Abstract
We describe a method for high-throughput typing of short tandem repeat (STR) polymorphisms. Current gel electrophoresis techniques allow only moderate throughput with long hands-on and analysis time, and the output is on a relative scale of electrophoretic mobility, prone to artifacts. Matrix-assisted laser- desorption/ionization mass spectrometry (MALDI-MS) enables an automated high throughput and delivers accurate data directly depicting the molecular nature of the analyte1. Analysis of large DNA fragments, however, is limited by adduct formation and fragmentation2,3, which result in peak broadening and low signal intensity. MALDI typing of polymorphic STRs has been reported for tri- and tetranucleotide repeats1,4,5,6 with sufficient resolution to distinguish alleles. For dinucleotide repeats, essential in animal genome studies, an enhanced resolution is necessary. Increased mass resolution was reported for RNA (ref. 7) and modified DNA (refs 8–10) due to substituents that disfavor intramolecular reactions leading to fragmentation. RNA transcripts can be synthesized enzymatically from PCR products containing a promoter sequence, requiring no specialty reagents or primer labels. Furthermore, RNA transcripts are single-stranded, a prerequisite for high-resolution mass spectrometry of nucleic acids. The 3′ heterogeneities produced by viral RNA polymerases, however, impede exact sizing of RNA runoff transcripts. Non-templated multiple-base extensions as well as premature termination have been reported7,11. PCR of dinucleotide repeats tends toward the deletion of repeat units12,13, generating a complex pattern of interleaved extensions (from RNA polymerase) and deletions (from PCR) that obscure the true allele size. We overcome this obstacle by adding a 3′ sequence encoding a catalytic RNA sequence, the so-called hammerhead ribozyme14, that cleaves itself co-transcriptionally, creating a homogeneous 3′ end.
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Change history
27 March 2018
This article was initially published with an incorrect DOI that did not match the registered version at Crossref. The DOI has been corrected in the article.
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Acknowledgements
The authors thank K. Wild (EMBL Heidelberg) for helpful discussions. This work was realized with financial support of Tierzuchtforschung e.V. München, Arbeitsgemeinschaft Deutscher Rinderzüchter and the German Federal Department of Education, Science and Research.
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Krebs, S., Seichter, D. & Förster, M. Genotyping of dinucleotide tandem repeats by MALDI mass spectrometry of ribozyme-cleaved RNA transcripts. Nat Biotechnol 19, 877–880 (2001). https://doi.org/10.1038/nbt0901-877
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DOI: https://doi.org/10.1038/nbt0901-877
