CN110512007A - A kind of pair of canine gene loci collective database - Google Patents
A kind of pair of canine gene loci collective database Download PDFInfo
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Abstract
The invention discloses a kind of pair of canine gene loci collective databases of canine gene loci intersection technical field, this intersection is the gene loci intersection of comprehensive survey for canine, it wherein altogether include 50811 mononucleotide polymorphism sites (SNP) and insertion and deletion (Indels) in 3.1 gene order of Canfam, affiliated gene has with canine blood lineage ancestral source, spiral trough, three broad aspect of hereditary disease risk significantly to be contacted.By carrying out two generation gene sequencing to canine and result and this gene loci intersection being compared, blood lineage's distribution by inspection dog, appearance (such as hair color, ear shape etc.), personality, hereditary disease risk isophenous can be predicted.
Description
Technical field
The present invention relates to canine gene loci intersection technical fields, specially a kind of pair of canine gene loci collective data
Library.
Background technique
SNP is writing a Chinese character in simplified form for single nucleotide polymorphism (Single Nucleotide Polymorphism), refers to genome
Upper single nucleotide acid variation, i.e. the mutual change of tetra- kinds of bases of A, T, C, G, forming same position on genome can be there are many base
Existing polymorphism.SNP is widely present in crowd, rich polymorphism, is good genetic marker.Especially high-throughput
After SNP detection method occurs, it is widely used in the analysis of bioinformatics；
NP Genotyping refers to determining the base-pair type of SNP, and in addition to situation is not detected, a total of 4*4=16 kind can
It can result.The difference of Genotyping, the phenotype that may cause sample are different；
Zu Yuan analysis refers to that from science of heredity angle, whom the ancestors for describing everyone are, comes from which group.And SNP's is more
State property can all have with very strong group's specificity because different group's history of evolution are different, can be used to reflection group
Hereditary feature.By calculating ratio pair, different ancestral's derived components ratios can get；
Currently, not retrieving the patent specifically for canine customization gene loci set.It is not found temporarily in the field at present
There is the same or similar research, the full-length genome of canine is in 2011 by US National Biotechnology Information center (NCBI) public affairs
Cloth, wherein including 39 chromosomes, 2,410,976,875 gene locis in total.However, how to be dug from billions of a sites
Useful site is excavated, and converts it into then to become for the valid data for analysis and GENE Assay analysis is carried out to canine
Priority and difficulty.For this purpose, it is proposed that a kind of pair of canine gene loci collective database and method.
Summary of the invention
The purpose of the present invention is to provide a kind of pair of canine gene loci collective databases, to solve in above-mentioned background technique
The problem of proposition.
To achieve the above object, the invention provides the following technical scheme: a kind of pair of canine gene loci collective database:
Specific technical solution:
1.) select suitably refer to gene order, it is intelligent dote on selected latest edition 3.1 sequence of Can Fam be with reference to sequence
Column.
2.) document relevant to canine gene sequencing is collected, search key includes such as [Canine Genome
Sequencing], [Canine Ancestry Gene], [Canine Hereditary Disease Gene] etc..Record is every
A piece be applicable in sequencing sequence recorded in the literature, sequence version, site information, by inspection dog Relevant phenotype.
3.) entity sample (dog mouth epithelial cells) are collected and carries out machine sequencing.Main application has two parts,
One: by authentic specimen verify document mentioned in site and the correlation of phenotype, for in actual test
It is filtered without significant associated site.
Two: never reporting the kind of dog class being sequenced, such as Tibetan mastiff, U.S. local tyrant dog etc., by increasing these on supplement document
The data of type improve the integrity degree of whole gene intersection (refer in particular to blood lineage ancestral source in terms of).
4.) sample analysis process is as follows:
A. the mouth epithelial cells sample using buccal swab acquisition target dog only, and record every phenotype by inspection dog only
Information.
B. to sample progress DNA extraction is collected, this process uses CommaXP Swab DNA Kit kit, concrete operations
It is executed according to operating guidance.
C. upper machine sequencing is carried out to DNA sample using two generation sequencing technologies, obtains the gene order raw data of sample.
