KR20170056726A - SNP marker for prediction of dog's food consumption and prediction method using the same - Google Patents
SNP marker for prediction of dog's food consumption and prediction method using the same Download PDFInfo
- Publication number
- KR20170056726A KR20170056726A KR1020150159303A KR20150159303A KR20170056726A KR 20170056726 A KR20170056726 A KR 20170056726A KR 1020150159303 A KR1020150159303 A KR 1020150159303A KR 20150159303 A KR20150159303 A KR 20150159303A KR 20170056726 A KR20170056726 A KR 20170056726A
- Authority
- KR
- South Korea
- Prior art keywords
- base
- seq
- polynucleotide
- consecutive bases
- complementary polynucleotides
- Prior art date
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
The present invention relates to a SNP marker for predicting dog feeding ability and a dog feeding ability predicting method using the SNP marker.
Eating capacity of dogs is one of the most representative phenotypes of health status and is associated with dog health status.
The ability of the dogs to ingest the dogs is important for their ability to feed on dogs for growth and resistance to disease, so the need for early prediction through genetic testing is emerging.
A preliminary report on genetic markers for the traits of dogs is "A final report on the development of small-scale slaughtering by molecular breeding (research institute: Kyungpook National University, 2004, sponsored by the Ministry of Agriculture and Forestry).
The use of gene markers related to the feeding ability of the dogs can satisfy the preference of the dogs to the feeding ability and can restrain the indiscriminate breeding for producing the feeding ability and the growth rate of the high or low individuals, It is anticipated that it will be possible to set up a culture of dogs and dogs, and it will be possible to acquire the academic value and basic medical information about the appetite and growth of animals by discovering genes related to dog feeding ability.
Under these technical backgrounds, the present inventors have made intensive efforts to develop gene markers for early diagnosis by finding genes related to the ability of feeding dogs.
Accordingly, it is an object of the present invention to provide a SNP marker composition for predicting dog feeding ability.
It is another object of the present invention to provide a method for early prediction of dog feeding ability and growth using the SNP marker.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
According to one aspect of the present invention, there is provided a polynucleotide comprising: (a) a polynucleotide having a 61st base of A or G in the polynucleotide of SEQ ID NO: 1, an inner nucleotide sequence of SEQ ID NO: 1, A polynucleotide consisting of 121 consecutive bases or a complementary polynucleotide thereof; (b) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide shown in SEQ ID NO: 2 and the 61rd base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof; (c) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 3 and the 61rd base is the base sequence of SEQ ID NO: 3, or Complementary polynucleotides thereof; d) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 4, and the 61rd base is the base sequence of SEQ ID NO: 4; Complementary polynucleotides; (e) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 5 and the 61rd base is the base sequence of SEQ ID NO: 5, or Complementary polynucleotides thereof; (f) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 6 and the 61rd base is the internal base sequence of SEQ ID NO: 6; or Complementary polynucleotides thereof; (g) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 7, and the 61rd base is the internal base sequence of SEQ ID NO: 7, or Complementary polynucleotides thereof; (h) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide of SEQ ID NO: 8 and the 61rd base is the base sequence of SEQ ID NO: 8, or Complementary polynucleotides thereof; (i) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 9 and the 61rd base is the internal base sequence of SEQ ID NO: 9, or Complementary polynucleotides thereof; (j) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 10 and the 61rd base is the internal base sequence of SEQ ID NO: 10; or Complementary polynucleotides thereof; (k) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 11 and the 61rd base is the internal sequence of SEQ ID NO: 11; or Complementary polynucleotides thereof; (l) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 12 and the 61rd base is the base sequence of SEQ ID NO: 12; or Complementary polynucleotides thereof; (m) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 13, and the 61rd base is the internal nucleotide sequence of SEQ ID NO: 13; or Complementary polynucleotides thereof; (n) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 14 and the 61rd base is the base sequence of SEQ ID NO: 14; or Complementary polynucleotides thereof; (o) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 15 and the 61rd base is the internal sequence of SEQ ID NO: 15; or Complementary polynucleotides thereof; (p) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 16 and the 61rd base is the base sequence of SEQ ID NO: 16; or Complementary polynucleotides thereof; (q) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide represented by SEQ ID NO: 17, and the 61rd base is the base sequence of SEQ ID NO: 17; Complementary polynucleotides thereof; And (r) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide represented by SEQ ID NO: 18 and the 61rd base is the base sequence of SEQ ID NO: 18 Or a complementary polynucleotide thereof. The SNP marker composition according to any one of claims 1 to 7,
According to another aspect of the present invention, there is provided a kit for predicting feeding ability of a dog comprising the composition.
