CN101705299A - Microsatellite loci marker combination and application thereof - Google Patents
Microsatellite loci marker combination and application thereof Download PDFInfo
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Abstract
The invention discloses a microsatellite loci marker combination which comprises 17 microsatellite loci markers, and simultaneously provides an efficient detection method based on the 17 microsatellite loci markers; the markers have high polymorphism, are not linked with each other and have proper fragments interval, and are easy to be detected simultaneously. The invention is improved in a detection technology, optimizes an experimental system by utilizing a multi-marker amplification technology and a fluorescent semi-automatic microsatellite typing method, thus establishing high flux, accurate and effective 17 cattle microsatellite marker compound amplification and a gene typing method.
Description
Technical field
The dna marker that the present invention relates in the cow genome group detects, and relates in particular to the specificity combination and the application thereof at microsatellite loci marker seat.
Background technology
Little satellite (microsatellite) is short, the high dna sequence dna of variability of a class length in the higher organism genome.Usually be that repeating unit is series connection repetition form and exists by 2～5 Nucleotide, thus be also referred to as simple repeated sequence (Simple Sequence Repeat, SSR) or tandem repetitive sequence (Short Tandem Repeat, STR).
Microsatellite marker is the polymorphic very widely dna marker of current application, has following distinguishing feature: 1) in the genome of higher organism, extensively distribute, just there is a microsatellite marker in about according to estimates every interval 10-50kb, and the ox genetic map of announcing in 2004 has comprised 3802 microsatellite markers; 2) polymorphism height, information content is abundant, and many little satellites all have 5 above allelotrope; 3) be codominant inheritance, be easy to distinguish homozygous and heterozygous; 4) by pcr amplification and electropherotyping, can directly obtain idiotype, detect easy; 5) most of microsatellite marker is to be positioned at the genome non-coding region, is not subjected to the influence of natural selection and artificial selection, is a kind of neutral mark.Therefore, in Genetic Detection such as human medical jurisprudence evaluation, the affirmation of animal pedigree, microsatellite marker has irreplaceable effect.This is because height polymorphism and codominant inheritance make its individual recognition probability and non-father get rid of the probability height on the one hand.In addition, it is few that little satellite detects required sample amount, sensitivity and success ratio height, and be applicable to the biological sample in various sources.At present, U.S.'s Applied Biotechnology company (AppliedBiosystems) has developed and has been used for the commercialization microsatellite marker detection kit that people, ox, horse and dog pedigree are confirmed.
The detection of microsatellite marker comprises pcr amplification and two steps of gene type.At paternity test, the individual evaluation or situation such as population genetic analysis of variance, need increase and detect a plurality of little satellites seat usually, workload is big, experimentation cost is high.Can significantly improve detection efficiency and reduce cost by composite amplification technology (multiplex PCR).Its basic thought is, adds manyly to primer in same reaction system, and a plurality of target sequences increase.So not only save dna profiling and test consumable, and the step that simplifies the operation, improved the detection efficiency of genetic marker.Yet in order to guarantee a plurality of purpose fragments while specific amplification in 1 PCR reaction, the selection of combination of primers and the optimization of reaction conditions are most important.This not only needs to analyze the compatibility between primer, thereby also need determine the primer concentration ratio by repetition test.
For little satellite gene type,, therefore must distinguish by the high resolution electrophoresis because the not isoallele of little satellite often only differs 2～5 bases.Classifying method be the earliest polyacrylamide gel electrophoresis in conjunction with radioautograph or cma staining, operate very complicated; Since last century end, fluorescent mark polyacrylamide gel electrophoresis and fluorescent mark capillary electrophoresis rise and alternative traditional method gradually.Detection accuracy, the corresponding raising of ease-to-operate.At a plurality of marks, when carrying out primer, modifies the fluorescence that adopts different colours, utilize genetic analyzer based on fluorescent scanning technique, binding molecule amount standard is calculated the allelotrope size automatically, can realize high-throughput and semi-automatic little satellite somatotype.
Current, commercial microsatellite loci marker detection kit only has the StockMarks for Cattle Genotyping Kit Bovine of U.S. AppliedBiosystems company, there are two outstanding problems in this test kit in the ox paternity identification is used: 1. this test kit mark seat is fixed as 11, can't need increase mark quantity at special detection; 2. this test kit costs an arm and a leg, and detects the cost height. these drawbacks limit the application of this test kit in ox breeding practice and scientific research.
