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  • Title: Rapid genotyping by MALDI-monitored nuclease selection from probe libraries.
    Author: Stoerker J, Mayo JD, Tetzlaff CN, Sarracino DA, Schwope I, Richert C.
    Journal: Nat Biotechnol; 2000 Nov; 18(11):1213-6. PubMed ID: 11062445.
    Abstract:
    Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information. Known methods for SNP analysis include restriction-fragment-length polymorphism polymerase chain reaction (PCR), allele-specific oligomer hybridization, oligomer-specific ligation assays, minisequencing, direct sequencing, fluorescence-detected 5'-exonuclease assays, and hybridization with PNA probes. Detection by mass spectrometry (MS) offers speed and high resolution. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products, mass-tagged oligonucleotides, DNA created by restriction endonuclease cleavage, and genomic DNA. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets. Here we use nuclease selections for genotyping by treating DNA to be analyzed with oligonucleotide probes representing known genotypes and digesting probes that are not complementary to the DNA. With phosphodiesterase I, the target-bound, complementary probe is largely refractory to nuclease attack and its peak persists in mass spectra (Fig. 1A). In optimized assays, both alleles of a heterozygote were genotyped with six nonamer DNA probes (> or = 125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).
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