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.

Single-nucleotide polymorphisms: analysis by mass spectrometry

“…This enzyme catalyzes the removal of 5¢ phosphate groups from nucleotides, DNA and RNA with higher efficiency than shrimp alkaline phosphatase, which has been used in published protocols [7][8][9] .…”

“…1, A), is based on chemical DNA modification [7][8][9] . In this method, no stringent purification is required, leading to a high-throughput and cost-efficient SNP-genotyping procedure, particularly for studies of candidate genes.…”

“…The template can be any nucleic acid that can be amplified by PCR. For SNP genotyping, that is mostly purified genomic DNA, but crude samples such as tissue samples digested with proteinase K can also be used 8 . In general, the efficiency of SNP-genotyping experiments depends very much on the quality and integrity of the template DNA.…”

Single nucleotide polymorphism genotyping: biochemistry, protocol, cost and throughput

“…Most solid-phase-mediated methods require a clean-up because the substrates of the allele-specific reaction can produce false-positive signals and complicate genotype scoring. Examples are mass spectrometry, 12,[16][17][18][19][20][21] zip-code technology, 22,23 Illumina color beads, 24,25 Orchid SNP-IT technology 11 and all hybridization based methods. 26,27 Product detection and identification can be performed in many different ways.…”

“…Since allelic discrimination reactions always change the weight of a molecule, mass spectrometry can be used to identify products from a reaction. 12,[16][17][18][19][20][21]75 Among the four bases that form DNA molecules, the smallest difference is between the A and the T bases (about 9 Da) and discriminating an A/T polymorphism can be difficult. A modification of SBE using tagged ddNTPs could make the difference easier to detect.…”