KR101823351B1

KR101823351B1 - SNP marker for prediction of dog's auditory ability and prediction method using the same

Info

Publication number: KR101823351B1
Application number: KR1020150159306A
Authority: KR
Inventors: 최봉환; 박종은; 임다정
Original assignee: 대한민국
Priority date: 2015-11-13
Filing date: 2015-11-13
Publication date: 2018-03-15
Also published as: KR20170056727A

Abstract

The present invention relates to SNP markers for predicting hearing ability of dogs and their use, and more specifically, the present invention relates to a SNP marker composition capable of predicting dog's hearing ability, A kit, a microarray, and a method for predicting hearing ability of a dog. Utilizing the SNP marker of the present invention, it is possible to quickly and accurately predict the hearing ability of the dog.

Description

SNP markers for predicting hearing ability and methods for predicting SNP markers using the same

The present invention relates to SNP markers for predicting the hearing ability of dogs and a method for predicting the hearing ability of dogs using the SNP markers.

Special-purpose dogs have auditory ability up to 100,000 times that of humans.

The hearing ability of special purpose dogs is related to the auditory cognitive ability as a special purpose dog in dogs or search dogs because of their individual differences, while the handler training special purpose dogs and dog breeders who give birth It is recognized as a phenotypic phenotype that is difficult to improve and 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).

Using the gene markers of the auditory ability, it is possible to meet the preferences of the special purpose observation institutions for the dog's hearing ability, to maximize the improvement effect on the dog's hearing ability, to improve the special purpose dog acceptance rate, It is anticipated that by digging out the genes, it will be possible to acquire the academic value and industrial basic information on the auditory-related genes of animals.

Under these technical backgrounds, the inventors have made intensive efforts to develop gene markers for early diagnosis by identifying genes related to the auditory ability of dogs.

Accordingly, it is an object of the present invention to provide a SNP marker composition for predicting the hearing ability of dogs.

It is another object of the present invention to provide a method for predicting the performance of dogs using SNP markers in an early stage.

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 G 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 G in the polynucleotide shown in SEQ ID NO: 3 and the 61rd base is the internal 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 T in the polynucleotide shown in SEQ ID NO: 5 and the 61rd base is an internal 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; (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; Complementary polynucleotides thereof; (s) 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: 19 and the 61rd base is the base sequence of SEQ ID NO: 19; or Complementary polynucleotides thereof; (t) 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: 20 and the 61rd base is the internal base sequence of SEQ ID NO: 20, or Complementary polynucleotides thereof; (u) 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: 21 and the 61rd base is the internal base sequence of SEQ ID NO: 21, or Complementary polynucleotides thereof; (v) 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: 22 and the 61rd base is the internal base sequence of SEQ ID NO: 22, or Complementary polynucleotides thereof; And (w) 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: 23 and the 61rd base is an internal base sequence of SEQ ID NO: 23 Or a complementary polynucleotide thereof. The present invention also provides SNP marker compositions for predicting hearing ability.

According to another aspect of the present invention, there is provided a kit for predicting hearing ability of a dog including the composition.

According to another aspect of the present invention, there is provided a microarray for predicting hearing ability of a dog including 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 23 in the separated nucleic acid molecule.

According to an embodiment of the present invention, it is possible to predict the hearing ability of the dog early, and it is possible to satisfy the preference of special purpose observation institutions for the hearing ability.

In addition, it is possible to maximize the improvement effect on the hearing ability and improve the special purpose dog acceptance rate.

Furthermore, it is anticipated that it will be possible to acquire academic value and industrial basic information on the auditory-related genes of animals by identifying genes related to the auditory ability of 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 G 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 G in the polynucleotide shown in SEQ ID NO: 3 and the 61rd base is the internal 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 T in the polynucleotide shown in SEQ ID NO: 5, and the 61rd base is the internal nucleotide 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; 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; (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; Complementary polynucleotides thereof; (s) 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: 19 and the 61rd base is the base sequence of SEQ ID NO: 19; or Complementary polynucleotides thereof; (t) 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: 20 and the 61rd base is the internal base sequence of SEQ ID NO: 20, or Complementary polynucleotides thereof; (u) 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: 21 and the 61rd base is the internal base sequence of SEQ ID NO: 21, or Complementary polynucleotides thereof; (v) 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: 22 and the 61rd base is the internal base sequence of SEQ ID NO: 22, or Complementary polynucleotides thereof; And (w) 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: 23 and the 61rd base is the internal base sequence of SEQ ID NO: 23 Or a complementary polynucleotide thereof. The present invention also provides SNP marker compositions for predicting hearing ability.

According to one embodiment of the present invention, the composition comprises an agent capable of detecting or amplifying the SNP marker.

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.