According to another aspect of the present invention, there is provided a microarray for predicting feeding ability of a dog comprising the composition.
According to yet another aspect of the present invention, there is provided a method for detecting a nucleic acid molecule comprising: a) separating a nucleic acid molecule from a dog; And b) identifying the base type of the SNP corresponding to the 61st base of the polynucleotide of any one of SEQ ID NOS: 1 to 18 in the separated nucleic acid molecule.
According to an embodiment of the present invention, dog feeding ability can be predicted early, so that the preference of dog owners for feeding ability can be met.
In addition, it is possible to limit the indiscriminate breeding to produce individuals with high or low feeding ability and growth rate, thus contributing to the protection and welfare policy of the dogs and establishing a culture of dogs.
Furthermore, it is anticipated that the researchers will be able to acquire academic values and basic medical information on the appetite and growth of animals by identifying genes related to the ability of feeding dogs.
Hereinafter, the present invention will be described in more detail.
According to one aspect of the present invention, there is provided a polynucleotide comprising: (a) a polynucleotide having a 61st base of A or G in the polynucleotide of SEQ ID NO: 1, an inner nucleotide sequence of SEQ ID NO: 1, A polynucleotide consisting of 121 consecutive bases or a complementary polynucleotide thereof; (b) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide shown in SEQ ID NO: 2 and the 61rd base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof; (c) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 3 and the 61rd base is the base sequence of SEQ ID NO: 3, or Complementary polynucleotides thereof; d) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 4, and the 61rd base is the base sequence of SEQ ID NO: 4; Complementary polynucleotides; (e) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 5 and the 61rd base is the base sequence of SEQ ID NO: 5, or Complementary polynucleotides thereof; (f) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 6 and the 61rd base is the internal base sequence of SEQ ID NO: 6; or Complementary polynucleotides thereof; (g) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 7, and the 61rd base is the internal base sequence of SEQ ID NO: 7, or Complementary polynucleotides thereof; (h) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide of SEQ ID NO: 8 and the 61rd base is the base sequence of SEQ ID NO: 8, or Complementary polynucleotides thereof; (i) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 9 and the 61rd base is the internal base sequence of SEQ ID NO: 9, or Complementary polynucleotides thereof; (j) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 10 and the 61rd base is the internal base sequence of SEQ ID NO: 10; or Complementary polynucleotides thereof; (k) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 11 and the 61rd base is the internal sequence of SEQ ID NO: 11; or Complementary polynucleotides thereof; (l) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 12 and the 61rd base is the base sequence of SEQ ID NO: 12; or Complementary polynucleotides thereof; (m) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 13, and the 61rd base is the internal base sequence of SEQ ID NO: 13; Complementary polynucleotides; (n) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 14 and the 61rd base is the base sequence of SEQ ID NO: 14; or Complementary polynucleotides thereof; (o) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 15 and the 61rd base is the internal sequence of SEQ ID NO: 15; or Complementary polynucleotides thereof; (p) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 16 and the 61rd base is the base sequence of SEQ ID NO: 16; or Complementary polynucleotides thereof; (q) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide represented by SEQ ID NO: 17, and the 61rd base is the base sequence of SEQ ID NO: 17; Complementary polynucleotides thereof; And (r) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide represented by SEQ ID NO: 18 and the 61rd base is the base sequence of SEQ ID NO: 18 Or a complementary polynucleotide thereof. The SNP marker composition according to any one of claims 1 to 7,
According to one embodiment of the present invention, the composition comprises an agent capable of detecting or amplifying the SNP marker.
According to another aspect of the present invention, there is provided a kit for predicting feeding ability of a dog comprising the composition.
In the present invention, the kit may be, but is not limited to, an RT-PCR kit or a microarray chip kit including a preparation capable of detecting or amplifying SNP markers.
The RT-PCR kit can comprise a respective pair of primers specific for the marker gene and can be used in combination with other test tubes or other appropriate containers, reagents necessary for PCR amplification, such as buffers, DNA polymerases (e.g., Thermus aquaticus Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermisflavus, Thermococcus literalis or Pyrococcus furiosus (Pfu)), DNA polymerase joins and dNTPs.
The kit may be made from a number of separate packaging or compartments containing the reagent components described above.
According to another aspect of the present invention, there is provided a microarray for predicting feeding ability of a dog comprising the composition.
