CZ34779U1 - Goat polymorphism (SNPs) detection kit in exon 7 and 9 of the beta casein gene (CSN2) in goats - Google Patents

Description

Kit for detection of polymorphism in exon 7 and 9 of the beta casein gene in goats

Field of technology

The solution concerns a multiplex kit for the detection of genetic polymorphisms (SNPs) in exons 7 and 9 of the beta casein gene (CSN2) in goats by the so-called mini-sequencing method using an automatic capillary sequencer.

Prior art

The main protein fraction in goat's milk is caseins, representing up to 80% of the total protein content in milk. The most abundant casein fraction in goat's milk is beta casein (CSN2), on average it contains about 10.6 g / l, which corresponds to about 50 to 55% of the total milk protein content. CSN2 is a key protein for milk coagulation and curd strength, so it fundamentally affects the technological conditions in milk processing. The genetic polymorphism of CSN2 is characterized by extensive variability, currently 9 alleles are described in goats: A, Al, B, C, Cl, D, E and two so-called "zero alleles" 0 and 0 '. The null alleles are characterized by mutations responsible for the premature stop codon (in the region of exon 7, at positions 8561 and 8915) and are associated with an undetectable amount of this protein in milk. Alleles A, Al, C, Cl and E are described at the DNA level and differ by nucleotide substitutions at positions 8561bp, 8913bp, 8915bp, 8946bp and 10562bp (GeneBank AJ011018.3). In contrast, alleles B and D are detected only at the protein level, i.e. they differ with a change in protein structure.

To detect genetic variants of the CSN2 gene, especially in the region of exon 7, the following techniques have been used so far: Sanger sequencing, SSCP, PCR - RFLP, TaqMan probes, etc. These are techniques commonly used in molecular genetics, but they are often technically demanding or require significant initial investment in equipment (SSCP, DGGE, DHPLC). The recently used KASPar method (based on the principle of FRET probes) is not suitable for change for multiplex PCR analysis, ie simultaneous analysis of multiple SNPs in one reaction. Genetic polymorphism of milk protein genes significantly affects the quantitative and technological properties of milk, and therefore new possibilities for the detection of these genetic variants are still being sought.

The essence of the technical solution

The above-mentioned shortcomings of the detection methods used so far are eliminated by a set of primers for the detection of polymorphism in exon 7 and 9 of the beta casein gene in goats by multiplex PCR and mini-sequencing - SnaPshot, according to the technical solution, which consists in containing primer sets of composition

Forward 5 '- TCCTTCTTTCCAggATgAACTCC - 3'

Reverse 5 '- ACTTACAAgAATAgggAAgggTCC - 3' with an amplified section length of 51 Ibp in exon 7, and

Forward 5 '- ATgggggTgAgATgAAgAgT - 3'

Reverse 5 '- ACAAgAAAACTTAAATATgCCTAAggg - 3' with a length of amplified 401bp region in exon 9,

- 1 CZ 34779 UI and a set of five extension primers (SBE primers) with the following sequences in region 7 of exon

SBE1 (8561): 5'- ATATATggATgAACTCCAggATAAA - 3 ', for detection of genetic polymorphism at position - 856Ibp (A / A),

SBE2 (8913): 5 - ATATATATATATATATATATTCCgTgCTgTCCCTTT - 3 ', for detection of genetic polymorphism at position - 8913bp (C / A),

SBE3 (8915): 5 '- AggCAgAACTTTgggCT - 3', for detection of genetic polymorphism at position - 8915bp (C / T),

SBE4 (8946): 5 - ATATATATATATATATATATTATCATATCTCTCTggggCACT - 3 ', to detect genetic polymorphism at position - 8946bp (C / T), and in region 9 of exon

SBE5 (10562): 5 '- ATATATATATATATATATATATATCCATATACTTTCAAAATg TTAATTCAA - 3', to detect genetic polymorphism at position - 10562bp (C / T).

The essence of the technical solution is that the determination of individual SNPs at positions 856 Ibp A / A, 8913bp C / A, 8915bp C / T, 8946bp C / T in the region of exon 7, and 10562bp C / T in exon 9 of the CSN2 gene in goats with performed in two consecutive steps, which is based on a two-step PCR reaction and subsequent capillary electrophoresis of fluorescently labeled length fragments in a capillary sequencer (SNP fragmentation analysis based on the mini-sequencing method). The first mPCR reaction involves amplification of target regions using new forward and reverse primers according to the technical solution.

