Kaetzke Annette, Eschrich Klaus
Institute of Biochemistry, Medical Faculty, University of Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Nucleic Acids Res. 2002 Nov 1;30(21):e117. doi: 10.1093/nar/gnf116.
All currently available DNA sequencing protocols rest fundamentally upon the homogeneity of the template. In this paper we describe the parallel DNA sequencing of various templates in one sample by a combination of the Sanger method and MALDI-TOF mass spectrometric analysis of the products. PCR-amplified hypervariable 16S rDNA fragments of the bacterium Escherichia coli DF1020 and cDNA of the 6-phosphofructo-1-kinase isoenzymes (PFK-1, EC 2.7.1.11) in rat brain were chosen as model systems for essentially heterogeneous templates. Avoiding cloning of the inhomogeneous PCR products we were able to read three sequences for both the 16S rDNA fragment of E.coli DF1020 and the cDNA of 6-phosphofructo-1-kinase from the peak lists of the Sanger sequencing reactions. Short sequences with a length between 21 and 25 nt were sufficient to reflect the heterogeneity of the 16S rDNA genes in E.coli and the existence of three isoenzymes of PFK-1 in rat brain.
目前所有可用的DNA测序方案基本上都依赖于模板的均一性。在本文中，我们描述了通过Sanger法和对产物进行基质辅助激光解吸电离飞行时间质谱分析相结合的方法，对一个样本中的各种模板进行平行DNA测序。大肠杆菌DF1020的PCR扩增高变16S rDNA片段和大鼠脑中6-磷酸果糖-1-激酶同工酶（PFK-1，EC 2.7.1.11）的cDNA被选作基本异质模板的模型系统。在不克隆不均一PCR产物的情况下，我们能够从Sanger测序反应的峰列表中读取大肠杆菌DF1020的16S rDNA片段和6-磷酸果糖-1-激酶cDNA的三个序列。长度在21至25个核苷酸之间的短序列足以反映大肠杆菌中16S rDNA基因的异质性以及大鼠脑中PFK-1三种同工酶的存在。
