MALDI-TOF mass spectrometry coupled with ligase reaction and nicking amplification for high-throughput multiplex SNP genotyping

Abstract

Single-nucleotide polymorphisms (SNPs) are single DNA sequence variations in specific site of genomic DNAs obtained from different individuals. The SNPs are the most common type of genetic variation among people. Humans have approximately one SNP per 1000 base pairs on average which causes individual difference in physical or physiological features. Besides, they can serve as biological signposts for genes associated with genetic disease, helping predict an individual’s genetic predisposition to disease. In this study, a novel multiplex mutation genotyping method was developed utilizing ligase reaction and nicking amplification based on mass spectrometry. To identify a mutated base in specific site, ligase reaction was performed using two ligation probes that flanked the mutation site. The primary ligation probe is designed to contain both a complementary base to that of mutation site at its 5′ end and a primer annealing site for nicking amplification at the 3’ end. The secondary probe is designed to have a nicking enzyme recognition site and a mass marker sequences at its 5’ end. Therefore, the primary and secondary ligation probes were linked only in the presence of mutant alleles in DNA sample. The ligation product was then utilized as a template for nicking amplification reaction. The universal primer was annealed to the 3’ end of the ligation product and extended during the nicking amplification reaction. As a result of the reaction, the cleaved short DNA segments used as a mass marker were generated and amplified. They were subjected to MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization - Time of Flight Mass Spectrometry) and the mass peak was detected only in the case of mutant samples. Using this strategy, we successfully identified 4 mutation sites in BRCA 1 gene in a single reaction tube. This new mass spectrometry-based strategy does not require thermal cycling due to isothermal nicking amplification of mass marker, resulting in rapid analy...