Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

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Abstract
Recently a facile method for genotyping single nucleotide polymorphisms (SNPs) using MALDI mass spectrometry, termed the GOOD assay, was developed. It does not require any purification and is performed with simple liquid handling, thermal incubation and cycling steps. Although this method is well suited to automation and high-throughput analysis of SNPs, it did not allow full flexibility due to lack of certain reagents. A complete set of β-cyanoethyl phosphoramidites is presented herein that give this SNP genotyping method full sequence and multiplex capabilities. Applications to SNP genotyping in the prion protein gene, the β-2-adrenergic receptor gene and the angiotensin converting enzyme gene using the GOOD assay are demonstrated. Because SNP genotyping technologies are generally very sensitive to varying DNA quality, the GOOD assay has been stabilised and optimised for low quality DNA. A template extraction method is introduced that allows genotyping from tissue that was taken while placing an ear tag on an animal. This dramatically facilitates the application of genotyping to animal agricultural applications, as it demonstrates that expensive and cumbersome DNA extraction procedures prior to genotyping can be avoided.