Rapid and accurate characterisation of short tandem repeats by MALDI-TOF analysis of endonuclease cleaved RNA transcripts.
We describe the application of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for the characterisation of short tandem repeat (STR) sequences by the analysis of endonuclease cleaved RNA transcripts. Several simple bovine STR loci as well as interrupted and compound microsatellites were chosen as model loci to evaluate the capabilities of MALDI-TOF MS for STR analysis. In short, the described approach consists of a PCR amplification of the investigated STR sequence, which then is transcribed into RNA and cleaved by G-specific RNase T1. Base-specific cleavage of the transcript results in high informative fragment patterns from both the repetitive core sequence and the flanking region. Since sequence specificity from endonuclease cleavage is combined with the accuracy of MALDI-TOF measurements, this technique allows for fast and reliable determination of simple repeat lengths as well as for further characterisation of STR allele sequences, which is of high interest especially in more complex STR loci.
Seichter
Doris
D
Lehrstuhl für Tierzucht und Allgemeine Landwirtschaftslehre, Tierärztliche Fakultät der Ludwig-Maximilians-Universität, Veterinärstrasse 13, D-80539 München, Germany. Doris.Seichter@gen.vetmed.uni-muenchen.de
Krebs
Stefan
S
Förster
Martin
M
D018900:Dinucleotide Repeats / Q000235:genetics*; D005838:Genotype; D012313:RNA / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D006163:Ribonuclease T1 / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014158:Transcription, Genetic / Q000235:genetics; D018911:Trinucleotide Repeats / Q000235:genetics*
D012313:RNA; D006163:Ribonuclease T1