Summary of the invention
The objective of the invention is to exist complicated operation, consuming time, repeatability shortcoming such as low and existing commercial kit at existing method on detection technique costs an arm and a leg, detect weak points such as cost height, on detection technique, improved, 17 microsatellite loci markers that specificity is chosen high-throughput, precise and high efficiency increase and somatotype, not chain and clip size suitably, is easy to detect simultaneously at interval between these mark polymorphism height, mark.
Another object of the present invention provides based on the detection method of these 17 microsatellite markers and application.The present invention further utilizes multiple labeling composite amplification technology and the semi-automatic little satellite classifying method of fluorescence, and the optimization experiment system has been set up 17 microsatellite loci marker composite amplifications and classifying method.
For achieving the above object, technical scheme of the present invention provides a kind of microsatellite loci marker combination, comprises following 17 microsatellite loci marker: TH10, ETH225, TGLA227, BM1818, TGLA126, BM1824, INRA023, TGLA53, BM2113, TGLA122, MM12, HEL9, INRA063, SPS115, ILSTS006, ETH152, CSRM060.
17 microsatellite markers of the present invention are selected for applicant's process creative work, and its primer sequence can inquire with the microsatellite marker combination (http://dad.fao.org/) that is used for the research of animal genetic diversity of ISAG (ISAG) combine recommendation in Food and Argriculture OrganizationFAO (FAO).It has following characteristics:
(1) polymorphism height
Through laboratory, applicant place Chinese He Sitanniu is detected, institute is underlined all to show amplification stability and polymorphism preferably.
(2) not chain between mark
17 indicia distribution are on 14 karyomit(e)s of ox.ETH10 and ETH152 are located on karyomit(e) No. 5, ETH225 and the MM12 karyomit(e) that coexists No. 9, TGLA227 and the INRA063 karyomit(e) that coexists No. 18, but these on the same karyomit(e) between mark genetic distance far away, the possibility that is in the linkage disequilibrium state is very low.
(3) clip size suitably, is easy to detect simultaneously at interval
The expanding fragment length of described 17 marks is between 79～296bp, and difference is suitable at interval for selected each mark lengths, utilizes the difference of the fluorescence color of modifying, and is easy to detect simultaneously, realizes high-pass typing.
The present invention further provides the reagent that is used to detect described microsatellite loci marker combination, it comprises the primer or the probe of described 17 microsatellite loci markers that are used to increase.Wherein preferred primer sequence (shown in SEQ ID NO.1～34) as follows:
Further, the invention provides the method that is used to detect described microsatellite loci marker combination, may further comprise the steps:
(1) DNA extraction: extract the ox cell DNA;
(2) pcr amplification: the special purpose fragment of described 17 microsatellite loci markers that increase;
(3) gene type: the described marker genotypes of electrophoretic analysis.
Aforesaid method, preferably described 17 microsatellite loci markers are divided into 4 groups and carry out composite amplification respectively, and the fluorescence that adopts different colours carries out primer and modifies, obtain the special purpose fragment of 17 marks through 4 polymerase chain reaction (PCR) amplification, described composite amplification grouping is modified as follows with fluorescence:
| The composite amplification combination | Mark | Karyomit(e) | Fragment length | Fluorescence is modified |
| Combination 1 | ??ETH10 | ??5 | ??208-224 | ??HEX |
| ??ETH225 | ??9 | ??137-156 | ??HEX | |
| ??TGLA227 | ??18 | ??79-104 | ??FAM | |
| ??BM1818 | ??23 | ??256-268 | ??FAM | |
| ??TGLA126 | ??20 | ??116-126 | ??HEX | |
| Combination 2 | ??BM1824 | ??1 | ??176-190 | ??HEX |
| ??INRA023 | ??3 | ??197-215 | ??FAM | |
| ??TGLA53 | ??16 | ??150-172 | ??FAM | |
| ??BM2113 | ??2 | ??123-137 | ??FAM | |
| ??TGLA122 | ??21 | ??138-183 | ??VIC | |
| Combination 3 | ??MM12 | ??9 | ??110-128 | ??HEX |
| ??HEL9 | ??8 | ??146-169 | ??FAM |
| The composite amplification combination | Mark | Karyomit(e) | Fragment length | Fluorescence is modified |
| ??INRA063 | ??18 | ??174-184 | ??HEX | |
| Combination 4 | ??SPS115 | ??15 | ??245-257 | ??FAM |
| ??ILSTS006 | ??7 | ??284-296 | ??HEX | |
| ??ETH152 | ??5 | ??189-205 | ??FAM | |
| ??CSRM060 | ??10 | ??90-102 | ??FAM |
Select primer institute mark fluorescent element according to the amplified production size, for fragment length the eclipsed mark is arranged especially, modify, little satellite of different colours is detected on a plate glue simultaneously, improve test efficiency with different fluorescence dyes.
