SNPs are important genetic markers for disease susceptibility and pharmacogenomics studies and high throughput methods are required for their analysis. SPC-SBE is a MALDI MS based technique that uses primer extension with biotinylated terminators for SNP genotyping. Briefly, it involves design of a library of primers that anneal one base upstream of desired SNP sites and their simultaneous extension using biotinylated terminators in an SBE reaction. Extended primers carry a biotin moiety at their 3' end and are selectively isolated using streptavidin coated magnetic beads followed by washing and release. They are then analyzed by MALDI MS simultaneously to reveal SNP identity based on unique mass of each extension product. Due to the removal of unextended primers and other reaction contaminants by washing, SPC-SBE provides sample cleanup prior to MALDI MS analysis. Besides, it allows analysis of larger number of extension products due to the absence of primers peaks in the spectrum.
However, the use of streptavidin coated magnetic beads for isolating biotinylated extended primers in the SPC step limits use of SPC-SBE for high throughput genotyping. The extremely high affinity between biotin and streptavidin requires harsh denaturing conditions for the release of biotinylated fragments and involves long procedure prior MALDI analysis.
To address these issues, we have fabricated devices with monomeric avidin (MA) coated beads to improve the SPC step and increase process throughput. MA coated beads were trapped in a pipette tip using a nylon mesh at its end to act as a filter. We have demonstrated isolation of biotinylated extended primers selectively from an SBE reaction with direct spotting on the MALDI plate utilizing the pipette tip device. The devices have been used for simultaneous analysis of many samples in combination with a multichannel pipette. We have been able to reuse each device a minimum of 5 times with a simple regeneration protocol thus reducing genotyping cost. This demonstrated feasibility of the device for selective isolation of biotinylated fragments. We used a similar approach to trap MA coated beads in a microchannel device and were able to repeat experiments done with the pipette tip device. This can be extended to an arrangement with multiple microchannels for semi-automated processing of multiple samples and be used in high throughput SNP genotyping studies.