Seichter Doris, Krebs Stefan, Förster Martin
Lehrstuhl für Tierzucht und Allgemeine Landwirtschaftslehre, Tierärztliche Fakultät der Ludwig-Maximilians-Universität, Veterinärstrasse 13, D-80539 München, Germany.
Nucleic Acids Res. 2004 Jan 20;32(2):e16. doi: 10.1093/nar/gnh017.
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.
我们描述了通过分析核酸内切酶切割的RNA转录本，利用基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）对短串联重复序列（STR）进行表征的方法。选择了几个简单的牛STR基因座以及中断型和复合型微卫星作为模型基因座，以评估MALDI-TOF MS进行STR分析的能力。简而言之，所描述的方法包括对研究的STR序列进行PCR扩增，然后将其转录为RNA并由G特异性核糖核酸酶T1切割。转录本的碱基特异性切割产生了来自重复核心序列和侧翼区域的高信息量片段模式。由于核酸内切酶切割的序列特异性与MALDI-TOF测量的准确性相结合，该技术能够快速、可靠地确定简单重复序列的长度，并进一步表征STR等位基因序列，这在更复杂的STR基因座中尤其具有重要意义。
