RU2528743C1 - Method of carrying out pcr-rflp for genotyping cattle on alleles a and k of gene dgat1 - Google Patents

Abstract

FIELD: veterinary medicine.

SUBSTANCE: method of carrying out PCR-RFLP for genotyping cattle on alleles A and K of gene DGAT1, which is different from the nearest prototype [1] in that at the stage of PCR the other sequences of the oligonucleotide primers: DGAT1-1: 5'- CCGCTTGCTCGTAGCTTTCGAAGGTAACGC-3' (SEQ ID NO:1): DGAT1-2: 5'-CCGCTTGCTCGTAGCTTTGGCAGGTAACAA-3' (SEQ ID NO:2): DGAT1-3: 5'-AGGATCCTCACCGCGGTAGGTCAGG-3' (SEQ ID NO:3) are used, and at the stage of RFLP the other restriction endonuclease - TaqI is used, with the generation of the genotype-specific fragments: the genotype AA=82/18 bp, the genotype KK=100 bp and the genotype AA=100/82/18 bp (Figures 1 and 2).

EFFECT: development of an efficient method of genotyping the cattle on DGAT1-gene based on PCR-RFLP analysis.

2 dwg, 2 tbl

Description

The invention relates to the field of genetics and breeding of farm animals, in particular to the assessment of allelic polymorphism of the DGAT1 gene (dacylglycerol O-acyltransferase 1) of cattle by molecular genetic research method.

There are various methods for PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) for genotyping cattle by the A and K alleles of the DGAT1 gene.

So, in the method proposed by J. Komisarek (2008) [1], primers F: 5 ^/ -TGCCGCTTGCTCGTAGCTTTTGGCC-3 ^/ and R: 5 ^/ -ACCTGGAGCTGGGTGAGGAACAGC-3 ^{/ are used} , which initiate the amplification of the DGAT1-gene locus with a length of 378 with a length of 378 of the b8 gene with a length of b8 by its endonuclease cleavage with the restriction enzyme BglI to generate and interpret the resulting genotype-specific fragments (genotype AA = 254/96/28 bp, genotype KK = 282/96 bp and genotype AK = 282/254/96/28 bp).

The purpose of the invention is the development of an effective method for genotyping cattle by the DGAT1 gene based on PCR-RFLP analysis.

We have developed a method for PCR-RFLP for genotyping cattle by the A and K alleles of the DGAT1 gene, which differs from the nearest prototype [1] in that the primers are used at the PCR stage:

and at the RFLP stage, the restriction endonuclease TaqI is used, with the generation of genotype-specific fragments: genotype AA = 82/18 bp, genotype KK = 100 bp and genotype AK = 100/82/18 bp (Figs. 1 and 2).

Reaction conditions

Nucleic acid was extracted from whole cattle blood samples, preserved with 10 mM EDTA-Na2, by the sorption method (DNA Sorb B, TsNIIEM, Russia).

PCR-RFLP was performed according to the protocol presented in table 1.

The detection of PCR-RFLP results was carried out by horizontal electrophoresis in 3% agarose gel in TBE buffer (pH 8.0) containing ethidium bromide at a concentration of 0.5 μg / ml, followed by visualization of amplified products in an ultraviolet transilluminator (λ = 310 nm) .

The sizes of DNA fragments were evaluated by mobility in comparison with standard DNA markers.

In the work, products were used for molecular biological studies of the production of SibEnzyme LLC, Russia.

Conclusion

According to the results of practical studies aimed at testing the method we developed for carrying out PCR-RFLP for genotyping cattle by the A and K alleles of the DGAT1 gene, we obtained the technical result provided by the claimed method, expressed in the effective identification of the desired genotypes (AA, KK, AK) in view of correct interpretation of the generated genotype-specific fragments (AA = 82/18 bp, KK = 100 bp and AK = 100/82/18 bp), where PCR-RFLP fragments with lengths of 100 bp and 82 bp are identification, and minor RFLP- 18 bp fragment - non-infor ativnost, because of its small size, it does not significantly reduce the accuracy of DNA analysis.

Information sources

1. Komisarek, J.A relationship between DGAT1 K232A polymporphism and selected reproductive traits in Polish Holstein-Friesian cattle / J. Komisarek, A, Michalak // Animal Science Papers and Reports. - 2008. - V.26. - N.2. - P.89-95.

Table 1 Protocol PCR-RFLP for genotyping on the A and K alleles of the DGAT1 gene PCR protocol Reagents Initial concentration Working concentration 1 sample (μl) 10 samples (μl) dH ₂ O 13.4 134 DMSO one hundred% 5% one 10 dNTP 2.5 mm 0.25 mm 2 twenty Buffer 10 × 1 × 2 twenty Taq DNA Polymerase 5 units 1 unit 0.2 2 SEQ ID NO: 1 50 μM 0.25 μM 0.1 one SEQ ID NO: 2 50 μM 0.25 μM 0.1 one SEQ ID NO: 3 50 μM 0.5 μM 0.2 2 DNA sample one TOTAL twenty

Primers

Amplification Mode

× 1: 94 ° C - 4 min

× 40: 94 ° C - 10 sec., 72 ° C - 10 sec.

× 1: 72 ° C - 5 min

STORAGE + 10 ° C

PCR product = 100 bp

RFLP Protocol

Reagents Initial concentration Working concentration 1 sample 10 samples dH ₂ O 1.25 12.5 BSA 10 mg / ml 0.1 mg / ml 0.25 2,5 SE buffer Y 10 × 1 × 2,5 25 Taqi 20 U 20 U one 10 PCR assay twenty TOTAL 25

Incubation at 65 ° C overnight

PCR-PDFR fragments:

Genotype AA = 82/18 bp

Genotype KK = 100 bp

Genotype AK = 100/82/18 bp

Electrophoresis in 3% agarose

A brief description of the graphic materials

Explanation of figure 1 - Flanked sections of the primers used for genotyping cattle by alleles A and K of the DGAT1 gene by the proposed method for PCR-RFLP.

Explanation of figure 2 - Electrophoregram of the technical result of the proposed method for PCR-RFLP for genotyping of cattle by alleles A and K of the gene DGAT1.

Designations:

M) DNA markers 100 bp + 50 bp (SibEnzyme).

1-4, 6, 8-11, 13, 15, 17-18) AK genotype of the DGAT1 gene

5, 16) AA genotype of the DGAT1 gene

7, 12. 14) KK genotype of the DGAT1 gene

Claims (1)

The method of PCR-RFLP for genotyping of cattle by alleles A and K of the DGAT1 gene, characterized in that the primers are used at the PCR stage:

and at the RFLP stage, the restriction endonuclease TaqI is used, with the generation of genotype-specific fragments: genotype AA = 82/18 bp, genotype KK = 100 bp and genotype AK = 100/82/18 bp.

Images