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- W2025888918 abstract "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." @default.
- W2025888918 created "2016-06-24" @default.
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- W2025888918 date "2003-04-01" @default.
- W2025888918 modified "2023-10-15" @default.
- W2025888918 title "RNaseCut: a MALDI mass spectrometry-based method for SNP discovery" @default.
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- W2025888918 doi "https://doi.org/10.1093/nar/gng037" @default.
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