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

Year 2020, Volume: 9 Issue: 1, 6 - 11, 25.06.2020

Mevlut Arslan

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

Cited By: 5

Abstract

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.

Keywords

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

Abstract

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.

Keywords

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

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