In the present invention, a microarray means a group of polynucleotides immobilized on a substrate at a high density, and the polynucleotide group means a microarray immobilized in a constant region. Such microarrays are well known in the art. The microarrays are described, for example, in U.S. Patent Nos. 5,445,934 and 5,744,305, the contents of which are incorporated herein by reference.
According to yet another aspect of the present invention, there is provided a method for detecting a nucleic acid molecule comprising: a) separating a nucleic acid molecule from a dog; And b) identifying the base type of the SNP corresponding to the 61st base of the polynucleotide of any one of SEQ ID NOS: 1 to 18 in the separated nucleic acid molecule.
According to an embodiment of the present invention, the separated nucleic acid molecule is amplified in step b).
According to an embodiment of the present invention, the amplified gene product may be purified to analyze the base sequence or hybridize with the SNP marker of the present invention.
Methods for amplifying the nucleic acid molecules include PCR, ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, , Strand displacement amplification or amplification through a Q [beta] replicase, or any other suitable method for amplifying nucleic acid molecules known in the art. Among them, PCR is a method of amplifying a target nucleic acid from a pair of primers that specifically bind to a target nucleic acid using a polymerase. Such PCR methods are well known in the art, and commercially available kits may be used. The PCR can be carried out using a PCR reaction mixture containing various components known in the art necessary for the PCR reaction.
In the present invention, hybridization refers to a process in which two complementary strands of a nucleic acid are combined to form a double stranded molecule (hybrid). In the method of the present invention, the hybridization is carried out under high stringency hybridization conditions.
To detect the degree of hybridization, the target sequence may be labeled with a detectable labeling substance. In one embodiment, the labeling material may be a fluorescent, phosphorescent or radioactive substance, but is not limited thereto. Preferably, the labeling substance is Cy-5 or Cy-3. When the target sequence is amplified, PCR is carried out by labeling the 5'-end of the primer with Cy-5 or Cy-3, and the target sequence may be labeled with a detectable fluorescent labeling substance. In addition, if the radioactive isotope such as P32 or S35 is added to the PCR reaction solution during the PCR, the amplification product may be synthesized and the radioactive substance may be incorporated into the amplification product and the amplification product may be labeled as radioactive.
According to one embodiment of the present invention, when the base type of the SNP is identified from the isolated nucleic acid molecule, when the 61st base is A in the polynucleotide of SEQ ID NO: 1; When the 61st base in the polynucleotide represented by SEQ ID NO: 2 is C; When the 61st base in the polynucleotide represented by SEQ ID NO: 3 is A; When the 61st base in the polynucleotide represented by SEQ ID NO: 4 is A; The polynucleotide of SEQ ID NO: 5 has the 61st base G; When the 61st base in the polynucleotide of SEQ ID NO: 6 is A; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 7; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 8; The polynucleotide of SEQ ID NO: 9 has the 61st base of G; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 10; When the 61st base is G in the polynucleotide shown by SEQ ID NO: 11; When the 61st base in the polynucleotide represented by SEQ ID NO: 12 is A; When the 61st base of the polynucleotide represented by SEQ ID NO: 13 is A; When the 61st base in the polynucleotide represented by SEQ ID NO: 14 is A; When the 61st base in the polynucleotide shown in SEQ ID NO: 15 is A; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 16; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 17; And when the 61st nucleotide of the polynucleotide represented by SEQ ID NO: 18 is A, a method is provided for determining that the feeding ability is relatively good.
Hereinafter, the present invention will be described in more detail with reference to Examples. It should be understood, however, that these examples are for illustrative purposes only and are not to be construed as limiting the scope of the present invention.
□ Expression traits and scoring methods for feeding ability
1 point: When the dog is actively feeding, if eating without consciousness by hand holding the insomnia.
2 points: When eating on the middle of a dog, feeding when the animal is placed on the floor
3 points: When the dog is neglected by eating, the animal is placed on the silk and fed when the test ground comes out.
4 points: If the dog is not fed, the test animal is put on the silk and the test ground comes out and does not feed within 1 minute
Example Canine 170K SNP chip analysis for 1. dog
DNA was extracted from the blood of 50 dogs using the Wizard Genomic DNA Purification Kit (Promega, Madison, Wis., USA) and SNP genotyping was performed using CanineHDBeadChip (Illumina, San Diego, CA, USA).
