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 Ligation Detection Reaction (LDR) and a Universal Array (UA) was used. Polymorphisms both in the coding and non coding sequences of αS1-casein, β-casein, κ-casein, and β-lactoglobulin genes were considered due to their well known effects on milk composition and cheese-making attitude. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR (mPCR) was developed in order to amplify simultaneously all the DNA target sequences. Secondly, 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 Polymorphisms (SSCP) 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 conventional typing methods. At the ê-casein gene, in fact, four samples were heterozygous for an allele coding for Thr136 and Ala148. Such variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The mPCR/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 the animal genotyping.
Development of a single nucleotide polymorphism genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms.
Stefania Chessa;Clarissa Consolandi;
2007
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 Ligation Detection Reaction (LDR) and a Universal Array (UA) was used. Polymorphisms both in the coding and non coding sequences of αS1-casein, β-casein, κ-casein, and β-lactoglobulin genes were considered due to their well known effects on milk composition and cheese-making attitude. A total of 22 polymorphic sites, corresponding to 21 different variants, were included in the diagnostic microarray. First, a multiplex PCR (mPCR) was developed in order to amplify simultaneously all the DNA target sequences. Secondly, 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 Polymorphisms (SSCP) 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 conventional typing methods. At the ê-casein gene, in fact, four samples were heterozygous for an allele coding for Thr136 and Ala148. Such variant, which can be considered as the wild type of the genus Bos, is not usually identifiable by the conventional typing methods used. The mPCR/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 the animal genotyping.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