D. initial data is compared with one's own department or unit point set database, i.e. call variant, and the sample is obtained after comparison
Variant sites data.
E. its Mutation site further carries out annotating with database and compare, i.e. annotation finally obtains this
The relevant phenotypic information of Mutation in sample.
5.) database file is constructed, according to the site information that unified format typing has been screened, and adds data base administration
Log records the operations such as the addition of database each time/revise.
6.) each data includes following several entries altogether in the site database included:
Site chromosome coordinate, affiliated gene, variation mode, reference sequences, corresponding phenotype, hereditary pattern, flanking sequence,
Allele.
By in August, 2019, one's own department or unit point data base has been included and related locus 50398, blood lineage ancestral source, hereditary disease correlation
Totally 159, site, macroscopic features related locus 200, behavioral trait related locus 46, drug safety 8, one's own department or unit point data
Library will persistently arrange update.
Gene order illustrates: A, C, G and T respectively represent four kinds of nucleotide --- adenine, cytimidine, the bird of composition DNA
Purine, thymidine, each letter represent a kind of base, and two bases form a base-pair, and the pairing rule of base-pair is
Fixed, be: A-T, C-G, typically they are nonseptate is arranged together, such as sequence AAAGTCTGAC, random length
A string of nucleotide greater than 4 are referred to as a sequence, as shown in Figure 1.
Used DNA extracts reagent from CommaXP Swab DNA Kit kit, concrete composition and operation
It is as follows:
Kit forms:
The technique includes the following steps:
Step 1: Preparatory work of experiment
A: for the first time before use, the dehydrated alcohol of designated volume is added in Buffer PD, Buffer PW；
B: prepare Tip head, the centrifuge tube of free nucleic acid and nuclease pollution；
C: prepare RNase A, the Poly Carrier RNA (1 μ g/ μ l) of 10mg/ml；
D: before use, whether observation solution precipitates, if having precipitating in Buffer GL, can add in 56 DEG C of water-baths
Heat of solution simultaneously reuses after being cooled to room temperature；
Step 2: the cotton swab wiped across in cheek is placed in 2ml centrifuge tube, with scissors by cotton swab part from its bar
It cuts, 300 μ l Buffer SA, vortex oscillation 15sec is added, 400 μ l Buffer GL are added, vortex oscillation is mixed to thorough
It is even, be then added 20 μ l Proteinase K solution, vortex 10sec is mixed, and 20min are placed in 65 DEG C of water-baths, during which every
5min oscillation mixes.
Note: a small amount of RNase solution A can be added in the genomic DNA polluted if you need to no RNA, vibrate 15sec, and room temperature is put
Set 5min.
Step 3: after brief centrifugation, drawing 400 μ l supernatants, isometric Buffer GB be added and is sufficiently mixed by inversion, and 65
DEG C place 15min, during which every 5min vibrate mix.
Step 4: may generate white precipitate when Buffer GB is added, and whens general 65 DEG C of placements can disappear, Bu Huiying
Ring subsequent experimental.
Step 5: adding 400 μ l dehydrated alcohols, be sufficiently mixed by inversion, and brief centrifugation is centrifuged the drop of cap wall to remove.
Note: being added after dehydrated alcohol it is possible that flocculent deposit, but does not influence DNA extraction.
Step 5: by previous step acquired solution in two times all be added an adsorption column C5 in (adsorption column C5 has been put into
In 2.0mL collecting pipe), 12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in
In collecting pipe.
Step 6: 500 μ l Buffer PD are added into adsorption column C5 and (is please first checked whether before use and anhydrous second has been added
Alcohol), 12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in collecting pipe.
Step 7: 600 μ l Buffer PW are added into adsorption column C5 and (is please first checked whether before use and anhydrous second has been added
Alcohol), 12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in collecting pipe.
Step 8: step 6 is repeated.
Step 9: 12,000rpm (~13,400 × g) are centrifuged 2min, abandon waste liquid.Adsorption column C5 is placed at room temperature for several points
Clock, thoroughly to dry rinsing liquid remaining in adsorbent material.