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 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 an embodiment of the present invention, when the base type of the SNP is identified from the separated nucleic acid molecule, the polynucleotide of SEQ ID NO: 1 and the 61st base are G; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 2; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 3; When the 61st base in the polynucleotide represented by SEQ ID NO: 4 is A; When the 61st base is T in the polynucleotide represented by SEQ ID NO: 5; 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; When the 61st base is A in the polynucleotide represented by SEQ ID NO: 9; When the 61st base in the polynucleotide of SEQ ID NO: 10 is A; When the 61st base is G in the polynucleotide shown by SEQ ID NO: 11; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 12; The polynucleotide of SEQ ID NO: 13 is G at the 61st base; When the 61st base in the polynucleotide represented by SEQ ID NO: 14 is A; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 15; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 16; When the 61st base in the polynucleotide represented by SEQ ID NO: 17 is A; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 18; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 19; When the 61st base in the polynucleotide represented by SEQ ID NO: 20 is A; When the 61st base is A in the polynucleotide represented by SEQ ID NO: 21; When the 61st base is G in the polynucleotide represented by SEQ ID NO: 22; And a polynucleotide of SEQ ID NO: 23 is A at position 61, the present invention provides a method for determining that the hearing 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.

□ How to measure and score expressive traits on hearing ability

To measure auditory cognitive abilities, the dogs were seated and allowed to stand, and then the dogs were divided into 4 groups according to the degree of the reaction by repeating two low-frequency clippers and one intermediate clippers at the back of the dog .

1 point: When all three clicks are heard

2 points: When you respond to all 2 clicks

3 points: When all the clicks are heard

4 points: If you do not respond to the click sound

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).

&Lt; 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.

A total of 23 predictable SNPs were selected for each of the 50 hearing aids through high-density SNP 170K chip analysis. The information on these is shown in Table 1.

The present invention can be used as a genetic marker for early identification of the dog's hearing ability by selecting 23 SNPs that are significantly associated with the auditory ability through high-density SNP 170K chip analysis and genotyping using these SNPs. As expected effects, it can satisfy the preferences of special purpose observation institutions for the hearing ability of dogs. By restricting indiscriminate breeding to produce individuals according to their hearing ability, they can contribute to the protection welfare policies of special purpose dogs, It is possible to establish an academic value and medical basic information about the growth of an animal by discovering related genes for the ability of the hearing ability of dogs.

