Lawrence A. Haff

Methidium intercalator inserted into synthetic oligonucleotides.

Abstract A new methidium intercalator phosphoramidite has been synthesized. Methidium incorporation into an oligonucleotide during the synthesis was confirmed by UV and MALDI TOF MS data. UV melting experiments showed enhanced stability of a duplex, containing internal methidium.

SNP Genotyping by MALDI-TOF Mass Spectrometry

The Sequazyme Pinpoint assay has successfully typed hundreds of different loci in many different laboratories. Major advantages of the technique are that primer selection is extremely simple and a high percentage of selected primers successfully genotype. The primers need contain no special labels or groups, so they are inexpensive and easily obtainable. Because the PCR and primer extension protocols are solution-based and the extension protocol is universal, the assay does not involve any of the relatively high infrastructure costs of DNA chips. Because MALDI-TOF is largely artefact-free and high in resolution, the base-calling accuracy is extremely high. The ability to detect and quantitate low relative abundance of the minor allele in pooled samples is largely unique to mass spectrometry, due to its high resolution compared to electrophoretic, chromatographic, fluorescent, and isotopic techniques. The ability to process several thousand samples a day, together with the ability to internally multiplex, enables throughputs of about 20,000 alleles a day, suitable for most medium to high throughput genotyping laboratories. Future developments include further miniaturisation to lower reagent costs, faster lasers to support higher throughput MALDI-TOF, and associated robotic systems to provide greater throughput.