Note: the purpose of this step is to remove rinsing liquid remaining in adsorption column, and the residual of ethyl alcohol can shadow in rinsing liquid
Ring subsequent enzyme reaction (digestion, PCR etc.) experiment.
Step 10: adsorption column C5 is transferred in a new 1.5ml collecting pipe, 30- is vacantly added dropwise to adsorbed film middle position
80 μ l Buffer TE are placed at room temperature for 2-5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, collect DNA solution.
Note: elution buffer Buffer TE volume should not be less than 30 μ l, the too small influence recovery efficiency of volume.To improve
The yield of genomic DNA can add centrifugation obtained solution in adsorption column C5,12,000rpm (~13,400 × g) from
Heart 2min.
Compared with prior art, the beneficial effects of the present invention are:
1. the research and development of kit middle through the invention of the invention, by kit extraction and analysis, so that genome includes altogether
50398 mononucleotide polymorphism sites (SNP) in 3.1 gene order of Canfam, affiliated gene and canine blood lineage
Zu Yuan, spiral trough, three broad aspect of hereditary disease risk have significant connection.By carrying out two generation gene sequencing to canine and will tie
Fruit is compared with this gene loci intersection, can predict by blood lineage's distribution of inspection dog, appearance (such as hair color, ear
Shape etc.), personality, hereditary disease risk isophenous；
2. the present invention can quickly, simply be isolated and purified from buccal swab total using the pellosil adsorption technology of specificity
DNA, remains the integrality and purity for extracting genomic DNA to greatest extent, and the DNA of general single buccal swab must measure as 1.0 μ
g-4.5μg.Various routine operations are applicable to using the genomic DNA that this kit extracts, such as digestion, PCR, Real-Time
The experiment of the downstreams such as PCR, library construction, molecular labeling；
3. the solution formula that the present invention optimizes, can simple and quick extraction buccal swab, exclusive silica gel membrane technology, in spy
Determine under solution environmental, it is strong to the adsorption capacity of nucleic acid, change condition, easily elution nucleic acid, without toxic reagents such as phenol, chloroforms.
Detailed description of the invention:
Fig. 1 is gene order table of the present invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Referring to Fig. 1, the present invention provides a kind of technical solution: a kind of pair of canine gene loci collective database,
Specific technical solution:
1.) select suitably refer to gene order, it is intelligent dote on selected latest edition 3.1 sequence of Can Fam be with reference to sequence
Column.
2.) document relevant to canine gene sequencing is collected, search key includes such as [Canine Genome
Sequencing], [Canine Ancestry Gene], [Canine Hereditary Disease Gene] etc..Record is every
A piece be applicable in sequencing sequence recorded in the literature, sequence version, site information, by inspection dog Relevant phenotype.
3.) entity sample (dog mouth epithelial cells) are collected and carries out machine sequencing.Main application has two parts:
One: by authentic specimen verify document mentioned in site and the correlation of phenotype, for in actual test
It is filtered without significant associated site.
Two: never reporting the kind of dog class being sequenced, such as Tibetan mastiff, U.S. local tyrant dog etc., by increasing these on supplement document
The data of type improve the integrity degree of whole gene intersection (refer in particular to blood lineage ancestral source in terms of).
4.) sample analysis process is as follows:
A. the mouth epithelial cells sample using buccal swab acquisition target dog only, and record every phenotype by inspection dog only
Information.
B. to sample progress DNA extraction is collected, this process uses CommaXP Swab DNA Kit kit, concrete operations
It is executed according to operating guidance.
C. upper machine sequencing is carried out to DNA sample using two generation sequencing technologies, obtains the gene order raw data of sample.
D. initial data is compared with one's own department or unit point set database, i.e. call variant, and the sample is obtained after comparison
Variant sites data.
E. its Mutation site further carries out annotating with database and compare, i.e. annotation finally obtains this
The relevant phenotypic information of Mutation in sample.
5.) database file is constructed, according to the site information that unified format typing has been screened, and adds data base administration
Log records the operations such as the addition of database each time/revise.
6.) each data includes following several entries altogether in the site database included:
Site chromosome coordinate, affiliated gene, variation mode, reference sequences, corresponding phenotype, hereditary pattern, flanking sequence,
Allele.
