Development of a Single Nucleotide Polymorphism Genotyping Microarray Platform for the Identification of Bovine Milk Protein Genetic Polymorphisms

Abstract

The objective of this study was to develop and validate a fast method for typing the main mutations of bovine milk protein genes by using microarray technology. An approach based on the ligation detection reaction (LDR) and a universal array (UA) was used. Polymorphisms in both the coding and noncoding sequences of alpha(S1)-casein, beta-casein, kappa-casein, and beta-lactoglobulin genes were considered because of their well-known effects on milk composition and cheese production. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR was developed to amplify all the DNA target sequences simultaneously. Second, the LDR-UA assay was implemented. The method was validated by analyzing 100 Italian Friesian DNA samples, which were also genotyped by conventional methods both at the protein level by means of milk isoelectrofocusing and at the molecular level using PCR-RFLP and PCR-single strand conformation polymorphism techniques. The genotypes obtained using the LDR-UA approach were in full agreement with those obtained by the conventional analyses. An important result of the LDR-UA assay was a more accurate genotyping of the different milk protein alleles than was found with conventional typing methods. At the kappa-casein gene, in fact, 4 samples were heterozygous (3 reference samples and 1 validation sample) for an allele coding for Thr(136) and Ala(148). This variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The multiplex PCR-LDR-UA approach developed provides for an accurate, inexpensive, and high-throughput assay that does not exhibit false positive or false negative signals, thus making it highly suitable for animal genotyping.

Marcadores SNP: conceitos básicos, aplicações no manejo e no melhoramento animal e perspectivas para o futuro

“…Metodologias de genotipagem de SNPs baseadas em microarranjos de DNA fabricados através da impressão de oligonucleotídeos em lâminas de vidro (Hacia et al 1999) trouxeram novos avanços, com aplicações sendo geradas inclusive para espécies de interesse pecuário (Kamiñski et al, 2005;Chessa et al, 2007;Figura 4A). Essa tecnologia permite a genotipagem de dezenas de marcadores no mesmo ensaio com uma diminuição significativa de mão-de-obra.…”

“…Essas aplicações requerem a genotipagem de algumas dezenas de marcadores em grandes números de amostras, demandando quesitos não oferecidos pelos ensaios Taqman e de minisequenciamento. As tecnologias geralmente utilizadas nessas aplicações foram as plataformas baseadas em espectrometria de massa (MALD-TOF, Heaton et al, 2005) e em microarranjos (Kaminski et al, 2005;Chessa et al, 2007).…”

“…Esforços significativos foram empregados para identificar microsatélites em bovinos (Fries et al, 1990;Moore et al, 1994;Vaiman et al, 1994;Stone et al, 1995;Kirkpatrick et al, 1995;Stone et al, 1997;Reed et al, 2001), suínos (Johansson et al, 1992;Miller et al, 1992;Coppieters et al, 1993;Fredholm et al, 1993;Alexander et al, 1996) e eqüinos (Johansson et al, 1992;Marklund et al, 1994;Hopman et al, 1999;Murphie et al 1999;Ruth et al, 1999). Tal metodologia trouxe muitos benefícios, associados, principalmente, às bases moleculares desse tipo de marcador (Tabela 1).…”

Potentials and limitations of molecular diagnostic methods in food safety

“…Finally, a LDR-UA based kit was also developed to detect mutations of bovine milk protein genes with the aim of improving the composition and quality of milk and milk derived products [8]. …”

“…In this way, the replacement of a malfunctioning probe or the addition of new probe sets is easily and quickly done at the cost of the new probes only. Furthermore, the probe/target annealing temperature is not constrained to a certain value, as it happens during regular array hybridization: the LDR can be performed separately according to each probe optimum temperature and the ligation products can be pooled together before hybridizing on the UA [8].…”