Identification of different allelic forms of PrP gene in Naeinian sheep using melting curve analysis technique

Genotyping using direct analysis of DNA has a considerable importance in plants and animals breeding, also for diagnosis and evaluation of genetic disorders in plants, animals and in forensic science. The melting curve analysis method is one of the most cost-effective genotyping methods that can be used in relevant studies. In order to evaluate the performance of this technique, the current study was performed for detection of single nucleotide polymorphisms (SNPs) PrP gene in sheep. The DNA was extracted from 145 blood samples of Naieni sheep from Esfahan province with modified salting out method. After amplification of the desired fragment by polymerase chain reaction (PCR) genotyping of samples was carried out by melting curve analysis technique. In the following, the two methods of PCR-SSCP and direct sequencing method were used to confirm the genotyping results. The results of the present study showed that nine genotypes were observed at PrP locus in studied sheep population. But, due to multiple SNP at the amplified fragment, it cannot possible to exactly predict each genotype by melting curve analysis. With the results obtained from this study it can be concluded that melting curve analysis technique easily separates genotypes occurred by a single nucleotide substitution in one amplified fragment (single marker site), but because of the complexity of the presence of several SNP in an amplified fragment (multiple marker sites), it is not easily possible to separate each genotypes specifically. According to the advantages of melting curve analysis technique for isolation of genotypes, it is suggested that more research should be done for resolving the limitations of this technique for amplified fragments carrying multiple marker sites.