By in August, 2019, one's own department or unit point data base has been included and related locus 50398, blood lineage ancestral source, hereditary disease correlation
Totally 159, site, macroscopic features related locus 200, behavioral trait related locus 46, drug safety 8, one's own department or unit point data
Library will persistently arrange update.
Gene order illustrates: A, C, G and T respectively represent four kinds of nucleotide --- adenine, cytimidine, the bird of composition DNA
Purine, thymidine, each letter represent a kind of base, and two bases form a base-pair, and the pairing rule of base-pair is
Fixed, be: A-T, C-G, typically they are nonseptate is arranged together, such as sequence AAAGTCTGAC, random length
A string of nucleotide greater than 4 are referred to as a sequence, as shown in Figure 1.
Used DNA extracts reagent from CommaXP Swab DNA Kit kit.
Conclusion:
Compared with single document, this gene loci intersection has greatly expanded the direction multiplicity of pet dog genetic analysis
Property, including blood lineage ancestral source, macroscopic features, hereditary disease risk, behavioral trait, drug safety.The bit number of points that it is included are also
Reach 50000 or more, analysis comprehensively is carried out to the gene data of pet dog for science and provides strong knowledge support.
Meanwhile subsequent actual samples and continue the novel blood lineage site of amended record make this site gather sophistication be continuously improved.
It although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of variations, modification, replacement can be carried out to these embodiments without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (1)
1. a kind of pair of canine gene loci collective database, it is characterised in that:
Specific technical solution:
1.) select suitably refer to gene order, it is intelligent dote on selected latest edition 3.1 sequence of Can Fam be reference sequences.
2.) document relevant to canine gene sequencing is collected, search key includes such as [Canine Genome
Sequencing], [Canine Ancestry Gene], [Canine Hereditary Disease Gene] etc..Record is every
A piece be applicable in sequencing sequence recorded in the literature, sequence version, site information, by inspection dog Relevant phenotype.
3.) entity sample (dog mouth epithelial cells) are collected and carries out machine sequencing.Main application has two parts,
One: by authentic specimen verify document mentioned in site and the correlation of phenotype, for do not have in actual test
Significant associated site is filtered.
Two: never reporting the kind of dog class being sequenced, such as Tibetan mastiff, U.S. local tyrant dog etc., by increasing these types on supplement document
Data improve the integrity degree of whole gene intersection (refer in particular to blood lineage ancestral source in terms of).
4.) sample analysis process is as follows:
A. the mouth epithelial cells sample using buccal swab acquisition target dog only, and record and believed by the every phenotype of inspection dog only
Breath.
B. to collect sample carry out DNA extraction, this process use CommaXP Swab DNA Kit kit, concrete operations according to
Operating guidance executes.
C. upper machine sequencing is carried out to DNA sample using two generation sequencing technologies, obtains the gene order raw data of sample.
D. initial data is compared with one's own department or unit point set database, i.e. callvariant obtains the variation of the sample after comparison
Site data.
E. its Mutation site further carries out annotating with database and compare, i.e. annotation finally obtains the sample
In the relevant phenotypic information of Mutation.
5.) database file is constructed, according to the site information that unified format typing has been screened, and adds data base administration day
Will records the operations such as the addition of database each time/revise.
6.) each data includes following several entries altogether in the site database included:
Site chromosome coordinate, affiliated gene, variation mode, reference sequences, corresponding phenotype, hereditary pattern, flanking sequence, equipotential
Gene.
By in August, 2019, one's own department or unit point data base has been included and related locus 50398, blood lineage ancestral source, hereditary disease related locus
Totally 159, macroscopic features related locus 200, behavioral trait related locus 46, drug safety 8, one's own department or unit point data base will
Lasting arrange updates.
Gene order illustrate: A, C, G and T respectively represent composition DNA four kinds of nucleotide --- adenine, cytimidine, guanine,
Thymidine, each letter represent a kind of base, and two bases form a base-pair, and the pairing rule of base-pair is fixed
, be: A-T, C-G, typically they are nonseptate is arranged together, such as sequence AAAGTCTGAC, and random length is greater than 4
A string of nucleotide be referred to as a sequence.
