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Simultaneous determination of different DNA sequences by mass spectrometric evaluation of Sanger sequencing reactions

机译:通过Sanger测序反应的质谱分析同时测定不同的DNA序列

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摘要

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.
机译:目前所有可用的DNA测序方案基本上都取决于模板的同质性。在本文中,我们通过Sanger方法和产品的MALDI-TOF质谱分析相结合,描述了一个样品中各种模板的平行DNA测序。选择大鼠脑中细菌DF1020细菌的PCR扩增高变16S rDNA片段和6-磷酸果糖-1-激酶同工酶(PFK-1,EC 2.7.1.11)的cDNA作为基本异质模板的模型系统。避免克隆不均一的PCR产物,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-激酶的cDNA的三个序列。长度在21到25 nt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-1三种同功酶的存在。

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