RNaseCut: a MALDI mass spectrometry-based method for SNP discovery

Nucleic Acids Res. 2003 Apr 1;31(7):e37. doi: 10.1093/nar/gng037.


MALDI mass spectrometry is an established platform for high-throughput genotyping of single nucleotide polymorphisms (SNPs). For many species and also for specific ethnic groups, the number of described SNPs is far from sufficient. Here we present a method for SNP discovery that can use existing MALDI genotyping platforms and is automation-compatible. The method is based on in vitro RNA transcripts from PCR products, that can be used to obtain highly informative sequence fingerprints by digestion with the guanosine- specific ribonuclease T1. In these fingerprints, a mutation can be detected as either a mass shift, absence of an existing peak or appearance of an additional peak. Due to mass-degeneracy of fragments and multiple presence of shorter fragments in a given sequence, a certain fraction of possible mutations will remain undetected with this method. Screening of both strands from one PCR product is possible by using T3- and T7-tailed primers and the respective RNA polymerases, and markedly decreases the probability of missing an existing SNP. The use of mass-shifted nucleotides can significantly reduce fragment overlaps and hence increase detectability. We have used a simulation of RNase digests of a set of randomly generated sequences to provide estimates for the general detection probability in dependence on PCR product length. A software package is provided that helps to design PCR primers by plotting out regions with a high SNP discovery score, calculates expected mass fingerprints and peaklists from the target sequence selected for screening and helps in interpretation of digest spectra.

MeSH terms

  • Animals
  • Caseins / genetics
  • Cattle
  • DNA / chemistry
  • DNA / genetics
  • Mannose-Binding Lectin / genetics*
  • Molecular Sequence Data
  • Point Mutation
  • Polymorphism, Restriction Fragment Length
  • Polymorphism, Single Nucleotide / genetics*
  • Prions / genetics
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • RNA / genetics
  • RNA / metabolism
  • Ribonuclease T1 / metabolism
  • Ribonuclease, Pancreatic / metabolism
  • Ribonucleases / metabolism*
  • Sequence Analysis, DNA
  • Sheep
  • Software*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / methods


  • Caseins
  • Mannose-Binding Lectin
  • Prions
  • Proto-Oncogene Proteins c-bcl-2
  • RNA
  • DNA
  • Ribonucleases
  • Ribonuclease T1
  • Ribonuclease, Pancreatic

Associated data

  • GENBANK/AF515847
  • GENBANK/AF515848