Aforesaid method, preferred fluorescent mark polypropylene amine gel electrophoresis or the described marker genotypes of fluorescent mark capillary electrophoresis analysis of adopting in the wherein said step (3).
According to foregoing description, a kind of specific operation process of method of the present invention is as follows:
(1) carries out the extraction that bovine blood DNA extraction or bull are frozen smart DNA according to ordinary method;
(2) pcr amplification:
The applicant optimizes amplification condition and adjusts primer concentration by repetition test, screens best PCR system composition, amplification condition, has set up the optimum composite amplification method of 17 marks.Make 17 mark seats just can amplify all purpose fragments by 4 PCR reactions.
The PCR reaction system
Composite amplification group 1
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??ETH10 | ??0.25μmol/L |
| ??ETH225 | ??0.25μmol/L |
| ??TGLA227 | ??0.25μmol/L |
| ??BM1818 | ??0.25μmol/L |
| ??TGLA126 | ??0.50μmol/L |
| Composition | Concentration |
| The Taq archaeal dna polymerase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 2
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??BM1824 | ??0.25μmol/L |
| ??INRA023 | ??0.25μmol/L |
| ??TGLA53 | ??0.25μmol/L |
| ??BM2113 | ??0.25μmol/L |
| ??TGLA122 | ??0.50μmol/L |
| The TaqDNA polysaccharase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 3
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??MM12 | ??0.25μmol/L |
| ??HEL9 | ??0.375μmol/L |
| ??INRA063 | ??0.50μmol/L |
| Composition | Concentration |
| The TaqDNA polysaccharase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 4
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??SPS115 | ??0.375μmol/L |
| ??ILSTS006 | ??0.375μmol/L |
| ??ETH152 | ??0.375μmol/L |
| ??CSRM060 | ??0.25μmol/L |
| The Taq archaeal dna polymerase | ??1U |
| Dna profiling | ??20～50ng |
The composite amplification program
(3) gene type:
Sample is prepared
The pcr amplification product of composite amplification group 1 and composite amplification group 2 is mixed, be called test set 1; The pcr amplification product of composite amplification group 3 and composite amplification group 4 mixes, and is called test set 2.Test set 1 comprises 10 mark seats, and test set 2 comprises 7 mark seats.In test set, different microsatellite marker seats can be distinguished, thereby all marker genotypes can be obtained by an electrophoresis according to fragment length and fluorescence color.
With mixed detected sample, will dilute 10～20 times with the deionized water of sterilizing.Get dilution back sample 1.5 μ L, add GeneScan-500LIZ Size Standard 0.25 μ L, instantaneous centrifugal behind the deionized formamide 9 μ L. mixings, 95 sex change 4 minutes are put at once in the ice chest and cooled off 3 minutes, change sample after the sex change over to 96 hole sample panel.
Capillary electrophoresis
The sample panel ABI PRISM3730XL genetic analyzer of packing into is carried out electrophoresis.
Interpretation of result
Utilize GeneMapper
TM3.0 software behind the setting molecular weight standard band, according to the color and the position of the collected electrophoresis fluorescence signal of genetic analyzer, calculates the genotype at each mark seat automatically.
Further, the present invention also provides a kind of detection kit that adopts described microsatellite loci marker combination, comprises DNA extraction reagent, pcr amplification reagent and electrophoresis reagent.
Technique scheme has following advantage:
1. number of labels is many: Applied Biosystems company test kit comprises 11 seats, and comprises 17 marks in the detection architecture of the present invention.Like this individuality evaluation, paternity test and population genetic analysis all had higher detection susceptibility and statistics effectiveness.