≪ Amplification >
○ Materials and equipment
1. Reagents
2. Organization
Centrifuge, Vortex, Illumina Hybridization oven
○ Experimental Method
1. Place 20 μl of MA1 on a MIDI plate (labeled MSA3 plate) with an MSA3 bar code.
2. Place 4 ul of DNA into MSA3 plate.
3. Record the DNA ID and the location of the MSA3 plate on the lab tracking form.
4. Add 4 ul of 0.1 N NaOH to each well of MSA3 plate containing MA1 and DNA.
5. Cover the MSA3 plate using a 96-well cap mat and vortex for 1 minute at 1600 rpm.
6. Centrifuge at 280 × g for 1 minute.
7. React at room temperature for 10 minutes.
8. Add 34ul MA2 to each well of the MSA3 plate containing the sample.
9. Add 38ul MSM to each well of the MSA3 plate containing the sample.
10. Cover the cap mat and centrifuge at 280 × g for 1 minute.
11. React for 20-24 hours in an Illumina Hybridization oven at 37 ° C.
(Amplification)
<Day 2 of experiment - Fragment>
○ Materials and equipment
○ Experimental Method
1. Remove the plate from the oven and centrifuge at 50 × g for 1 minute.
2. Place 25 ul of FMS into each well containing sample.
3. Cover the MSA3 plate with a cap mat and vortex for 1 minute at 1600 rpm.
4. Remove the plate and centrifuge at 50 × g for 1 min.
5. Allow to react for 1 hour at 37 ℃ heat block.
<Day 2 - Precipitation>
○ Materials and equipment
○ Experimental Method
1. Remove the cap mat and place 25 ul of PM1 in each well containing the sample.
2. Cover the cap mat and centrifuge at 1600 rpm for 1 minute.
3. React at 37 ° C for 5 minutes.
4. Remove the plate and centrifuge at 50 × g for 1 min.
5. Remove the cap mat and place 155 ul of 2-propanol in each well containing the sample.
6. Cover the plate using a new cap mat, turn over 10 times, mix and store at 4 ° C for 30 minutes.
7. Centrifuge at 4 ° C at 3,000 rpm for 20 minutes and immediately remove the MSA3 plate from the centrifuge.
8. Immediately remove the cap mat and turn it over quickly to discard the supernatant.
9. Tap on the absorbent pad (kitchen towel, kim towel, etc.) 10 times.
10. Put the inverted plate on the tube rack and dry naturally for 1 hour.
<Day 2 - Resuspend >
○ Materials and equipment
○ Experimental Method
1. Place 23ul of RA1 in each well containing DNA pellet and store the remaining RA1 for XStain HD Bead Chip (freeze storage).
2. Place the foil seal on the MSA3 plate and seal it by pressing the heat-sealer block for 5 seconds.
3. React for 1 hour in an Illumina Hybridization oven at 48 ° C
4. Vortex for 1 minute at 1800 rpm.
5. Centrifuge at 280 × g for 1 minute.
<Day 2 - Hybridization>
○ Materials and equipment
○ Experimental Method
1. MSA3 plate is denatured for 20 minutes at 95 ℃ heat block.
2. After 20 minutes, remove the MSA3 plate from the heat block and allow to cool to room temperature for 30 minutes.
3. Insert the HybChamber Gaskets into the HybChamber as the plate is nearly 30 minutes cool.
4. Place 400ul of PB2 in 8 humidifying buffer reservoirs in HybChamber, close HybChamber lid and place at room temperature.
5. After cooling the DNA for 30 min at room temperature, centrifuge the MSA3 plate at 280 × g for 1 min.
6. Take the stored chips one by one from the refrigerator to get the chips guarantee, align the barcode of the HybChamber insert with the bar code of the chips, and load the samples on both sides of the chips after 15ul per sample using a multi-channel pipette.
7. As soon as the sample loading of each chip is finished, place it in the HybChamber and repeat the following chips in the same way.
8. When the chamber is filled, close the chamber lid and place in the Illumina Hybridization oven at 48 ° C. Set it to speed 5 and react for 16-24 hours.
<Day 3 - Washing bead chips>
○ Materials and equipment
1. Reagents
2. Organization
- Multi-sample beadChip Alignment fixture
- Te-Flow Flow-Through chambers (black frames, spacers, glass back plate and clamps)
- Wash Dish
- Wash Rack
○ Experimental Method
1. Remove the Hyb chamber from the Hybridization oven.
2. Open the lock of the Hyb chamber and take out one insert at a time in the chamber.
3. Pull the seal on the chip to remove it from the chip.
4. Insert the chip with the seal removed into the Wash Rack and dip into the WB1 Wash dish.
5. Once all chips are in the WB1, wash the Wash Rack in a dish for 1 minute, transfer the Wash Rack to another Wash Dish containing PB1 and repeat this process for 1 minute.