It is a multiplex classical PCR amplification reaction with exponential growth of amplificates. Purification of the amplification mixture is followed by a second PCR reaction with a set of five extensively unlabeled specific primers.

The kit according to the technical solution contains primers which are suitable for sensitive, rapid and accurate detection of SNPs in exon 7 and exon 9 of the beta casein gene (CSN2) in goats based on mPCR and subsequently SNaPshottzv. mini-sequencing.

The polymerase chain reaction (PCR) was prepared in the medium of genomic DNA, Taq DNA polymerase, primers, magnesium chloride and buffer. Amplification was performed in a standard thermocycler. The initial denaturation at high temperature is followed by several dozen temperature cycles at a temperature about 20 to 30 ° C lower than during denaturation. The quality of the PCR product and the PCR products obtained were separated by means of an agar gel electrophoretic system, and the DNA was stained with ethidium bromide. The SNaPshot reaction was prepared in an SNaPshot MultiplexReady Reaction mix, a mixture of extension primers, mPCR product and deionized water. The SNaPshot reaction takes place in a standard thermocycler. The obtained products were then analyzed on an ABI3130 Genetic Analyzer (Applied Biosystems; Foster City, CA, USA) using an E5 - Matrix Standard Set DS-02 (dRHO, dRGG, dTAMRA, dROX, LIZ) and POP7 polymer.

Detection is performed by two sets of primers according to the technical solution based on the principle of multiplex polymerase chain reaction (PCR) and subsequent mini-sequencing, which was developed by the inventors on the basis of the published sequence GeneBank AJ011018. This set of newly designed primers allows the simultaneous determination of all the above-mentioned SNPs. The set of primers and extension primers according to the technical solution enable sensitive, fast, specific and accurate

-2 CZ 34779 UI determination of five SNPs at position 8561bp, 8913bp, 8915bp, 8946bp and 10562bp (GeneBank AJ011018) in exon 7 and 9 of the beta casein gene (CSN2) in goats.

Explanation of drawings

In FIG. 1 shows electrophoretic patterns (visualizations) of PCR products in the exon 7 and 9 region of the beta casein gene (CSN2) in goats and subsequently the result of multiplex PCR (mPCR) in individual samples.

In FIG. Figures 2 to 8 show the electrophoretograms of the five SNPs detected and the resulting genotype.

Giant. 2

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: CC;

10562bp sub-genotype: CC.

The resulting genotype of CSN2 is in this case AA and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 3

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: CT;

10562bp sub-genotype: CC.

The resulting genotype of CSN2 is in this case AC and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 4

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: CT;

10562bp sub-genotype: CT.

The resulting genotype of CSN2 is in this case AC1 and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 5

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: TT;

10562bp sub-genotype: CC.

The resulting genotype of CSN2 is in this case CC and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 6

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: TT; 10562bp sub-genotype: CT.

-3 CZ 34779 UI

The resulting genotype of CSN2 is in this case CC1 and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 7

8915bp sub-genotype: CC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: TT;

10562bp sub-genotype: TT.

The resulting genotype of CSN2 is in this case C1C1 and arises from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

Giant. 8

8915bp sub-genotype: TC; 8561bp sub-genotype: AA;

8913bp sub-genotype: CC; 8946bp sub-genotype: TT;

10562bp sub-genotype: TT.

The resulting genotype of CSN2 is in this case CIO 'and results from a combination of partial genotypes of variants of a total of 5 analyzed SNPs.

The following examples of the detection of SNPs by a set according to the technical solution only illustrate the present solution, without limiting it in any way.

Examples of technical solution

Example 1

Detection of SNPs in the region of exon 7 and 9 of the beta casein gene (CSN2) in goats using two-step PCR and subsequent SNaPshot mini-sequencing was performed by the inventors in the molecular genetics laboratories of the Research Institute of Animal Production, v. I., Prague - Uhříněves, CZ

The μΐ PCR mix contained 2 μΐ genomic DNA (10 to 100 ng), 10 pM of both primers, and 1x concentrated PCR hot-start master mix (Uabmark, Czech Republic).