2. the present invention utilizes the composite amplification method, and can increase by 4 polymerase chain reactions obtains the specific fragment at 17 mark seats, has avoided the repetitive operation of the microsatellite marker that increases one by one in the traditional method.
3. the present invention designs the difference of utilizing not amplified production size and entrained fluorescence between the isolabeling seat, adopts the fluorescent mark capillary electrophoresis method to carry out allelic gene typing, the feasible genotype that can obtain 17 mark seats by 2 capillary electrophoresis.Than the experimental implementation of complicated and time consumption such as traditional polyacrylamide gel electrophoresis, cma staining, it is not only simple to operate, highly sensitive that fluorescent signal detects, and stability, good reproducibility as a result.
4. the present invention can prepare a cover test kit; being used for the individual sibship of ox infers and the population genetic analysis; be specially adapted to DNA such as individuality is reviewed, paternity test, hybridization individual recognition and identify that value is widely used in fields such as ox genetic improvement, conservation of resources and legal medical expert's evaluation.
Description of drawings
Fig. 1 is the capillary electrophoresis fluoroscopic examination signal that detects combination 1 (10 microsatellite markers) in the embodiment of the invention 4;
Fig. 2 is the capillary electrophoresis fluoroscopic examination signal that detects combination 2 (7 microsatellite markers) in the embodiment of the invention 4;
Wherein X-coordinate is represented fragment length, ordinate zou expression signal intensity.
Embodiment
Below in conjunction with embodiment, the specific embodiment of the present invention is described in further detail.Following examples are used to illustrate the present invention, but are not used for limiting the scope of the invention.
Embodiment 1: the primer sequence at little satellite seat
According to following primer sequence synthetic oligonucleotide primer thing, utilizing sterilization deionized water dilution primer is 5 μ mol/L.
Table 117 a microsatellite marker primer sequence and fluorescence are modified
Embodiment 2:DNA extracts
(1) bovine blood DNA extraction
Adopt the biochemical DP318 of the biotech company test kit of day root to extract genomic dna from clot, concrete steps are as follows:
1) take out sample, after the thawing, clip 200 μ L are in the 2mL centrifuge tube;
2) add 600 μ L cell pyrolysis liquid CL, shake up;
3) the centrifugal 1min of 10000rpm discards the garnet supernatant;
4) repeated for 2 and 3 steps once;
5) add 200 μ L damping fluid GS, with the vortex instrument clot particle that fully suspends;
6) add 20 μ L Proteinase Ks, 220 μ L damping fluid GB fully rock and shake up;
7) digest more than 3 hours in 56 ℃ of baking ovens, digestion is early stage, put upside down mixing for several times, until solution becomes clarification, no clot particle;
8) add 200 μ L dehydrated alcohols, rock mixing gently, transfer in the adsorption column, the centrifugal 30s of 12000rpm discards the sap green waste liquid in the collection tube;
9) add 500 μ L damping fluid GD in adsorption column, the centrifugal 30s of 12000rpm discards waste liquid in the collection tube;
10) add 700 μ L rinsing liquid PW in adsorption column, the centrifugal 30s of 12000rpm discards waste liquid;
11) add 500 μ L rinsing liquid PW, the centrifugal 30s of 12000rpm discards waste liquid;
12) the centrifugal 2min of 12000rpm;
13) adsorption column is transferred in the new 1.5mL centrifuge tube opening airing;
14) add the elution buffer TB of 100 μ L, leave standstill 2min 64 ℃ of preheatings;
15) the centrifugal 2min of 12000rpm abandons adsorption column.Genomic dna is present in the centrifuge tube in the solution, preserves standby or-20 ℃ of prolonged preservation for 4 ℃.
