A New Primer Designing for PCR-RFLP Analysis of A and B Genetic Variants of Bovine Kappa-Casein

Yıl 2020, Cilt: 9 Sayı: 1, 6 - 11, 25.06.2020

Mevlut Arslan

https://doi.org/10.31196/huvfd.651821

Cited By: 5

Öz

Bovine milk contains about 3.4% high-quality protein and it is very essential for human nutrition. Kappa (κ) casein
is one of the milk proteins and encoded by the CSN3 gene. Studies have shown that κ-casein has an important influence on
milk properties and the manufacturing of milk. κ-casein takes role as a stabilizing factor during the curdling of milk, which
makes it desired in the cheese factory. A and B genetic variants of κ-casein are well-known and well-studied. For the
genotyping of bovines, the PCR-RFLP approach has been used. For this aim, different primer pairs have been used to
amplify the polymorphic region of the CSN3 gene. In a previous study, the HinfI enzyme digestion of the polymorphic region
resulted in short and similar length fragments. In agarose gel electrophoresis, separation of very similar DNA fragments is
almost impossible and interpretation of the short DNA fragment sometimes causes challenges. Therefore, in this study, a
new primer design was described. Using the new primer, longer DNA fragment was amplified successfully and HinfI
digestion of the PCR product let to a longer and very different lengths of DNA fragments, which can be easily separated and
interpreted in agarose gel electrophoresis. The primer described in this study can be used in further studies related to allele
frequency researches and breeding strategies.

Anahtar Kelimeler

Milk, κ-casein, CSN3, Polymorphism, PCR-RFLP, Primer design

Sığır Kappa-Kazeinin A ve B Genetik Varyantlarının PCR-RFLP Analizi için Yeni Bir Primer Dizaynı

Öz

İnek sütü yaklaşık %3,4 oranında yüksek kaliteli protein içerir ve insan beslenmesi için oldukça elzemdir. Kappa (κ)
kazein, süt proteinlerinden birisidir ve CSN3 geni tarafından kodlanmaktadır. Çalışmalar κ-kazeinin sütün özellikleri ve
işlenmesi üzerine önemli bir etkiye sahip olduğunu göstermektedir. κ-kazein sütün kesilmesi sürecinde stabilize edici bir
faktör olarak rol almaktadır; bu durum, κ-kazeini peynir fabrikalarında istenilir hale getirmektedir. Kappa (κ) kazeinin A ve B
genetik varyantları iyi bilinmekte ve yoğun çalışılmaktadır. Sığırların genotiplenmesi için PCR-RFLP yöntemi kullanılmaktadır.
Bu amaçla, farklı primer çiftleri CSN3 geninin polimorfik bölgesini çoğaltmak için kullanılmaktadır. Önceki bir çalışmada,
polimorfik bölgenin HinfI enzimi ile kesimi kısa ve benzer uzunlukta fragmentler oluşturdu. Agaroz jel elektroforezinde çok
benzer DNA fragmentlerinin ayrımı neredeyse imkansızdır ve kısa DNA fragmentlerin yorumlanması bazen zorluklara sebep
olmaktadır. Bu sebeple, bu çalışmada yeni bir primerin dizaynı tanımlandı. Yeni primerin kullanılamasıyla daha uzun DNA
fragmenti başarılı bir şekilde çoğaltıldı ve bu PCR ürünlerinin HinfI kesimi, agaroz jel elektroforezinde kolaylıkla ayrılabilen ve
yorumlanabilen çok farklı uzunlukta DNA fragmentlerinin oluşmasını sağladı. Bu çalışmada tanımlanan primer, allel frenkans
araştırmaları ve yetiştirme stratejileri ile ilgili gelecek çalışmalarda kullanılabilir.

