Pacini, Federico (2007) Sviluppo di Metodologie Molecolari per la Qualit√† dei Prodotti di Origine Animale. [Tesi di dottorato]
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The objectives of this study were to develop and optimize molecular techniques based on the DNA melting analysis for the detection of allele variants or SNPs in genes involved in the quality of animal products.
For the dairy industry the detection of the cow k-casein B allele (CSN3*B) is very important for the role of its codifying protein during the cheese making. It is known that the k-casein B allele plays a major role in cheese technology with great effects in the quality and quantity of cow milk. The availability of accurate and reliable protocols for the identification of the most common alleles is of great interest in breeding projects.
In the present study a denominated denaturant DNA melting analysis was developed using a new buffer to quickly identify A and B k-casein allelic variants. The DNA melting analysis is fast and suitable for the identification of A and B alleles.
The development of this denaturant buffer allows to enhance the discrimination power of the instrumentation. The same approach was used to detect SNPs in two important meat gene quality using an ad-hoc instrument for the melting analysis
The RYR1 and PRKAG3 genes have an important role in the meat quality, influencing the pH, water content, lean meat content and colour. The frequencies of their alleles were investigated in Bayern, Australian ans Sudtiroler pigs in order to test a high resolution DNA melting analysis protocol, which allows to detect a total of 6 genotypes in simple manner and in few minutes trough a post multiplex PCR.
It was possible to compare the sensibility obtained with the no-denaturant approach against the denaturant approach.
The observed frequencies were: 0.86, 0.13 and 0.01 for the N/N, N/n e n/n respectively for RYR1 gene and 0.17, 0.54, 0.29 for I/I, I/V and V/V for the PRKAG3 gene respectively.
Its is interesting to note that even if the pigs tested belonged to breed with attitude for high meat content and quality, a relatively high genotype frequency N/n and a relatively low genotype frequency I/I were found. In these productions, where meat quality is a relevant attribute, could be possible to enhance the favourable SNPs in bores and sows.
The denaturant buffer developed allows to detect with major detection level the homo/hetero genotypes of the RYR1 and PRKAG3 genes. This approach for the detection of known SNP is very fast, reliable and could substitute the specific enzymatic cleavage for the detection of specific substitutions.
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