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Rapid genotyping by MALDI-monitored nuclease selection from probe libraries

Nature Biotechnology volume 18, pages 1213–1216 (2000)Cite this article

Abstract

Data on five single-nucleotide polymorphisms (SNPs) per gene are estimated to allow association of disease risks or pharmacogenetic parameters with individual genes1. Efficient technologies for rapidly detecting SNPs will therefore facilitate the mining of genomic information2. 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 probes3,4,5,6. Detection by mass spectrometry (MS) offers speed and high resolution7,8. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS) can detect primer extension products9,10,11, mass-tagged oligonucleotides12, DNA created by restriction endonuclease cleavage13, and genomic DNA14. We have previously reported MALDI-TOF-monitored nuclease selections of modified oligonucleotides with increased affinity for targets15. 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 (≥125 fmol each) and asymmetrically amplified DNA from exon 10 of the cystic fibrosis transmembrane regulatory gene (CFTR).

(A) Schematic representation of the processes underlying genotyping by selection. A library of probes is annealed to the target DNA, followed by treatment with a single-strand specific nuclease. MALDI-TOF mass spectra from the reaction solution, acquired under matrix conditions that lead to the dissociation of duplexes, allow detection of the selected, surviving probe, whereas peaks of the digested probes disappear from the mass region of interest. (B–D) Representative MALDI-TOF mass spectra of a four-component probe library (47 pmol/μl per probe) used to perform sequence analyses of CFTR 40-mer targets by monitored nuclease selection. (B) Spectrum at the beginning of the assay. (C,D) After 120 min reaction time with target 40-mers. (C) One equivalent of I506S target DNA. (D) Half an equivalent each of normal and Dual Poly (see Table 1) target DNA (simulated heterozygote).

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Figure 2: Genotyping of locus 2 on exon 10 of the CFTR gene.
Figure 3: Genotyping locus 1 in the CFTR gene with a six-probe library under optimized conditions.

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Acknowledgements

The authors thank Dr. Margery Beinfeld (Tufts University) for access to her equipment, Dr. W. Edward Highsmith (University of Maryland) for PCR primers and patient DNA, and Tufts University for support.

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  1. Department of Chemistry, Tufts University, Medford, 02155, MA

    Jay Stoerker, Jason D. Mayo, Charles N. Tetzlaff, David A. Sarracino, Ina Schwope & Clemens Richert

  2. Department of Pharmacology and Experimental Therapeutics, Boston, 02111, MA

    Jay Stoerker, Jason D. Mayo, Charles N. Tetzlaff, David A. Sarracino, Ina Schwope & Clemens Richert

  3. Department of Chemistry, University of Constance, Konstanz, D-78457, Germany

    Jason D. Mayo, Charles N. Tetzlaff & Clemens Richert

  4. Bruker Daltonics, Manning Park, Billerica, 01821, MA

    Jay Stoerker

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Correspondence to Clemens Richert.

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Stoerker, J., Mayo, J., Tetzlaff, C. et al. Rapid genotyping by MALDI-monitored nuclease selection from probe libraries . Nat Biotechnol 18, 1213–1216 (2000). https://doi.org/10.1038/81226

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