Simultaneous determination of different DNA sequences by mass spectrometric evaluation of Sanger sequencing reactions.
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.
Kaetzke
Annette
A
Institute of Biochemistry, Medical Faculty, University of Leipzig, Liebigstrasse 16, D-04103 Leipzig, Germany.
Eschrich
Klaus
K
D000818:Animals; D001921:Brain / Q000201:enzymology; D004247:DNA / Q000032:analysis* / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000032:analysis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics; D010732:Phosphofructokinase-1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D051381:Rats; D017422:Sequence Analysis, DNA / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013698:Templates, Genetic
D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D007527:Isoenzymes; D012336:RNA, Ribosomal, 16S; D004247:DNA; D010732:Phosphofructokinase-1