Rapid genotyping by MALDI-monitored nuclease selection from probe libraries

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with in dividual genes(1). Efficient technologies for rapidly detecting SNPs will t herefore facilitate the mining of genomic information(2). Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase c hain reaction (PCR), allele-specific oligomer hybridization, oligomer-speci fic ligation assays, minisequencing, direct sequencing, fluorescence-detect ed 5'-exonuclease assays, and hybridization with PNA probes(3-6). Detection by mass spectrometry (MS) offers speed and high resolution(7,8). Matrix-as sisted laser desorption/ionization rime-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products(9-11), mass-tagged oligonucleo tides(12), DNA created by restriction endonuclease cleavage(13), and genomi c DNA(14). We have previously reported MALDI-TOF-monitored nuclease selecti ons of modified oligonudeotides with increased affinity for targets(15). He re we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting pro bes that are not complementary to the DNA. With phosphodiesterase I, the ta rget-bound, complementary probe is largely refractory to nuclease attack an d its peak persists in mass spectra (Fig. 1A). In optimized assays, both al leles of a heterozygote were genotyped with six nonamer DNA probes (greater than or equal to 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).