Used DNA extracts reagent from CommaXP Swab DNA Kit kit, and concrete composition and operation are as follows:
Kit forms:
The technique includes the following steps:
Step 1: Preparatory work of experiment
A: for the first time before use, the dehydrated alcohol of designated volume is added in Buffer PD, Buffer PW；
B: prepare Tip head, the centrifuge tube of free nucleic acid and nuclease pollution；
C: prepare RNase A, the Poly Carrier RNA (1 μ g/ μ l) of 10mg/ml；
D: it before use, whether observation solution precipitates, if having precipitating in Buffer GL, can be heated in 56 DEG C of water-baths molten
It solves and is reused after being cooled to room temperature；
Step 2: the cotton swab wiped across in cheek is placed in 2ml centrifuge tube, is cut cotton swab part from its bar with scissors
Under, 300 μ l Buffer SA, vortex oscillation 15sec are added, 400 μ l Buffer GL are added, vortex oscillation is mixed to thorough,
Then 20 μ l Proteinase K solution are added, vortex 10sec is mixed, and 20min is placed in 65 DEG C of water-baths, is during which shaken every 5min
Swing mixing.
Note: a small amount of RNase solution A can be added in the genomic DNA polluted if you need to no RNA, vibrate 15sec, be placed at room temperature for
5min。
Step 3: after brief centrifugation, 400 μ l supernatants is drawn, isometric Buffer GB is added and is sufficiently mixed by inversion, 65 DEG C put
15min is set, during which vibrates and mixes every 5min.
Step 4: may generate white precipitate when Buffer GB is added, and whens general 65 DEG C of placements can disappear, after will not influence
Continuous experiment.
Step 5: adding 400 μ l dehydrated alcohols, be sufficiently mixed by inversion, and brief centrifugation is centrifuged the drop of cap wall to remove.
Note: being added after dehydrated alcohol it is possible that flocculent deposit, but does not influence DNA extraction.
Step 5: by previous step acquired solution in two times all be added an adsorption column C5 in (adsorption column C5 be put into 2.0mL collection
In pipe), 12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in collecting pipe.
Step 6: 500 μ l Buffer PD (please first check whether before use and dehydrated alcohol has been added) being added into adsorption column C5,
12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in collecting pipe.
Step 7: 600 μ l Buffer PW (please first check whether before use and dehydrated alcohol has been added) being added into adsorption column C5,
12,000rpm (~13,400 × g) are centrifuged 30sec-1min, abandon waste liquid, adsorption column C5 is placed back in collecting pipe.
Step 8: step 6 is repeated.
Step 9: 12,000rpm (~13,400 × g) are centrifuged 2min, abandon waste liquid.Adsorption column C5 is placed at room temperature for several minutes, with
Thoroughly dry rinsing liquid remaining in adsorbent material.
Note: the purpose of this step is to remove rinsing liquid remaining in adsorption column, after the residual of ethyl alcohol will affect in rinsing liquid
Continuous enzyme reaction (digestion, PCR etc.) experiment.
Step 10: adsorption column C5 is transferred in a new 1.5ml collecting pipe, and 30-80 μ l is vacantly added dropwise to adsorbed film middle position
Buffer TE is placed at room temperature for 2-5min, and 12,000rpm (~13,400 × g) are centrifuged 2min, collects DNA solution.
Note: elution buffer Buffer TE volume should not be less than 30 μ l, the too small influence recovery efficiency of volume.To improve gene
The yield of group DNA can add the solution that centrifugation obtains in adsorption column C5,12,000rpm (~13,400 × g) centrifugation
2min。
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| CN110931082A (en) * | 2019-12-12 | 2020-03-27 | 爱尔生基因医学科技有限公司 | Method and system for gene detection and evaluation |
| CN113667763A (en) * | 2020-09-02 | 2021-11-19 | 北京中科昆朋生物技术有限公司 | Biomarker, kit and method for identifying dogs with pickup behaviors |
| CN113921089A (en) * | 2021-11-22 | 2022-01-11 | 北京安智因生物技术有限公司 | Method and system for confirming updating frequency of IVD gene annotation database |
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