6. Immerse again in PB1 wash dish, wash Wash Rack in dish for 1 minute, transfer Wash Rack to another Wash Dish containing PB1 and repeat this process for 1 minute.
7. After washing, place the back frame on the BeadChips Alignment fixture, place the chip one by one in the direction of the barcode, and then fit the space of the transparent part separated from the white part to the top and bottom of the alignment fixture.
8. Raise the space, place the Alignmet bar on the top part of the chip (no barcode), cover the glass with the end of the glass facing the bar, and insert the clip.
(Flow-through chamber assembly completed)
9. After inserting the clip, remove the alignment bar and cut off the space at both ends of the flow-through chamber assembly with scissors.
<Day 3 - XStain Beadchips >
○ Materials and equipment
1. Reagents
2. Organization
- Water circulator
- Chamber Rack
- 2 wash dishes, staining rack, tube rack
○ Experimental Method
1. When the chamber temperature reaches 44 ° C, insert the flow-through chamber assembly into the chamber rack.
2. Add 150ul of RA1 to each chips and let it react for 30 seconds. Repeat this process five more times.
3. Add 450ul of XC1 to each chip and incubate for 10 minutes.
4. Add 450ul of XC2 to each chip and incubate for 10 minutes.
5. Add 200 ul of TEM to each chip and incubate for 10 minutes.
6. Add 450 μl of 95% formamide / 1 mM EDTA to each chip and incubate for 1 minute.
7. React for 5 minutes.
8. Check the temperature on the label of the LTM tube and change the temperature of the chamber rack according to the temperature.
9. Add 450ul of XC3 to each chip and allow to react for 1 minute, then insert again and wait until the temperature reaches 8 times.
10. Add 250 ul of LTM to each chip and let it react for 10 minutes.
11. Add 450ul of XC3, add one more time after 1 minute, and react for 5 minutes.
12. Add 250 ul of ATM to each chip and let it react for 10 minutes.
13. Add 450ul of XC3, add one more time after 1 minute, and react for 5 minutes.
14. Add 250 ul of LTM to each chip and let it react for 10 minutes.
15. Add 450ul of XC3, add one more time after 1 minute, and react for 5 minutes.
Add 250ul of ATM to each chip and let it react for 10 minutes.
17. Add 450ul of XC3, add one more time after 1 minute, and react for 5 minutes.
18. Add 250 ul of LTM to each chip and react for 10 minutes.
19. Add 450ul of XC3, add one more time after 1 minute, and react for 5 minutes.
20. At the end of this process, immediately remove the chamber rack from the flow-through chamber, move it to a room temperature laboratory table and place it flat.
21. Place 310 ml of PB1 in the wash container and immerse the dyeing rack in the container.
22. Using a tool, remove the clips from the chamber rack, lift the glass block, and remove the space on both ends of the bead of chips.
23. Once all attachments on the chip have been removed, immerse them in PB1 of the staining rack contained in PB1. Process all chips in the same way.
24. Slowly douse the chip by moving the dye rack up or down about 10 times and immerse for 5 minutes.
25. Fill 310 ml of XC4 in another flushing vessel, quench 10 times in the same manner as 24, and soak for 5 minutes.
26. After 5 minutes, remove the dyeing rack from the cleaning container and place it on the tube rack as shown in the following figure.
27. Using the forceps, carefully remove the chips from the rack and place them on the tube rack.
28. Carefully put the tube rack on the Chips into a vacuum dryer and allow to dry for 55-55 minutes under a vacuum of 508 mm Hg (0.68 bar).
29. Once the chips are dried, use a kim wipe moistened with ethanol to close the edges of the chip. Be careful not to touch the bead.
30. Beadchips allows you to image with a Scanner within 72 hours after completion of the experiment.
Table 1 shows the SNPs of 18 dogs predictable for their feeding capacities through high density SNP 170K chip analysis for 50 dogs.
The present invention can be used as a genetic marker which can select 18 SNPs significantly correlated with the feeding ability of dogs through high density SNP 170K chip analysis and enable early prediction of dog feeding ability by genotype analysis using these SNPs. The expected effect is to satisfy the dogs' preference for the feeding ability of dogs. By restricting indiscriminate breeding to produce dogs with high or low ability to feed dogs, By discovering genes related to feeding ability of dogs, academic value and basic medical information on animal growth can be obtained.
While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.
