Briefly, it involves PCR amplification of SNP containing regions from genomic DNA. PCR products are then used as templates for single base extension (SBE) of multiple SBE primers at corresponding SNP sites with biotinylated dideoxynucleotides. Extension products are terminated with a biotin moiety and captured simultaneously on a streptavidin-coated surface followed by their analysis with MALDI-TOF mass spectrometry. The difference in mass between extension products and corresponding primers is used for inferring SNP genotypes. Elimination of primers prior to MALDI-TOF analysis eases spectrum interpretation and allows the analysis of larger number of SNPs. Recently, we have used this approach for concurrent analysis of 50 SNPs from the CYP2A13 gene including all known exonic SNPs. This has demonstrated the capacity of SPC-SBE for highly multiplexed SNP genotyping. However, despite the advance in levels of multiplexing, current implementation of SPC-SBE is limited in its capacity for genotyping multiple samples rapidly due to the use of streptavidin for isolating biotinylated fragments.
We report here an approach that allows rapid isolation of biotinylated fragments for multiple samples simultaneously. We have fabricated a microbead-pipette tip device and used it for isolating biotinylated oligonucleotides generated during SPC-SBE followed by direct spotting on a MALDI-TOF sample plate for analysis. In combination with a multi-channel pipettor, the device has been used for parallel analysis of many samples thus increasing throughput. We used the device for concurrent genotyping of 50 SNPs of CYP2A13 using SPC-SBE and were able to discern genotypes for all SNPs unambiguously. Results of the study indicate that the system is robust and improves process throughput substantially. Further, the system can be automated and used in other SNP genotyping applications.