(2) bull is frozen the extraction of smart DNA
1) the smart branch of freezing of bull tubule packing and two kinds of forms of granule packaging are arranged.Seminal fluid is taken out from tubule, perhaps directly put into the 2mL centrifuge tube freezing smart particle;
2) add 800 μ L physiological saline, abundant mixing, flush away freeze a large amount of protein ingredients such as yolk diluent in the essence and seminal plasma;
3) 12, the centrifugal 1min of 000rpm carefully removes waste liquid and keeps white precipitate;
4) repeat the operation of 2 and 3 steps once;
5) add the Proteinase K 5 μ L that freeze smart DNA extract 600 μ L and 20mg/mL,, fully shake up with vortex instrument suspended particle;
6) 56 ℃ of digestion of constant temperature are spent the night, and to the solution clear, can't see muddy particle;
7) add 600 μ L phenol, rotate mixing 10min gently;
8) 12, the centrifugal 10min of 000rpm;
9) carefully draw supernatant, transfer in another new centrifuge tube, add 300 μ L phenol and 300 μ L chloroforms, rotate mixing 10min gently;
10) 12, the centrifugal 10min of 000rpm;
11) suct clearly, add the iced dehydrated alcohol of 800mL, overturn gently and turn to DNA and assemble agglomerating;
12) with rifle head (clean syringe needle) DNA group is chosen, 70% washing with alcohol is put into new centrifuge tube, airing;
13) add 500 μ L ddH
2O, fully dissolving is preserved standby or-20 ℃ of prolonged preservation for 4 ℃.
Embodiment 3:PCR amplification
The PCR reaction system
Composite amplification group 1
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??ETH10 | ??0.25μmol/L |
| ??ETH225 | ??0.25μmol/L |
| ??TGLA227 | ??0.25μmol/L |
| ??BM1818 | ??0.25μmol/L |
| ??TGLA126 | ??0.50μmol/L |
| The Taq archaeal dna polymerase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 2
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Composition | Concentration |
| Primer | |
| ??BM1824 | ??0.25μmol/L |
| ??INRA023 | ??0.25μmol/L |
| ??TGLA53 | ??0.25μmol/L |
| ??BM2113 | ??0.25μmol/L |
| ??TGLA122 | ??0.50μmol/L |
| The TaqDNA polysaccharase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 3
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??MM12 | ??0.25μmol/L |
| ??HEL9 | ??0.375μmol/L |
| ??INRA063 | ??0.50μmol/L |
| The Taq archaeal dna polymerase | ??1U |
| Dna profiling | ??20～50ng |
Composite amplification group 4
| Composition | Concentration |
| ??Mg 2+ | ??2.0μmol/L |
| Composition | Concentration |
| ??dNTP | ??200μmol/L |
| Primer | |
| ??SPS115 | ??0.375μmol/L |
| ??ILSTS006 | ??0.375μmol/L |
| ??ETH152 | ??0.375μmol/L |
| ??CSRM060 | ??0.25μmol/L |
| The TaqDNA polysaccharase | ??1U |
| Dna profiling | ??20～50ng |
The composite amplification program
Embodiment 4: gene type
1, sample is prepared
The pcr amplification product of composite amplification group 1 and composite amplification group 2 is mixed, be called test set 1; The pcr amplification product of composite amplification group 3 and composite amplification group 4 mixes, and is called test set 2.Test set 1 comprises 10 mark seats, and test set 2 comprises 7 mark seats (seeing Fig. 1 and Fig. 2 respectively).In test set, can distinguish, thereby can obtain all marker genotypes by an electrophoresis according to fragment length and fluorescence color.
With mixed detected sample, will dilute 10～20 times with the deionized water of sterilizing.Get dilution back sample 1.5 μ L, add GeneScan-500LIZ Size Standard 0.25 μ l, deionized formamide 9 μ l.Instantaneous centrifugal behind the mixing, 95 sex change 4 minutes are put at once in the ice chest and cooled off 3 minutes, change sample after the sex change over to 96 hole sample panel.
2, capillary electrophoresis
The sample panel ABI PRISM3730XL genetic analyzer of packing into is carried out electrophoresis.
3, interpretation of result
Utilize GeneMapper
TM3.0 software behind the setting molecular weight standard band, according to the color and the position of the collected electrophoresis fluorescence signal of genetic analyzer, calculates the genotype at each mark seat automatically.