<110> RURAL DEVELOPMENT ADMINISTRATION <120> SNP marker for prediction of dog's food consumption and prediction method using the same <130> NPF-28993 <160> 18 <170> PatentIn version 3.2 <210> 1 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 1 cctcctgccc tgctaccact gatgacccag ggctggacga ctaaccccaa ctgaacccac 60 agcttctctc tcctgggaat ttggaattag aacaaggact gttgtgggat gggaggctgt 120 g 121 <210> 2 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 2 cacatgtcct cactgagaac ctcccagcac ctgccgccct acatgccacc tggtcactgt 60 aagctctgct tgttctcgaa gagctgcttt tatgtgcagg taggtagccc tgctgtcctg 120 g 121 <210> 3 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 3 ccatctcctt tgggcctctt gtccccctga catccccagt tgtaacatgt gaacatttgc 60 ataggaccag cggtgtcccc ccaagaagcc cctcatccca ccagacaagg gcacctcctc 120 t 121 <210> 4 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 4 acagctcctg tcactgtcac tattaccagg agggcactcc tgggttatgg ggtacttctg 60 aactttgggt gaggccacaa agagagggag gacttgggtc ttagattatt aggtttgagc 120 c 121 <210> 5 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 5 tgttctctgg tttggaagtt taagttatat atttgttaag taatgagcag catttagtaa 60 aagataaagt tactatatag tttatatgcc tattgcaacc acagaatatt tgcttttcca 120 t 121 <210> 6 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 6 tcagatgttc ccgagtcaga ccacacaaag ttgggagtga tttagcatga aawaaaggaa 60 aggggatgga ttctacattc actttatttc tccttacggt acaacttaac tgggagatgt 120 t 121 <210> 7 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 7 cccctccaca tggtccctat acctgccctt ctgctctcac trgcccctcc ttgccctgcc 60 aggcttacag tcaactgttg agcgatgact ttccttcttg atgaaggttt ctgcctcctt 120 t 121 <210> 8 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 8 aacccctctg tttctctact ttgaaatgtc tgtccccatg ttcttttatg gtttagtgtc 60 aagagtttgg gcttgggaat cagtgcttag cacagcgctt aaagcatgta tatgtgagct 120 g 121 <210> 9 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 9 gatgctgagg gcaaagtaca cagctgtgag gatgatggtc cctttgaact tgtggaatag 60 aaggttgacc aagccagcct ggaagacgaa ggtgttgaag aacatgagga aaatgatgat 120 g 121 <210> 10 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 10 gccttgtccc caagaagcat ttggaatgtg ctgtgagaac aggcaatcgg ccctctccac 60 agtcaggatc ctccctggac actgcggcca gccttttgaa gaaggtctgg gccactgagg 120 a 121 <210> 11 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 11 gcttacagaa tccctctgac aaggatcaag tccatgcgag aaaacctcag ggagaaggac 60 aagctgaaag attatctaga gaagcatcct tacaacctgg cctacaagtt tgttgagtct 120 g 121 <210> 12 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 12 ttcttttgaa atgtgttggt atccctcccc agtgagaata caagactatc ctctcatggc 60 acattctgaa aatcctggtg ctcagtacac ttcctagggc atggcagggc tgagggccat 120 g 121 <210> 13 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 13 aattctgaaa tcagtgagct ggagcagctc caccagccgg tccaactgga cagccaccag 60 aaccacttct aacagaggct cacagaagac aaactgtgac agtggaaaga actggcgaat 120 t 121 <210> 14 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 14 aagcaaagga cctgacggca aaagtcaggt tatgggtttt ggttcaggga atgattccgg 60 aatggagctg cagaggagac ccctcagaca gggwttatct cacatgccat tttctgttgt 120 t 121 <210> 15 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 15 acattcgccc ctttaaatca ttggtgattt tgagggcatt ctcacatctt cccccatgaa 60 attgttgcat atggttcact cttttgaagt tcaattcagc tcagcaaaca cttcctgaag 120 c 121 <210> 16 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 16 ctcactctta ccagattctt acaggcttct cttacattac tcaggagcat tattaatcac 60 attaaatgaa taaatcactg catcttaaca ctagaaagca tacttcattc actatgtgaa 120 a 121 <210> 17 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 17 ccccacatgc tcaagttcta tggtattttc actccatcta actaagaaat gtgaatcaac 60 aacaaatgct cactagcaga atgttcactc tgtctctttg cacaggagaa acaagaaacc 120 c 121 <210> 18 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 18 ctgtgcccca cgtgccttac acaggaaggc accagcaccc catttaatga ggggagcccc 60 attttagagg cccagatggt tgctggctgc agttcaggta ggcgcctcct gcatgtgtgg 120 g 121
Claims (7)
(b) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide shown in SEQ ID NO: 2 and the 61rd base is the internal base sequence of SEQ ID NO: 2, or Complementary polynucleotides thereof;
(c) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 3 and the 61rd base is the base sequence of SEQ ID NO: 3, or Complementary polynucleotides thereof;
(d) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 4 and the 61rd base is the base sequence of SEQ ID NO: 4; or Complementary polynucleotides thereof;
(e) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or C in the polynucleotide of SEQ ID NO: 5 and the 61rd base is the base sequence of SEQ ID NO: 5, or Complementary polynucleotides thereof;
