Genotyping of dinucleotide tandem repeats by MALDI mass spectrometry of ribozyme-cleaved RNA transcripts.
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 analyte. Analysis of large DNA fragments, however, is limited by adduct formation and fragmentation, which result in peak broadening and low signal intensity. MALDI typing of polymorphic STRs has been reported for tri- and tetranucleotide repeats 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 reported. PCR of dinucleotide repeats tends toward the deletion of repeat units, 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 ribozyme, that cleaves itself co-transcriptionally, creating a homogeneous 3' end.
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Institute for Animal Breeding, Veterinary Medicine, Ludwig-Maximilian-University, Veterinärstr. 13, 80713 Munich, Germany. Stefan.Krebs@gen.vetmd.uni-muenchen.de
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D000483:Alleles; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D005821:Genetic Techniques; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry*; D016337:RNA, Catalytic / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D016337:RNA, Catalytic; D012313:RNA