SNP Analysis by Single Base Extension of Primers

Several modes of detecting a single base extension have been investigated, including measuring the incorporation of fluorescent, haptenated, or radioactive ddNTPs [6, 7] or gel electrophoresis based-detection of fluorescent primers extended by non-fluorescent nucleotides [8]. Recently, the Applied Biosystems SNapshot lYimer Extension Kit was introduced. In this assay, a primer is extended by one or more fluorescent- labelled dideoxynucleotides with subsequent detection in a fluorescent-based DNA sequencer. Several primers can be analysed within one lane of the DNA sequencer. 

MALDI-TOF mass spectrometry has recently proven an attractive means to analyse single base extensions. Several thousand samples can be analysed each day, because each analysis requires only a few seconds. Furthermore, within each sample, multiple independent loci can be simultaneously analysed. With this internal multiplexing, tens of thousands of SNP genotypes can be obtained each day. Reagent costs are comparatively low, because there are no signal molecules in the assay and the primers are unlabeled and comparatively inexpensive. Because 

Genotyping primer anneals to one strand of PCR product and is extended by a length of one base. Nucleotides(s) complementary to the iiolvmornhic site are incornorated. 

MALDI-TOF mass spectrometry is very high in resolution, the available degree of multiplexing is much higher than using fluorescent detection, which is typically limit due to a great deal of spectral overlap between different dyes. Allele determinations by mass spectrometry are nearly artefact-free, because the process measures the intrinsic mass of the bases added to the primer. These mass measurements are not affected by secondary considerations, such as the conformation oftheDNA, which can confound gel-based techniques. 

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