Ilie D. E. et. al./Scientific Papers: Animal Science and Biotechnologies, 2013, 46 (1) 102 High-Resolution Melting Assay as a Tool for Identification of CSN3 Genotypes in Cattle Population Daniela Elena Ilie 1,2* , Ada Cean 1,2 , Oana Isabella Gavriliuc 2,3 , Ana Claudia Carstea 3 , Andrei Cristian Grădinaru 4 1 Research and Development Station for Bovine - Arad, 310059, Bodrogului 32, Arad, Romania 2 Banat’s University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotechnologies, 300645, Calea Aradului 119, Timisoara, Romania 3 University of Medicine and Pharmacy "Victor Babes" Timisoara, 300041, Eftimie Murgu 2, Romania 4 University of Agricultural Sciences and Veterinary Medicine of Iasi, 700490, Sadoveanu Alley 3, Iasi, Romania Abstract High-resolution melting (HRM) assay is a novel tool for polymorphism analysis. We describe here the application of high- resolution melting analysis for genotyping the main mutation of kappa-casein (CSN3) gene. The milk protein gene CSN3 is relevant in relation to milk production parameters and milk protein quality. The CSN3 gene encodes milk protein that is important for the structure and stability of casein micelles. The B allele of CSN3 is associated with higher milk protein content and milk quality. We tested 90 animals with known genotypes for CSN3 that were previously genotyped by PCR-RFLP and IEF methods, and then examined the sensitivity of mutation detection using quantitative PCR (qPCR) followed by HRM curve analysis. Known CSN3 variants were readily detectable using HRM assay. Thus, the qPCR-HRM assay is a sensitive in-tube methodology that allowed the rapid and sensitive identification of CSN3 genotypes. The results demonstrate that qPCR-HRM method is a fast, simple and reliable assay for CSN3 allele detection. Keywords: CSN3, genotyping, high-resolution melting. 1. Introduction  Kappa-casein (CSN3) is a protein with an important polymorphism, its genetic variants being able to influence the yield and composition of milk and its processing properties [1-5]. The major A and B CSN3 variants differ by two amino acid substitutions, i.e., Thr136/Ile and Asp148/Ala [6]. These two alleles may be distinguished by the presence or absence of HindIII restriction nuclease recognition site [7,8]. Moreover, the change in the GAT triplet coding for Asp at a.a. position 148 abolishes a HinfI site of the CSN3 B allele [9]. Up to now, many methods were used to study  * Corresponding author: Daniela E. Ilie, Tel. +40257339130, Email danailie@yahoo.com milk protein polymorphisms in different cattle breeds. Several genetic tests, such as Polyacrylamide Gel Electrophoresis (PAGE), PCR-RFLP, PCR-SSCP, were described to identify the mutation and type single-nucleotide polymorphisms (SNPs). Most of these techniques require processing, separation steps or allele- specific primers, or probes, which make them less favorable for high-throughput assays [10]. The new approach reported here is based on a High-resolution melting (HRM) assay that was introduced as a homogeneous closed-tube system, which allows post-PCR analysis of genetic mutations or variance without the need of separation steps. This method is based on the properties of double-stranded DNA dissociation (melting), which is subsequently examined by melting curve analysis. Different melting profiles are obtained from the transition of double-