Application Example of the present invention
Embodiment 1: paternity test
1. sample:
(1) cow I7 blood sample, volume are No. 1.The woman
(2) bull 96100 is frozen smart sample, and volume is No. 2.The father
2. identify and require: DNA check, paternity test
3. check and the result
Extract above-mentioned sample DNA, 17 microsatellite genetic markers are carried out pcr amplification after, on ABI PRISM3730XL genetic analyzer, detect, the result is as follows:
| Sample | ??BM1818 | ??BM1824 | ??BM2113 | ??ETH10 | ??ETH225 |
| ??1 | ??262，264 | ??178，182 | ??125，125 | ??212，216 | ??137，145 |
| ??2 | ??264，264 | ??188，188 | ??125，135 | ??218，218 | ??147，149 |
| Sample | ??INRA023 | ??TGLA122 | ??TGLA126 | ??TGLA227 | ??TGLA53 |
| ??1 | ??205，213 | ??138，149 | ??120，122 | ??98，104 | ??154，154 |
| ??2 | ??209，213 | ??142，163 | ??116，118 | ??98，100 | ??156，164 |
| Sample | ??CSM060 | ??ETH152 | ??HEL9 | ??ILSTS006 | ??INRA063 |
| ??1 | ??90，100 | ??195，195 | ??161，163 | ??292，294 | ??176，176 |
| ??2 | ??96，98 | ??194，201 | ??163，169 | ??288，292 | ??176，176 |
| Sample | ??MM12 | ??SPS115 | |||
| ??1 | ??116，128 | ??249，251 | |||
| ??2 | ??116，116 | ??245，249 |
4. expert's conclusion
In 17 detected microsatellite markers, having 8 not meet parent-offspring's mode of inheritance, is the biology father of cow X so get rid of bull Y.
Embodiment 2: consistence is identified
1. sample and sample:
1) bull is frozen smart 1, pipe number " 2128 ", and volume is No. 1.
2) bull is frozen smart 1, pipe number " 2043 ", and volume is No. 2.
2. identify and require: the DNA check, consistence is identified
3. check and the result
Adopt the cracking digestion method to extract above-mentioned sample DNA, 9 microsatellite genetic markers are carried out pcr amplification after, on ABI PRISM3730XL genetic analyzer, detect, the result is as follows:
| Sample | ??BM1818 | ??BM1824 | ??BM2113 | ??ETH10 | ??ETH225 |
| ??1 | ??260，260 | ??178，188 | ??133，133 | ??216，218 | ??147，149 |
| ??2 | ??260，260 | ??178，188 | ??133，133 | ??216，218 | ??147，149 |
| Sample | ??INRA023 | ??TGLA122 | ??TGLA126 | ??TGLA227 | ??TGLA53 |
| ??1 | ??201，209 | ??173，183 | ??118，124 | ??98，98 | ??156，158 |
| ??2 | ??201，209 | ??173，183 | ??118，124 | ??98，98 | ??156，158 |
| Sample | ??CSM060 | ??ETH152 | ??HEL9 | ??ILSTS006 | ??INRA063 |
| ??1 | ??90，98 | ??195，195 | ??161，167 | ??288，288 | ??174，176 |
| ??2 | ??90，98 | ??195，195 | ??161，167 | ??288，288 | ??174，176 |
| Sample | ??MM12 | ??SPS115 | |||
| ??1 | ??128，128 | ??245，253 | |||
| ??2 | ??128，128 | ??245，253 |
4. expert's conclusion
On 17 detected microsatellite markers, two parts of samples all have identical genotype, and the probability of their genetic identities is greater than 99.99%.
The above only is a preferred implementation of the present invention; should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Sequence table
＜110〉China Agricultural University
＜120〉microsatellite loci marker combination and application thereof
<130>KHP09113369.2
<160>34
<170>PatentIn?version?3.5
<210>1
<211>23
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<210>23
<211>20
<212>DNA
<213>MM12-F
<400>23
caagacaggt?gtttcaatct?????????20
<210>24
<211>20
<212>DNA
<213>MM12-R
<400>24
atcgactctg?gggatgatgt????20
<210>25
<211>20
<212>DNA
<213>HEL9-F
<400>25
cccattcagt?cttcagaggt????20
<210>26
<211>20
<212>DNA
<213>HEL9-R
<400>26
cacatccatg?ttctcaccac????20
<210>27
<211>23
<212>DNA
<213>INRA063-F
<400>27
atttgcacaa?gctaaatcta?acc?23
<210>28
<211>22
<212>DNA
<213>INRA063-R
<400>28
aaaccacaga?aatgcttgga?ag??22
<210>29
<211>22
<212>DNA
<213>ETH152-F
<400>29
tactcgtagg?gcaggctgcc?tg?????22
<210>30
<211>23
<212>DNA
<213>ETH152-R
<400>30
gagacctcag?ggttggtgat?cag????23
<210>31
<211>24
<212>DNA
<213>SPS115-F
<400>31
aaagtgacac?aacagcttct?ccag????24
<210>32
<211>24
<212>DNA
<213>SPS115-R
<400>32
aacgagtgtc?ctagtttggc?tgtg????24
<210>33
<211>20
<212>DNA
<213>ILSTS006-F
<400>33
tgtctgtatt?tctgctgtgg?????????20
<210>34
<211>20
<212>DNA
<213>ILSTS006-R
<400>34
acacggaagc?gatctaaacg????20
Claims (10)
1. a microsatellite loci marker combination is characterized in that, comprises following 17 microsatellite loci marker: TH10, ETH225, TGLA227, BM1818, TGLA126, BM1824, INRA023, TGLA53, BM2113, TGLA122, MM12, HEL9, INRA063, SPS115, ILSTS006, ETH152, CSRM060.