(f) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 6 and the 61rd base is the internal base sequence of SEQ ID NO: 6; or Complementary polynucleotides thereof;
(g) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 7, and the 61rd base is the internal base sequence of SEQ ID NO: 7, or Complementary polynucleotides thereof;
(h) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide of SEQ ID NO: 8 and the 61rd base is the base sequence of SEQ ID NO: 8, or Complementary polynucleotides thereof;
(i) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 9 and the 61rd base is the internal base sequence of SEQ ID NO: 9, or Complementary polynucleotides thereof;
(j) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 10 and the 61rd base is the internal base sequence of SEQ ID NO: 10; or Complementary polynucleotides thereof;
(k) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 11 and the 61rd base is the internal sequence of SEQ ID NO: 11; or Complementary polynucleotides thereof;
(l) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 12 and the 61rd base is the base sequence of SEQ ID NO: 12; or Complementary polynucleotides thereof;
(m) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 13, and the 61rd base is the internal nucleotide sequence of SEQ ID NO: 13; or Complementary polynucleotides thereof;
(n) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 14 and the 61rd base is the base sequence of SEQ ID NO: 14; or Complementary polynucleotides thereof;
(o) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 15 and the 61rd base is the internal sequence of SEQ ID NO: 15; or Complementary polynucleotides thereof;
(p) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 16 and the 61rd base is the base sequence of SEQ ID NO: 16; or Complementary polynucleotides thereof;
(q) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide represented by SEQ ID NO: 17, and the 61rd base is the base sequence of SEQ ID NO: 17; Complementary polynucleotides thereof; And
(r) a polynucleotide consisting of 5 to 121 consecutive bases, wherein the 61st base is A or G in the polynucleotide shown in SEQ ID NO: 18 and the 61rd base is the base sequence of SEQ ID NO: 18; A complementary polynucleotide of SEQ ID NO: 2, and a complementary polynucleotide thereof.
And a formulation capable of detecting or amplifying the SNP markers for predicting the feedability of the dog.
b) identifying the base type of the SNP corresponding to the 61st base of the polynucleotide of any one of SEQ ID NOS: 1 to 18 in the separated nucleic acid molecule.
Wherein the isolated nucleic acid molecule is amplified in step b).
When the 61st base in the polynucleotide represented by SEQ ID NO: 1 is A;
When the 61st base in the polynucleotide represented by SEQ ID NO: 2 is C;
When the 61st base in the polynucleotide represented by SEQ ID NO: 3 is A;
When the 61st base in the polynucleotide represented by SEQ ID NO: 4 is A;
The polynucleotide of SEQ ID NO: 5 has the 61st base G;
When the 61st base in the polynucleotide of SEQ ID NO: 6 is A;
When the 61st base is G in the polynucleotide represented by SEQ ID NO: 7;
When the 61st base is G in the polynucleotide represented by SEQ ID NO: 8;
The polynucleotide of SEQ ID NO: 9 has the 61st base of G;
When the 61st base is G in the polynucleotide represented by SEQ ID NO: 10;
When the 61st base is G in the polynucleotide shown by SEQ ID NO: 11;
When the 61st base in the polynucleotide represented by SEQ ID NO: 12 is A;
When the 61st base of the polynucleotide represented by SEQ ID NO: 13 is A;
When the 61st base in the polynucleotide represented by SEQ ID NO: 14 is A;
When the 61st base in the polynucleotide shown in SEQ ID NO: 15 is A;
When the 61st base is G in the polynucleotide represented by SEQ ID NO: 16;
When the 61st base is G in the polynucleotide represented by SEQ ID NO: 17; And
When the 61st base in the polynucleotide represented by SEQ ID NO: 18 is A;
Wherein the at least one selected from the group consisting of: < RTI ID = 0.0 > a < / RTI >
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150159303A KR101823355B1 (en) | 2015-11-13 | 2015-11-13 | SNP marker for prediction of dog's food consumption and prediction method using the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020150159303A KR101823355B1 (en) | 2015-11-13 | 2015-11-13 | SNP marker for prediction of dog's food consumption and prediction method using the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| KR20170056726A true KR20170056726A (en) | 2017-05-24 |
