Köster H, Tang K, Fu D J, Braun A, van den Boom D, Smith C L, Cotter R J, Cantor C R
University of Hamburg, Department of Biochemistry and Molecular Biology, Germany.
Nat Biotechnol. 1996 Sep;14(9):1123-8. doi: 10.1038/nbt0996-1123.
Two methods of solid-phase Sanger DNA sequencing followed by detection with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry are demonstrated. In one method, sequencing ladders generated on an immobilized synthetic template were resolved up to the 63-mer including the primer. Detection sensitivity and resolution were sufficient for sequence analysis in the given range. This approach is particularly suitable for comparative (diagnostic) DNA sequencing. A second method that has the potential for high throughput de novo DNA sequencing is also presented; it uses immobilized duplex probes with five-base single-stranded overhangs to capture an unknown DNA template serving as primers for Sanger DNA sequencing. The power of mass spectrometry is demonstrated not only by its very high speed, but also by its ability to identify sequences that are not readable using gel electrophoresis.
展示了两种固相桑格DNA测序方法，随后用基质辅助激光解吸/电离飞行时间质谱法进行检测。在一种方法中，在固定化合成模板上产生的测序梯可解析至包括引物在内的63聚体。检测灵敏度和分辨率足以在给定范围内进行序列分析。这种方法特别适用于比较（诊断）DNA测序。还介绍了第二种具有高通量从头DNA测序潜力的方法；它使用具有五个碱基单链突出端的固定化双链探针来捕获未知DNA模板，作为桑格DNA测序的引物。质谱法的强大功能不仅体现在其非常高的速度上，还体现在其识别使用凝胶电泳无法读取的序列的能力上。