Amplification was performed in a standard thermocycler (Biometra, TAdvanced, BioTech, Czech Republic) according to the following program: initial denaturation was at 95 ° C for 4 min; followed by 35 cycles: 95 ° C for 60 s, 59 ° C for 60 s, 72 ° C for 60 s, with final elongation at 72 ° C for 5 min. The quality and size of the individual PCR products (51 Ibp and 401bp) and multiplex PCR (mPCR) were verified on a 3% agarose gel stained with ethidium bromide (Fig. 1). The result was visualized by UV light.

Purification of PCR reaction: The obtained mPCR product (2.5 μΐ) was subsequently purified with an enzymatic mixture (0.5 U FastAP Thermosensitive Alkaline Phosphatase and 10 U Exonuclease I (Fermentas Biogen, Czech Republic)) to remove PCR primers and unincorporated ddTPs.

Design of Sh primers: Extension (SBE) primers are designed to specifically anneal 3 'or 5' to the polymorphic site of the fragment being analyzed. To design them, we used the theoretical sequence of the PCR fragment of interest, which contained the described polymorphic sites. The primers used in response to the multilocus assay must differ significantly in length (schematically shown in Figures 2 to 3) to avoid overlapping SNaPshot products. Therefore, the length of the primers is modified at the 5 'end by the addition of non-homologous polynucleotides (eg poles dT, poles dA, poles dC, or dGACT).

-4 CZ 34779 UI

Sequencing reaction preparation: 1.5 μl of purified PCR product, 2.5 μΐ ABI PRISM SNaPshotTM Multiplex Kit - ready Reaction Mix (Applied Biosystems; Foster City, CA, USA), all primers used in the SNaPshot reaction are sufficient to prepare the SNaPshot reaction alone. they must be mixed to a final concentration of 0.05 μΜ to 1.00 μΜ for each SBE primer and water to make up the reaction volume to 5.5 μΐ. Due to the sensitivity of the chemicals, the reaction is prepared on ice.

Thermal cycling and removal of unincorporated ddNTPs after cycling: SNaPshot mini-sequencing (Biometra, TAdvanced, BioTech, CR, thermal cycler) was performed under the following conditions: 25 cycles (96 ° C for 10 s, 50 ° C for 5 s, 60 ° C for 30 s). After PCR and sequencing, purification and purification are performed. To remove PCR primers and unincorporated ddNTPs, 1 U (0.5 μΐ) of FastAP Thermosensitive Alkaline Phosphatase enzyme and 1 U (0.5 μΐ) of Exonuclease I (Fermentas, Biogen, Czech Republic) were added to the mixture and the mixture was incubated for 30 min at 37 ° C. After purification, the enzyme was subsequently inactivated by incubating at 85 ° C for 15 minutes.

Electrophoresis: A mixture of 9 μΐ Hi-Di formamide (Applied Biosystems; Foster City, CA, USA), 0.5 μΐ GeneScan-120 LIZ size standard (Applied Biosystems; Foster City, CA, USA) and 0.5 μΐ SNaPshot product. After subsequent denaturation (95 ° C for 5 min), the samples were stored on ice until analysis. Capillary gel electrophoresis was performed in an ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems; Foster City, CA, USA) using a matrix standard E5 - Matrix Standard Set DS-02 (dRl 10, dRGG, dTAMRA, dROX, LIZ) and POP7. polymer.

Data analysis: GeneMapper v. 4.0 software (Applied Biosystems; Foster City, CA, USA) was used to analyze and evaluate the data.

Industrial applicability

A new kit for the detection of polymorphisms (SNPs) in the beta casein (CSN2) gene in goats enables sensitive, accurate, fast and efficient determination of SNPs in exon 7 and exon 9. Detection of these variants is important especially for breeding dairy goat breeds for milk production traits , qualitative and technological parameters of goat 's milk.

The kit is based on single nucleotide elongation of unlabeled specific primers (SBE primers), combining the specificity of di-deoxy-nucleotide incorporation by DNA polymerase and fluorescence sensitivity. Its introduction into the laboratory saves time and money, as it allows the analysis of five SNP polymorphisms with known localization in multiple DNA templates (genes) simultaneously and is applicable even when the PCR-RFLP method cannot be used. The SNPs detection method is reliable, sensitive, fast, with well reproducible results.

