DNA sequence analysis by MALDI mass spectrometry
DNA sequence analysis by MALDI mass spectrometry
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DOI：
10.1093/nar/26.11.2554
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发表时间：
1998-06-01
影响因子：
14.9
通讯作者：
Hillenkamp, F
中科院分区：
文献类型：
--
作者：
Kirpekar, F;Nordhoff, E;Hillenkamp, F
常规的DNA测序是基于测序产物的凝胶电泳分离，凝胶浇铸和电泳是时间限制步骤，并且凝胶分离偶尔由于某些片段的异常迁移率而不完美，导致错误的序列测定。此外，非法终止的产物经常不能与正确终止的产物区分开，这种现象也模糊了数据解释。本工作为MALDI质谱用于临床样本扩增DNA测序奠定了基础，明确和快速鉴定杂合携带者扩增DNA中的缺失和替换实际上表明MALDI质谱作为常规测序程序的未来替代方法用于高通量突变筛查。该方法的独特之处是通过对一个DNA片段进行测序来证明的，该DNA片段由于凝胶电泳条带压缩和存在多个非特异性终止产物而不能被常规测序。利用MALDI质谱提供的精确质量信息，推断序列，并确定非特异性终止的性质。本文描述的方法增加了DNA测序的保真度，是快速的，与标准DNA测序程序兼容，并适合自动化。
Conventional DNA sequencing is based on gel electrophoretic separation of the sequencing products, Gel casting and electrophoresis are the time limiting steps, and the gel separation is occasionally imperfect due to aberrant mobility of certain fragments, leading to erroneous sequence determination, Furthermore, illegitimately terminated products frequently cannot be distinguished from correctly terminated ones, a phenomenon that also obscures data interpretation. Ire the present work the base of MALDI mass spectrometry for sequencing of DNA amplified from clinical samples is implemented, The unambiguous and fast identification of deletions and substitutions in DNA amplified from heterozygous carriers realistically suggest MALDI mass spectrometry as a future alternative to conventional sequencing procedures for high throughput screening for mutations. Unique features of the method are demonstrated tay sequencing a DNA fragment that could not be sequenced conventionally because of gel electrophoretic band compression and the presence of multiple non-specific termination products. Taking advantage of the accurate mass information provided by MALDI mass spectrometry, the sequence was deduced, and the nature of the non-specific termination could be determined. The method described here increases the fidelity ire DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.