2. be used for the reagent that test right requires the combination of 1 described microsatellite loci marker, it is characterized in that, comprise the primer or the probe of described 17 microsatellite loci markers that are used to increase.
3. composition as claimed in claim 2 is characterized in that, the primer sequence of described 17 microsatellite loci markers is shown in SEQ ID NO.1～34.
4. be used for the method that test right requires 1 described microsatellite loci marker combination, it is characterized in that, may further comprise the steps:
(1) DNA extraction: extract the ox cell DNA;
(2) pcr amplification: the special purpose fragment of described 17 microsatellite loci markers that increase;
(3) gene type: the described marker genotypes of electrophoretic analysis.
5. method as claimed in claim 4 is characterized in that, in the described step (2) during amplification, described 17 microsatellite loci markers is divided into following 4 groups carries out composite amplification respectively, and adopt the fluorescence of different colours to carry out primer and modify:
6. method as claimed in claim 5 is characterized in that, adopts fluorescent mark polypropylene amine gel electrophoresis or the described marker genotypes of fluorescent mark capillary electrophoresis analysis in the described step (3).
7. be used for the test kit that test right requires 1 described microsatellite loci marker combination, comprise DNA extraction reagent, pcr amplification reagent and electrophoresis reagent.
8. the described microsatellite loci marker of claim 1 is combined in the application in the individual evaluation of ox, paternity test and the population genetic analysis.
9. the application of the described reagent of claim 2-3 on microsatellite loci marker detects.
10. the application of the described reagent of claim 2-3 in the individual evaluation of ox, paternity test and population genetic are analyzed.
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| CN 200910236820 CN101705299B (en) | 2009-10-30 | 2009-10-30 | Microsatellite loci marker combination and application thereof |
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| CN 200910236820 CN101705299B (en) | 2009-10-30 | 2009-10-30 | Microsatellite loci marker combination and application thereof |
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| CN101705299A true CN101705299A (en) | 2010-05-12 |
| CN101705299B CN101705299B (en) | 2013-11-06 |
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| CN103484457A (en) * | 2013-09-24 | 2014-01-01 | 山东省农业科学院畜牧兽医研究所 | Lilu cattle microsatellite molecular marker primer pair and application thereof |
| CN105039325A (en) * | 2015-07-10 | 2015-11-11 | 中国农业科学院北京畜牧兽医研究所 | Microsatellite markers for identifying parent-child relationships of Chinese Simmental cattle and application of microsatellite markers |
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| CN105779629A (en) * | 2016-05-09 | 2016-07-20 | 中国农业科学院农业质量标准与检测技术研究所 | Short-tandem-repeats (STR) primer combination for source-traced identification on beef product and detection kit |
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| US7083925B2 (en) * | 2002-01-11 | 2006-08-01 | Schnabel Robert D | Method of parentage testing in North American bison, Bison bison and domestic cattle using microsatellite loci |
| CN100587079C (en) * | 2007-04-09 | 2010-02-03 | 中国农业大学 | Offspring-parent discrimination method for Chinese Hesitan milk cow and its special-use primer and kit |
| CN101509033B (en) * | 2008-12-29 | 2012-02-22 | 西南民族大学 | Method for identifying single-parent water buffalo parental right, primer and reagent kit thereof |
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| CN101705299B (en) | 2013-11-06 |
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