male, otherwise is female.
Example 3
Identify the method for sex with the embryo
1, embryo's evaluation and mensuration size
The embryo who examines under stereomicroscope puts in the sterilisable chamber for 3 times with clean Xian of PBS, leaves standstill 0.5h, in nutrient solution, evaluates its developmental stage, embryo age and rank.
2, embryo's processing
Collect 1 ~ 2 cell of mammal embryo, earlier with 0.145mol/L NaCl towards Xian 2 times, add embryo's treatment solution (10mmol/LTris-HCl (pH8.3), 50mmol/L KCl, 2mmol/L MgCl again
2, 0.45%NP-40,0.45%Tween-20,0.1 μ g/ μ L Proteinase K), making cumulative volume is 20 μ L, with embryo and treatment solution thereof at 55 ℃ of water-bath effect 60min, then with this embryo's solute directly as the template of PCR reaction.
3, amplified reaction
Get PCR reaction solution n * 29. μ L, Taq archaeal dna polymerase n * 0.4 μ L and mix in a centrifuge tube by waiting to expand sample number n (the n=embryo handles sample number+2), strike with finger and severally make its mixing down, with the reaction tubes mark good after, press every pipe 30 μ L packing.Blank 50 μ L are added in the reaction tubes, get each embryo again and handle in the corresponding reaction tubes of sample (20 μ L) adding (reaction system is 50 μ L), every pipe adds 20 μ L sterilization Valelinum Liquidum, increases on the of short duration centrifugal rearmounted amplification instrument.Reaction parameter: behind 95 ℃ of pre-sex change 5min, by 95 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min programs are carried out 35 circulations, and 9min is stretched in last 72 ℃ of courts of a feudal ruler, finish reaction in 4 ℃.
4, quality testing is surveyed
Get pcr amplification product 10 μ L, add 2 μ L sample-loading buffers, fully point sample behind the mixing uses PCR Marker as molecular weight standard.Finish electrophoresis with containing EB painted 2% agarose gel electrophoresis 1 h (3-4V/cm) back.Glue is placed observation under the ultraviolet lamp, and what special band occurs is male, otherwise is female.
Example 4
Can carry out the sex identification of pig, dog, other animal of sheep with aforesaid method, method is with example 1 or example 2 or example 3.
Claims (3)
1, a kind of pcr amplification primer is the zone of 163bp on a pair of primer amplification Sry gene, and base sequence is as follows:
5 ' end upstream primer (SRY5): 5 '-TGAACGCCTTCATTGTGTGGTC-3 '
3 ' end upstream primer (SRY3): 5 '-GCTAGTAGTCTCTGTGCCTCCT-3 '.
2, the test kit that contains claim 1 primer, its main moiety are (50 reaction):
1. DNA extraction damping fluid (DNA extract buffer) 1mL, its main component is embryo's treatment solution, the composition of embryo's treatment solution is: 10mmol/LTris-HCl pH8.3,50mmol/L KCl, 2mmol/LMgCl
2, 0.45%NP-40,0.45%Tween-20,0.1 μ g/ μ L Proteinase K;
2. Taq archaeal dna polymerase (Taq DNA polymerase) 20 μ L are total to 100IU;
3. PCR reaction solution (PCR reaction buffer) 1500 μ L include 2.533mmol/LMgCl
2, 0.337 μ mol/L primer, 0.168mmol/L dNTPs;
4. blank (negative contrast) 250 μ L;
5. paraffin oil (paraffin liquid) 1mL;
6. sample-loading buffer (loading buffer) 100 μ L include 0.2% tetrabromophenol sulfonphthalein, 50% sucrose solution.
3, the application of test kit on the PCR mammalian sex is identified as claimed in claim may further comprise the steps:
1. sample preparation,
Collect 1 ~ 2 cell of mammal embryo, earlier with 0.145mol/L NaCl towards Xian 2 times, add embryo's treatment solution again: 10mmol/LTris-HCl pH8.3,50mmol/L KCl, 2mmol/L MgCl
2, 0.45%NP-40,0.45%Tween-20,0.1 μ g/ μ L Proteinase K, making cumulative volume is 20 μ L, with embryo and treatment solution thereof at 55 ℃ of water-bath effect 60min, then with this embryo's solute directly as the template of PCR reaction,
2. amplified reaction,
By waiting to expand sample number n, the n=embryo handles sample number+2, gets PCR reaction solution n * 29.6 μ L, Taq archaeal dna polymerase n * 0.4 μ L and mixes in a centrifuge tube, strike with finger and severally down make its mixing, with the reaction tubes mark good after, press every pipe 30 μ L packing.Blank 50 μ L are added in the reaction tubes, get each embryo's processing sample 20 μ L again and add in the corresponding reaction tubes, reaction system is 50 μ L, and every pipe adds 20 μ L sterilization Valelinum Liquidum, increases on the of short duration centrifugal rearmounted amplification instrument; Reaction parameter: behind 95 ℃ of pre-sex change 5min, by 95 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min programs are carried out 35 circulations, and 9min is stretched in last 72 ℃ of courts of a feudal ruler, finish reaction in 4 ℃;
3. product detects,