Abbreviations used in the text bp - base pair

DGGE - (denaturing gradient gel electrophoresis) - denaturing gradient gel electrophoresis

DHPLC - (Denaturing high performance liquid chromatography) - denaturing high performance liquid chromatography

KASP - (KBioscience Competitive Allele- Specific Polymerase chain reaction) - detection method developed by Bioscience

- 5 CZ 34779 UI mPCR - (multiplex polymerase chain reaction) - multiplex polymerase chain reaction

PCR - RFLP - (polymerase chain reaction - restriction fragment length polymorphism) - method of detection using polymerase chain reaction-restriction fragment length polymorphism

SBE primer - (single base extension primer) - extension / extension of the primer by one nucleotide base

SNP - (single nucleotide polymorphism) - single nucleotide polymorphism characterized by the exchange of one base pair for another

SSCP - (single strand conformation polymorphism) - single strand conformational polymorphism

List of used literature:

Chessa, S., E. Budelli, F. Chiatti, A. M. Čito, P. Bolla, and A. Caroli. 2005. Short communication: Predominance of beta casein (CSN2) C allele in goat breeds reared in Italy. J. Dairy Sei. 88: 1878 - 1881.

Chianese, L., S. Caira, G. Garro, M. Quarto, R. Mauriello, and F. Addeo. 2007. Occurrence of genetic polymorphism at goat β-CN locus. 5th Int. Symp. Challenge to Sheep and Goats Milk Sectors, Alghero 2007, Sardinia, Italy.

Cosenza, G., A. Pauciullo, D. Gallo, D. Di Berardino, and L. Ramunno. 2005. A SspI PCR-RFLP detecting a silent allele at the goat CSN2 locus. J. Dairy Res. 72: 456-459.

Persuy, M. A., C. Printz, J. F. Medrano, and J. C. Mercier. 1999. A single nucleotide deletion resulting in a premature stop codon is associated with marked reduction of transcripts from a goat beta-casein null allele. Anim. Genet. 30: 444-451.

Ramunno, L., P. Mariani, M. Pappalardo, A. Rando, M. Capuano, P. Di Gregorio, and G. Cosenza. 1995. A magician in effect on the content of caseinab in the latte di capra. XI Convegno ASPA, pages 185-186, Grado (GO) Italy.

Rando, A. M. 1998. Gen.Bank Accession no. AJ011018 (Capra hircus csn2 gene, exons 1 to 9 alleleA) http://www.ncbi.nlm.nih.gov/Accessed Feb. 21, 2005.

Rando, A. M., Pappalardo, M., Capuano, P, Di Gregorio, P., Rammuno, L. 1996. Two mutations might be responsible for the absence of β-casein in goat milk. Anim. Genet. 27, 31.

Claims (1)

CLAIMS FOR PROTECTION A kit for the detection of polymorphisms in exon 7 and exon 9 of the beta casein gene in goats by the mini-sequencing method - SNaPshot, characterized in that it comprises primer sets of the composition Forward 5 '- TCCTTCTTTCCAggATgAACTCC - 3' Reverse 5 '- ACTTACAAgAATAgggAAgggTCC - 3' with an amplified section length of 51 Ibp in exon 7, and Forward 5 '- ATgggggTgAgATgAAgAgT - 3' Reverse 5 '- ACAAgAAAACTTAAATATgCCTAAggg - 3' with the length of the amplified 401bp region in exon 9, and a set of five extension primers (SBE primers) with the following sequences in region 7 of exon SBE1 (8561): 5'- ATATATggATgAACTCCAggATAAA - 3 ', for detection of genetic polymorphism at position - 856Ibp (A / A), SBE2 (8913): 5 - ATATATATATATATATATATTCCgTgCTgTCCCTTT - 3 ', for detection of genetic polymorphism at position - 8913bp (C / A), SBE3 (8915): 5 '- AggCAgAACTTTgggCT - 3', for detection of genetic polymorphism at position - 8915bp (C / T), SBE4 (8946): 5 - ATATATATATATATATATATTATCATATCTCTCTggggCACT - 3 ', to detect genetic polymorphism at position - 8946bp (C / T), and in region 9 of exon SBE5 (10562): 5 '- ATATATATATATATATATATATATCCATATACTTTCAAAATg TTAATTCAA - 3 ', to detect genetic polymorphism at position - 10562 bp (C / T).