Nucleic Acids Research, 2003, Vol. 31, No. 7 e37
© 2003 Oxford University Press
RNaseCut: a MALDI mass spectrometry-based method for SNP discovery
ugoracInstitute for Animal Breeding, Veterinary Medicine, Ludwig-Maximilian-University, Veterinärstrasse 13, 80539 Munich, Germany
*To whom correspondence should be addressed. Tel: +49 89 21805886; Fax: +49 89 344925; Email: stefan.krebs{at}gen.vetmed.uni-muenchen.de
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.
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