Get pcr amplification product 10 μ L, add 2 μ L sample-loading buffers, fully point sample behind the mixing uses PCR Marker as molecular weight standard; With containing the painted 2% agarose gel electrophoresis 1h of EB, finish electrophoresis behind the 3-4V/cm, glue is placed under the ultraviolet lamp observe, what special band occurs is male, otherwise is female.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410014222 CN1664110A (en) | 2004-03-03 | 2004-03-03 | PCR amplification primer, kit and use thereof in identification of mammal sex |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410014222 CN1664110A (en) | 2004-03-03 | 2004-03-03 | PCR amplification primer, kit and use thereof in identification of mammal sex |
Publications (1)
Publication Number | Publication Date |
---|---|
CN1664110A true CN1664110A (en) | 2005-09-07 |
Family
ID=35035420
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200410014222 Pending CN1664110A (en) | 2004-03-03 | 2004-03-03 | PCR amplification primer, kit and use thereof in identification of mammal sex |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1664110A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101701257B (en) * | 2009-11-13 | 2011-12-21 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
CN101701256B (en) * | 2009-11-13 | 2011-12-21 | 中国检验检疫科学研究院 | Primer and method for PCR identification of maize |
CN112094840A (en) * | 2019-06-17 | 2020-12-18 | 南京尧顺禹生物科技有限公司 | Rapid preparation method of trace animal sample genome DNA template for genome segment amplification |
CN115851967A (en) * | 2022-09-09 | 2023-03-28 | 海南省农业科学院畜牧兽医研究所 | Substance for detecting SRY gene and PCR detection method for sex identification of pig, goat and cattle |
-
2004
- 2004-03-03 CN CN 200410014222 patent/CN1664110A/en active Pending
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101701257B (en) * | 2009-11-13 | 2011-12-21 | 中国检验检疫科学研究院 | PCR authenticating primer and method of oryza punctata |
CN101701256B (en) * | 2009-11-13 | 2011-12-21 | 中国检验检疫科学研究院 | Primer and method for PCR identification of maize |
CN112094840A (en) * | 2019-06-17 | 2020-12-18 | 南京尧顺禹生物科技有限公司 | Rapid preparation method of trace animal sample genome DNA template for genome segment amplification |
CN115851967A (en) * | 2022-09-09 | 2023-03-28 | 海南省农业科学院畜牧兽医研究所 | Substance for detecting SRY gene and PCR detection method for sex identification of pig, goat and cattle |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN103045727B (en) | SNP (Single Nucleotide Polymorphism) marker related with Chinese Holstein cow milk production property and somatic cell score and application thereof | |
CN106636072B (en) | General DNA extraction method and kit for animal traditional Chinese medicine molecular identification | |
CN102676514A (en) | Single nucleotide polymorphism (SNP) mark relevant with milk production traits of Chinese Holstein dairy cattle and application thereof | |
CN107385079B (en) | Method for detecting components of cattle, sheep, chicken, duck and pork by PCR (polymerase chain reaction) and RPA (reverse transcription amplification) amplification modes, primer group and kit | |
WO2014079350A1 (en) | Cho cell dna detection method | |
WO2012155577A1 (en) | Method for separating and purifying rna from biomaterial | |
CN104498599A (en) | Microsporidium molecule universal detection primers and kit thereof | |
CN109837345B (en) | Primer and method for detecting residual DNA of mouse cells | |
CN1721552A (en) | Fluorescent quantitative PT-PCR detection kit for detecting BCR-ABL fusion gene (P210bcr/abl)mRNA | |
CN101063169A (en) | PCR-RFLP method for checking goat luteotropin gene mononucleotide polymorphism | |
CN1664110A (en) | PCR amplification primer, kit and use thereof in identification of mammal sex | |
CN1693479A (en) | Kit with fluorescent quantitative RT-PCR detection technique used for cell keratin 19(Ck19)mRNA | |
CN1506468A (en) | PCR test kit for hygrophilous aeromonad and its test method | |
CN1824801A (en) | Real-time fluorescent PCR testing primer, probe and immobilized kit for citrus ulcer bacteria and testing process thereof | |
CN101029336A (en) | Reagent kit for predicting serotonin re-uptake inhibitor medicine effect | |
CN1900314A (en) | Fluorescence labelling ABO gene typing method and its reagent kit | |
CN1814785A (en) | Method for detecting Brucellosis and primer thereof | |
CN1196755A (en) | Methods for determining coat colour genotype of pig | |
CN1687458A (en) | FQ-PCR diagnosis kit of WSSV and test method | |
CN100344758C (en) | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application | |
JPWO2019240073A1 (en) | Quantitative PCR probe | |
CN104498509B (en) | HMG1 gene and application of HMG1 gene in silkworm microsporidia molecular detection | |
CN109355379B (en) | Kit for detecting autosomal dominant hereditary deafness family-induced deafness gene mutation | |
CN107130037B (en) | Method and special kit for auxiliary detection of cattle growth and carcass traits through TNNI1 gene | |
CN202671540U (en) | SNP (single nucleotide polymorphism) typing reagent kit for BMP (bone morphogenetic protein) 15 genes related to egg laying character of chicken |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20050907 |