Kirpekar F, Nordhoff E, Larsen L K, Kristiansen K, Roepstorff P, Hillenkamp F
Department of Molecular Biology, Odense University, Campusvej 55, DK-5230 Odense M, Denmark.
Nucleic Acids Res. 1998 Jun 1;26(11):2554-9. doi: 10.1093/nar/26.11.2554.
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. In the present work the use 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 by 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 in DNA sequencing, is fast, compatible with standard DNA sequencing procedures, and amenable to automation.
传统的DNA测序是基于测序产物的凝胶电泳分离。凝胶制备和电泳是耗时的步骤，并且由于某些片段的异常迁移，凝胶分离偶尔会不完美，从而导致错误的序列测定。此外，非法终止的产物常常无法与正确终止的产物区分开来，这种现象也会模糊数据解释。在本研究中，实现了使用基质辅助激光解吸电离飞行时间质谱（MALDI-MS）对从临床样本中扩增的DNA进行测序。从杂合子携带者扩增的DNA中明确且快速地鉴定缺失和替换，切实表明MALDI-MS作为未来常规测序程序用于高通量突变筛查的替代方法。通过对一个因凝胶电泳条带压缩和存在多个非特异性终止产物而无法用传统方法测序的DNA片段进行测序，证明了该方法的独特特性。利用MALDI-MS提供的精确质量信息，推导了序列，并确定了非特异性终止的性质。这里描述的方法提高了DNA测序的保真度，速度快，与标准DNA测序程序兼容，并且易于自动化。
