Development of multiplex genotyping methods of genetic variations based on mass spectrometry

Abstract

We herein describe new mass spectrometry-based methods for multiplex SNP genotyping by utilizing allele-specific ligation and isothermal amplification reaction. In these method, allele-specific ligation is first performed to discriminate base sequence variations at the SNP site. The primary ligation probe is extended by a universal primer annealing site while the secondary ligation probe has base sequences as an overhang with a nicking enzyme recognition site and complementary mass marker sequence. The ligation probe pairs are ligated by DNA ligase only at specific alleles in the target DNA, and the resulting ligated product serves as a template to promote the isothermal amplification reaction using a universal primer, which isothermally amplifies short DNA fragments, called mass markers, to be analyzed by mass spectrometry. In chapter 2, we employed strand displacement amplification (SDA) reaction for isothermal amplification to amplify mass markers and by varying the sizes of the mass markers, the multiple SNP sites are simultaneously identified based on peak positions on the mass spectrum. In chapter 4, we employed graphene oxide (GO)-assisted exponential amplification reaction (EXPAR) for isothermal amplification whose detection capability on mass spectrometry is confirmed in chapter 3. By utilizing these methods, we successfully demonstrated the multiplex SNP genotyping capability of these methods by reliably identifying several BRCA mutations in a multiplex manner with mass spectrometry.