Anahtar Kelimeler

süt, k-kazein, CSN3, polimorfizm, PCR-RFLP, Primer dizayn

Kaynakça

• Aleandri R, Buttazzoni LG, Schneider JC, Caroli A, Davoli R, 1990: The Effects of Milk Protein Polymorphisms on Milk Components and Cheese-Producing Ability. J. Dairy Sci., 73, 241-255.
• Alexander LJ, Stewart AF, Mackinlay AG, Kapelinskaya TV, Tkach TM, Gorodetsky SI, 1988: Isolation and characterization of the bovine kappa-casein gene. Eur. J. Biochem., 178, 395-401.
• Arber W, 1978: Restriction endonucleases. Angew. Chem. Int. Ed. Engl., 17, 73-9.
• Asif M, Mehboob-Ur-Rahman, Mirza JI, Zafar Y, 2008: High resolution metaphor agarose gel electrophoresis for genotyping with microsatellite markers. Pak. J. Agri. Sci, 45, 75-79.
• Azevedo AL, Nascimento CS, Steinberg RS, Carvalho MR, Peixoto MG, Teodoro RL, Verneque RS, Guimaraes SE, Machado MA, 2008: Genetic polymorphism of the kappa-casein gene in Brazilian cattle. Genet. Mol. Res., 7, 623-30.
• Breslauer KJ, Frank R, Blocker H, Marky LA, 1986: Predicting DNA duplex stability from the base sequence. Proc. Natl. Acad. Sci. U. S. A., 83, 3746-50.
• Caroli AM, Chessa S, Erhardt GJ, 2009: Invited review: milk protein polymorphisms in cattle: effect on animal breeding and human nutrition. J. Dairy Sci., 92, 5335-52.
• Comin A, Cassandro M, Chessa S, Ojala M, Dal Zotto R, De Marchi M, Carnier P, Gallo L, Pagnacco G, Bittante G, 2008: Effects of composite beta- and kappa-casein genotypes on milk coagulation, quality, and yield traits in Italian Holstein cows. J. Dairy Sci., 91, 4022-7.
• Dong Y, Zhu H, 2005: Single-strand conformational polymorphism analysis: basic principles and routine practice. Methods Mol. Med., 108, 149-57. Eigel WN, Butler JE, Ernstrom CA, Farrell HM, Harwalkar VR, Jenness R, Whitney RM, 1984: Nomenclature of Proteins of Cow's Milk: Fifth Revision1. J. Dairy Sci., 67, 1599-1631.
• Farrell HM, Jr., Jimenez-Flores R, Bleck GT, Brown EM, Butler JE, Creamer LK, Hicks CL, Hollar CM, Ng-Kwai-Hang KF, Swaisgood HE, 2004: Nomenclature of the proteins of cows' milk--sixth revision. J. Dairy Sci., 87, 1641-74.
• Fiat AM, Jolles P, 1989: Caseins of various origins and biologically active casein peptides and oligosaccharides: structural and physiological aspects. Mol. Cell. Biochem., 87, 5-30.
• Fodde R, Losekoot M, 1994: Mutation detection by denaturing gradient gel electrophoresis (DGGE). Hum. Mutat., 3, 83-94.
• Franca LT, Carrilho E, Kist TB, 2002: A review of DNA sequencing techniques. Q. Rev. Biophys., 35, 169-200.
• Lai E, Birren BW, Clark SM, Simon MI, Hood L, 1989: Pulsed field gel electrophoresis. Biotechniques, 7, 34-42.
• Lee PY, Costumbrado J, Hsu C-Y, Kim YH, 2012: Agarose gel electrophoresis for the separation of DNA fragments. Journal of visualized experiments : JoVE, 3923.
• Li J, Yang Y, Mao Z, Huang W, Qiu T, Wu Q, 2016: Enhanced Resolution of DNA Separation Using Agarose Gel Electrophoresis Doped with Graphene Oxide. Nanoscale research letters, 11, 404-404.
• Mann T, Humbert R, Dorschner M, Stamatoyannopoulos J, Noble WS, 2009: A thermodynamic approach to PCR primer design. Nucleic Acids Res., 37, e95-e95.
• Marziali AS, Ng-Kwai-Hang KF, 1986: Effects of Milk Composition and Genetic Polymorphism on Cheese Composition: Minutes of the Executive Committee Meetings and Summary Reports of ADSA Committees and Representatives. J. Dairy Sci., 69, 2533-2542.
• Mclean DM, Graham ER, Ponzoni RW, Mckenzie HA, 1984: Effects of milk protein genetic variants on milk yield and composition. J. Dairy Res., 51, 531-46. Ota M, Fukushima H, Kulski JK, Inoko H, 2007: Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism. Nat. Protoc., 2, 2857-64.
• Otaviano AR, Tonhati H, Sena JaD, Cerón Muñoz MF, 2005: Kappa-casein gene study with molecular markers in female buffaloes (Bubalus bubalis). Genet. Mol. Biol., 28, 237-241.
• Owczarzy R, Tataurov AV, Wu Y, Manthey JA, Mcquisten KA, Almabrazi HG, Pedersen KF, Lin Y, Garretson J, Mcentaggart NO, Sailor CA, Dawson RB, Peek AS, 2008: IDT SciTools: a suite for analysis and design of nucleic acid oligomers. Nucleic Acids Res., 36, W163-W169.
• Patel RK, Chauhan BJ, Singh MK, Soni KJ, 2007: Allelic Frequency of Kappa-Casein and Beta-Lactoglobulin in Indian Crossbred (Bos taurus×Bos indicus) Dairy Bulls. Turk J Vet Anim Sci, 31, 399-402.
• Prinzenberg EM, Krause I, Erhardt G, 1999: SSCP analysis at the bovine CSN3 locus discriminates six alleles corresponding to known protein variants (A, B, C, E, F, G) and three new DNA polymorphisms (H, I, A1). Anim. Biotechnol., 10, 49-62.
• Rachagani S, Gupta ID, 2008: Bovine kappa-casein gene polymorphism and its association with milk production traits. Genet. Mol. Biol., 31, 893-897.
• Rouzina I, Bloomfield VA, 1999: Heat capacity effects on the melting of DNA. 1. General aspects. Biophys. J., 77, 3242-3251.
• Saiki RK, Gelfand DH, Stoffel S, Scharf SJ, Higuchi R, Horn GT, Mullis KB, Erlich HA, 1988: Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-91.
• Smith HO, Wilcox KW, 1970: A restriction enzyme from Hemophilus influenzae. I. Purification and general properties. J. Mol. Biol., 51, 379-91.
• Stephenson F, 2003: The Polymerase Chain Reaction. In "Calculations for Molecular Biology and Biotechnology", Ed; Stephenson F, Academic Press, Burlington.
• Sugimoto N, Nakano S, Yoneyama M, Honda K, 1996: Improved thermodynamic parameters and helix initiation factor to predict stability of DNA duplexes. Nucleic Acids Res., 24, 4501-4505.
• Tsiaras AM, Bargouli GG, Banos G, Boscos CM, 2005: Effect of kappa-casein and beta-lactoglobulin loci on milk production traits and reproductive performance of Holstein cows. J. Dairy Sci., 88, 327-34.
• Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S, Madden TL, 2012: Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics, 13, 134.