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

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

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 veişlenmesi üzerine önemli bir etkiye sahip olduğunu göstermektedir. κ-kazein sütün kesilmesi sürecinde stabilize edici birfaktör olarak rol almaktadır; bu durum, κ-kazeini peynir fabrikalarında istenilir hale getirmektedir. Kappa (κ) kazeinin A ve Bgenetik 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 çokbenzer DNA fragmentlerinin ayrımı neredeyse imkansızdır ve kısa DNA fragmentlerin yorumlanması bazen zorluklara sebepolmaktadır. Bu sebeple, bu çalışmada yeni bir primerin dizaynı tanımlandı. Yeni primerin kullanılamasıyla daha uzun DNAfragmenti başarılı bir şekilde çoğaltıldı ve bu PCR ürünlerinin HinfI kesimi, agaroz jel elektroforezinde kolaylıkla ayrılabilen veyorumlanabilen çok farklı uzunlukta DNA fragmentlerinin oluşmasını sağladı. Bu çalışmada tanımlanan primer, allel frenkansaraştırmaları ve yetiştirme stratejileri ile ilgili gelecek çalışmalarda kullanılabilir.

Keywords:

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

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