Development of a cost effective direct DNA sequencing method for rapid SNP detection and genotyping of candidate genes

Abstract

The search for novel polymorphisms which can either have an impact on animal health, or be utilized for the identification of genotype/phenotype correlations is of paramount importance in the area of animal genetics and genomics. Although high throughput SNP detection and genomic selection tools are now available for production animals such as cattle and sheep (Matukumalli et al., 2009), these methods are very expensive mainly due to high instrumentation and operating costs and hence uneconomical for the evaluation of many regional breeds of sheep around the world. The candidate gene approach alone or in combination with data from genome wide association studies however requires DNA sequencing from at least twenty individual animals for SNP identification. Common direct DNA sequencing methodologies used for SNP identification within a gene first require the PCR amplification of the region of interest followed by electrophoresis and gel extraction prior to performing actual DNA sequencing, thereby increasing the processing costs and limiting the sample throughput rate. This study therefore aimed at the development of a rapid and cost effective direct sequenced based genotyping method that would not require gel extraction or PCR clean up kits prior to sequencing. To illustrate the effectiveness of the technique, we have analysed the entire coding region of the ovine prolactin gene consisting of 5 exons, including both the 5′ and 3′ UTR