| KR101823355B1 KR101823355B1 (en) | 2018-03-15 |
Family
ID=59051699
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| KR1020150159303A KR101823355B1 (en) | 2015-11-13 | 2015-11-13 | SNP marker for prediction of dog's food consumption and prediction method using the same |
Country Status (1)
| Country | Link |
|---|---|
| KR (1) | KR101823355B1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102185440B1 (en) * | 2019-10-01 | 2020-12-01 | 대한민국 | Composition for early predicting or diagnosing hypercholesterolemia in dog |
| KR102194878B1 (en) * | 2019-10-08 | 2020-12-23 | 대한민국 | Composition for early predicting or diagnosing mental stability in dog |
| KR102194881B1 (en) * | 2019-10-11 | 2020-12-24 | 대한민국 | SNP marker for prediction of dog's obesity and prediction method using the same |
-
2015
- 2015-11-13 KR KR1020150159303A patent/KR101823355B1/en active IP Right Grant
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102185440B1 (en) * | 2019-10-01 | 2020-12-01 | 대한민국 | Composition for early predicting or diagnosing hypercholesterolemia in dog |
| KR102194878B1 (en) * | 2019-10-08 | 2020-12-23 | 대한민국 | Composition for early predicting or diagnosing mental stability in dog |
| KR102194881B1 (en) * | 2019-10-11 | 2020-12-24 | 대한민국 | SNP marker for prediction of dog's obesity and prediction method using the same |
Also Published As
| Publication number | Publication date |
|---|---|
| KR101823355B1 (en) | 2018-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR101816608B1 (en) | SNP marker for prediction of dog's body weight and prediction method using the same | |
| JP2009505651A (en) | Method for detection of microbial and antibiotic resistance markers and nucleic acid oligonucleotides therefor | |
| KR101823355B1 (en) | SNP marker for prediction of dog's food consumption and prediction method using the same | |
| JP2007530026A (en) | Nucleic acid sequencing | |
| JP2016520326A (en) | Molecular bar coding for multiplex sequencing | |
| KR20180034868A (en) | SNP Markers in PLCz gene for Discriminating Quality of Pig Frozen Semen and Their Uses | |
| KR101823361B1 (en) | SNP marker for prediction of dog's chest depth and prediction method using the same | |
| KR101796218B1 (en) | SNP marker for prediction of dog's body length and prediction method using the same | |
| KR101816610B1 (en) | SNP marker for prediction of dog's body height and prediction method using the same | |
| KR101353083B1 (en) | SNP markers and methods for highly fetile pig | |
| KR101891557B1 (en) | SNP markers for prediction of low birth weight pigs size and methods for prediction of low birth weight pigs size using the same | |
| KR101890350B1 (en) | SNP maker for predicting meat quality of pig and use thereof | |
| KR20170055763A (en) | SNP Markers in ESR gene for Discriminating Quality of Pig Frozen Semen and Their Uses | |
| US20110276525A1 (en) | Reference markers for biological samples | |
| KR101960427B1 (en) | SNP marker for prediction of dog's tail shape and prediction method using the same | |
| KR101823351B1 (en) | SNP marker for prediction of dog's auditory ability and prediction method using the same | |
| KR101823352B1 (en) | SNP marker for prediction of dog's olfactory ability and prediction method using the same | |
| KR101843432B1 (en) | Composition comprising SNP genetic marker of cow mitochondria DNA for discriminating cow breed and cow discrimination method using the same | |
| KR101821552B1 (en) | SNP marker for prediction of pig's nipple number and method for prediction of highly fertile pig using the same | |
| KR101385764B1 (en) | Markers for Dog hair and diagnosing method using the same | |
| KR101766273B1 (en) | Composition for Breed Discrimination of Dog comprising mitochondria SNP Markers and Diagnosing Method using the Same | |
| KR101855666B1 (en) | SNP markers for prediction of pigs total litter size and methods for prediction of pigs total litter size using the same | |
| KR101855665B1 (en) | SNP markers for prediction of pigs weaning number and methods for prediction of pigs weaning number using the same | |
| KR101845711B1 (en) | SNP marker from OCIAD2 gene for identificating genomic impriting and method for identificating genomic impriting using the same | |
| KR102019993B1 (en) | Composition for discrimination of sex and breed for cattle, and discriminating method using the same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A201 | Request for examination | ||
| E902 | Notification of reason for refusal | ||
| E701 | Decision to grant or registration of patent right | ||
| GRNT | Written decision to grant |