Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP) analysis of goat α_s2-casein

Abstract

Goat caseins show high genetic polymorphism, with several mutations affecting the specific protein expression. To date at least seven alleles have been found at goat α gene (CSN1S2). Five of them, CSN1S2*A, CSN1S2*B, CSN1S2*C, CSN1S2*E, and CSN1S2*F, code for a normal content of α (about 2.5 g/l), whereas CSN1S2*D and CSN1S2*0 are associated respectively with a reduced α content and with the absence of this specific protein in the milk. Four different Restriction Fragment Length Polymorphism (RFLP) - Polymerase Chain Reaction (PCR) tests are needed to detect all these seven alleles at the DNA level. The aim of this study was to develop a new tool for the genetic study of CSN1S2 variability in goat.

A protocol for the simultaneous genotyping of CSN1S2*A, CSN1S2*B, CSN1S2*C, and CSN1S2*E alleles was developed by PCR – Single Strand Conformation Polymorphism (SSCP) technique. A 247 bp fragment containing exon 9 and a 273 bp fragment containing exon 16 of the goat CSN1S2 gene were simultaneously amplified by PCR, and subsequently analysed by SSCP. The test was validated by screening the CSN1S2 variability in the following goat breeds: Orobica (n = 81), Camosciata (n = 112), and Saanen (n = 76).

DNA was extracted from blood or milk by a commercial kit. Milk samples were analysed by isoelectric focusing (IEF) for α typing at the protein level. The DNA samples were analysed both by the novel PCR-SCCP protocol as well as by two PCR-RFLP tests already available, respectively for the detection of CSN1S2*A, CSN1S2*B, CSN1S2*C and for the detection of CSN1S2*E. Full agreement was found in the typing results obtained by the PCR-SSCP protocol developed, the two PCR-RFLP methods, and the IEF analysis which allows the simultaneous detection of the four variants at the protein level.

In all cases, a specific DNA test is needed to detect the mutation characterizing the CSN1S2*F allele. The PCR-SSCP technique developed is a cheep alternative to the two PCR-RFLP methods for CSN1S2 typing at the DNA level, allowing to reduce to three the number of tests necessary to identify the seven CSN1S2 alleles described.