RNaseCut: a MALDI mass spectrometry-based method for SNP discovery.
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.
Krebs
Stefan
S
Institute for Animal Breeding, Veterinary Medicine, Ludwig-Maximilian-University, Veterinärstrasse 13, 80539 Munich, Germany. stefan.krebs@gen.vetmed.uni-muenchen.de
Medugorac
Ivica
I
Seichter
Doris
D
Förster
Martin
M
D000818:Animals; D002364:Caseins / Q000235:genetics; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000235:genetics; D037601:Mannose-Binding Lectin / Q000235:genetics*; D008969:Molecular Sequence Data; D017354:Point Mutation; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011328:Prions / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000378:metabolism; D006163:Ribonuclease T1 / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D012260:Ribonucleases / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D012756:Sheep; D012984:Software; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D002364:Caseins; D037601:Mannose-Binding Lectin; D011328:Prions; D019253:Proto-Oncogene Proteins c-bcl-2; D012313:RNA; D004247:DNA; D012260:Ribonucleases; D006163:Ribonuclease T1; D012259:Ribonuclease, Pancreatic