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 auditory ability and prediction method using the same <130> NPF-28995 <160> 23 <170> PatentIn version 3.2 <210> 1 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 1 aagactgttt ttctctcagc ctacccccca caaaaatcaa agcaaaccaa gagtaccatc 60 acatatacac aaaagagccc tgcctcccag ttgstgttca agtgagccct gctagctatt 120 c 121 <210> 2 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 2 acagtcacat attagacaca gtctttagtc cattccattg tacctaaata tttaagaatt 60 acccatacaa aaagaatcag ggagctggtg gtgtttgtag agaggatgac agtgacaatg 120 a 121 <210> 3 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 3 cctcatctct taagtaaatt tttggtgata agaattccaa acacctatac agctctattt 60 agaatacatg gacttctgac tggactcagg ataaccatct actacactta aaaaaaaaag 120 g 121 <210> 4 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 4 agtcttttgg ctctttaatt tgggaagcag atgcttgatt gcttgtttaa agactgtgta 60 aaaagcccag cctagccttt cgaaagctca aaatattctt aagcagggat gtcaattaca 120 t 121 <210> 5 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 5 sacacaccaa gggaatgttg gataaagaaa agttggaaat actgatattg ctggaaaata 60 agactctcaa ctcagtacat agggttgaaa aaggcttgga ttaccccaga cttgagcaag 120 c 121 <210> 6 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 6 ctcaaactct ttgtggaaaa ctctgcttat gggaagattg taaacctgga cctttcaggg 60 atctgaaggg cccatggagt tggtaccagg tctccaaacc aggaaggtta atgataatatt 120 t 121 <210> 7 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 7 tgctctgtta attgcagtca ttttttccag aactctgctc ttcagcagcc tttgggggat 60 aactgacttc cttacccaga gccaagccag agaacaacac actgttaaca ctgtacaaca 120 a 121 <210> 8 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 8 cagaatccaa aggagagaaa ccctgttgcc tcgataagag gtacacaaaa gaagtcagat 60 atgaaggagc aaggaggtca tgggtcagtt atcatttaag acccaggcta aatgctggga 120 t 121 <210> 9 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 9 attttttttg gcttctctty cgtagcaggg atgggatgaa acatgcgtac aagcccgtct 60 aactctctag acgttaccta ggtctgccac acagagcacg ggccaccttt trtaagctac 120 g 121 <210> 10 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 10 ttgacttaac atgttgcttt tcacatttgg ggatcaatga agttggaaaa tctcagaggc 60 attactagct tgtggaaagg cactccactc agatggactg twggcctttg tcatgtgtga 120 t 121 <210> 11 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 11 tgccttaaaa actttttaga aggtatactg ctctaaacac gtttccatta tgcacagagt 60 acatttcctc tttgctgtct tgcctcagca acttaacaca ggtagactcc tatttataat 120 a 121 <210> 12 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 12 tgttctaaac aaaggatttt gtaattggtt ctacttaaga aggatataac aagtgactct 60 agggaagatg aggaggaggg ttgcaggaat ctataacaat tctcttgtgc ccttccattc 120 a 121 <210> 13 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 13 ataagtacag gacttgggaa atcttttcaa ttactttgta gctagagaag ctagtgtgag 60 aaaatgtctt gaaagaggaa gccattcagg gccttagtct tgaacattga gtctggaact 120 g 121 <210> 14 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 14 taacattact gaaaagctga ttggaaaaaa gcagctcctg taccacccat gcaaaatatc 60 agccaacaca tctggatgtt ttccctaccc tcactgtatc agttccatga ggcagactaa 120 t 121 <210> 15 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 15 ctcccaaatg acagtaaatg acaccaccac ttacacrgtt gttcaggctc agtaaatgac 60 accaccactt atgcagttgt tcaggctgat agattggtca ggtaaccaat tatctatacc 120 t 121 <210> 16 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 16 cctaatgtcc aacatttccc aaataatcta agtactctaa ccttttagcc ctcacccgac 60 atgcctactc actctaatca tctctgctct ctgcagtctt ccaagtcctg tctcctttac 120 t 121 <210> 17 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 17 gtgtactcaa ttggctttgc tcaagcgttc attatctcta catgggcttc caagtttgca 60 acttcctcaa acccatcttt catgcagcct ccagatggaa ttaaaactcc acttgcttaa 120 a 121 <210> 18 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 18 gaaaaggatg tgaagacacc agaagtagaa tagttttcat acctagatat atttttgtaa 60 agatttttgg ccctttgata taatttgtcc tatttcataa tagttggtga tgtttcactg 120 a 121 <210> 19 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 19 tttgcaagta tttaaacaga tccaccacca cttcccccct ccaagtgatg aggttagtag 60 acaggatttt aatggacaac agttctgcag attttgtttt aatcttattg aactagcaat 120 a 121 <210> 20 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 20 taaaaggttt aattcacatc ctgtttctaa ccaatggtga cctaaacatc ttgccatttt 60 agacttgcat ttttatcgtt tgtgtattag aggaaataat ttttgtcttg tttcctcaaa 120 g 121 <210> 21 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 21 aactattata gggatggtat attcctaaat agctaccttt ctttgaaaga ttacctctcc 60 atttatcatt tagaggatgc atcttcttaa tgcacagttg ttattagtat caagaatggg 120 t 121 <210> 22 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 22 gaagaactca ccctcatttc tagtttgttc caaccaataa ctctcttcca catggactga 60 atccactgag ttttggtcac tcttccatga caggcaagga gaagcttctc atttagagtg 120 a 121 <210> 23 <211> 121 <212> DNA <213> Canis lupus familiaris <400> 23 aactattata gggatggtat attcctaaat agctaccttt ctttgaaaga ttacctctcc 60 atttatcatt tagaggatgc atcttcttaa tgcacagttg ttattagtat caagaatggg 120 t 121

Claims

(a) 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: 1 and the 61rd base is the base sequence of SEQ ID NO: 1; or Complementary polynucleotides thereof;
(b) 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: 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 G in the polynucleotide shown in SEQ ID NO: 3 and the 61rd base is the internal 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 T in the polynucleotide shown in SEQ ID NO: 5, and the 61rd base is the internal nucleotide 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; 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;
(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; Complementary polynucleotides thereof;
(s) 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: 19 and the 61rd base is the base sequence of SEQ ID NO: 19; or Complementary polynucleotides thereof;
(t) 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: 20 and the 61rd base is the internal base sequence of SEQ ID NO: 20, or Complementary polynucleotides thereof;
(u) 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: 21 and the 61rd base is the internal base sequence of SEQ ID NO: 21, or Complementary polynucleotides thereof;
(v) 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: 22, and the 61rd base is the internal base sequence of SEQ ID NO: 22; or Complementary polynucleotides thereof; And
(w) 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: 23 and the 61rd base is the base sequence of SEQ ID NO: 23, or A complementary polynucleotide of SEQ ID NO: 2, and a complementary polynucleotide thereof.

The method according to claim 1,
And an agent capable of detecting or amplifying the SNP marker for predicting the hearing ability of the dog.

A kit for predicting hearing ability in dogs comprising the composition according to claim 1 or 2.

A microarray for predicting hearing ability of a dog comprising the composition according to any one of claims 1 and 2.

a) separating the nucleic acid molecule from the 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 23 in the separated nucleic acid molecule.

6. The method of claim 5,
Wherein said step b) amplifies and identifies said isolated nucleic acid molecule.

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