Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods
ABSTRACT: The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potent...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-11-01
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| Series: | Journal of Dairy Science |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S0022030222005136 |
| _version_ | 1828322115155132416 |
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| author | V. Vigolo M. Franzoi F. Cendron G. Salvadore M. Penasa M. Cassandro M. De Marchi |
| author_facet | V. Vigolo M. Franzoi F. Cendron G. Salvadore M. Penasa M. Cassandro M. De Marchi |
| author_sort | V. Vigolo |
| collection | DOAJ |
| description | ABSTRACT: The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale. |
| first_indexed | 2024-04-13T18:36:55Z |
| format | Article |
| id | doaj.art-50dd58b0644844ad98d22f30ff98b516 |
| institution | Directory Open Access Journal |
| issn | 0022-0302 |
| language | English |
| last_indexed | 2024-04-13T18:36:55Z |
| publishDate | 2022-11-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Dairy Science |
| spelling | doaj.art-50dd58b0644844ad98d22f30ff98b5162022-12-22T02:34:51ZengElsevierJournal of Dairy Science0022-03022022-11-011051189468955Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methodsV. Vigolo0M. Franzoi1F. Cendron2G. Salvadore3M. Penasa4M. Cassandro5M. De Marchi6Department of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), ItalyDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), ItalyDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy; Corresponding authorDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), ItalyDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), ItalyDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), Italy; Federazione delle Associazioni Nazionali di Razza e Specie, Via XXIV Maggio 44, 00187 Roma, ItalyDepartment of Agronomy, Food, Natural resources, Animals and Environment, University of Padova, Viale dell'Università 16, 35020 Legnaro (PD), ItalyABSTRACT: The 2 major subvariants of β-casein (A1 and A2), coded by CSN2 gene, have received great interest in the last decade both from the scientific community and the dairy sector due to their influence on milk quality. The consumption of the A1 variant, compared with the A2 variant, has a potential negative effect on human health after its digestion but, at the same time, its presence improves the milk technological properties. The aim of the present study was to compare the best method in terms of time required, costs, and technical engagement for the identification of β-casein A1 and A2 variants (homozygous and heterozygous animals) in milk to offer a reliable service for large-scale screening studies. Two allele-specific PCR procedures, namely RFLP-PCR and amplification refractory mutation system (ARMS-PCR), and one biochemical technique (HPLC) were evaluated and validated through sequencing. Manual and automated DNA extraction protocols from milk somatic cells were also compared. Automated DNA extraction provided better yield and purity. Chromatographic analysis was the most informative and the cheapest method but unsuitable for large-scale studies due to lengthy procedures (45 min per sample). Both allele-specific PCR techniques proved to be fast and reliable for differentiating between A1 and A2 variants but more expensive than HPLC analysis. Specifically, RFLP-PCR was the most expensive and labor-demanding among the evaluated techniques, whereas ARMS-PCR was the fastest while also requiring less technical expertise. Overall, automated extraction of DNA from milk matrix combined with ARMS-PCR is the most suitable technique to provide genetic characterization of the CSN2 gene on a large scale.http://www.sciencedirect.com/science/article/pii/S0022030222005136casein polymorphismRFLP-PCRARMS-PCRHPLCmilk |
| spellingShingle | V. Vigolo M. Franzoi F. Cendron G. Salvadore M. Penasa M. Cassandro M. De Marchi Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods Journal of Dairy Science casein polymorphism RFLP-PCR ARMS-PCR HPLC milk |
| title | Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods |
| title_full | Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods |
| title_fullStr | Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods |
| title_full_unstemmed | Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods |
| title_short | Characterization of the genetic polymorphism linked to the β-casein A1/A2 alleles using different molecular and biochemical methods |
| title_sort | characterization of the genetic polymorphism linked to the β casein a1 a2 alleles using different molecular and biochemical methods |
| topic | casein polymorphism RFLP-PCR ARMS-PCR HPLC milk |
| url | http://www.sciencedirect.com/science/article/pii/S0022030222005136 |
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