Full flexibility genotyping of single nucleotide polymorphisms by the GOOD assay

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 ss-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 ss-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.

Figure 1

Scheme of A^NH2, G^NH2, C^NH2 and U^NH2 phosphoramidites.

Figure 2

The top trace shows the MALDI-MS analysis of an individual homozygous for G at SNP 129 of the prion protein gene. The resulting product of the GOOD assay d(GptA^CTptGptdG) has a mass of 1458 Da. In the middle, the analysis of an individual homozygous for A with the resulting product d(GptA^CTptGptdA) and corresponding mass 1442 Da is shown. At the bottom, the analysis of heterozygous DNA is shown.

Figure 3

The GOOD assay applied to SNP 129 of the prion protein gene on bovine DNA. The assay started with tissue samples that were only proteinase K digested and not purified.

Figure 4

MALDI-MS genotyping of SNPs –20 and 79 in the human β-2-adrenergic receptor gene. Product masses of the alleles are d(GptG^CTptTptdA), 1496 Da and d(GptG^CTptTptdG), 1512 Da for SNP –20 and d(CptA^CTptGptdG), 1481 Da and d(CptA^CTptGptdC), 1441 Da for SNP 79. At 1437 and 1168 Da residual primers of the primer extension reaction are observed. The three spectra show a homozygous individual for either allele and a heterozygous individual.

Figure 5

MALDI-MS genotyping of SNP 33710 in ACE. Product masses of the alleles are d(TptC^CTptCptdA), 1417 Da and d(TptC^CTptCptdG), 1433 Da. At 1090 Da residual primer of the primer extension reaction is found. The three spectra show a homozygous individual for either allele and a heterozygous individual.