Krebs S, Seichter D, Förster M
Institute for Animal Breeding, Veterinary Medicine, Ludwig-Maximilian-University, Veterinärstr. 13, 80713 Munich, Germany.
Nat Biotechnol. 2001 Sep;19(9):877-80. doi: 10.1038/nbt0901-877.
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.
我们描述了一种用于短串联重复序列（STR）多态性高通量分型的方法。当前的凝胶电泳技术通量适中，操作和分析时间长，且输出结果是基于电泳迁移率的相对尺度，容易产生假象。基质辅助激光解吸/电离质谱（MALDI-MS）能够实现自动化高通量，并直接提供准确数据，描绘分析物的分子性质。然而，对大DNA片段的分析受到加合物形成和片段化的限制，这会导致峰展宽和低信号强度。已有报道称，MALDI对三核苷酸和四核苷酸重复序列进行多态性分型时具有足够的分辨率来区分等位基因。对于动物基因组研究中至关重要的二核苷酸重复序列，需要更高的分辨率。据报道，由于取代基不利于导致片段化的分子内反应，RNA（参考文献7）和修饰DNA（参考文献8 - 10）的质量分辨率有所提高。RNA转录本可以从含有启动子序列的PCR产物中酶促合成，无需特殊试剂或引物标记。此外，RNA转录本是单链的，这是核酸高分辨率质谱分析的前提条件。然而，病毒RNA聚合酶产生的3'异质性阻碍了RNA径流转录本的精确大小测定。已有报道称存在非模板化的多碱基延伸以及提前终止。二核苷酸重复序列的PCR倾向于删除重复单元，产生由交错延伸（来自RNA聚合酶）和缺失（来自PCR）组成的复杂模式，从而掩盖了真实的等位基因大小。我们通过添加一个编码催化RNA序列（即所谓的锤头状核酶）的3'序列来克服这一障碍，该核酶在转录过程中自我切割，产生一个均匀的3'末端。
