Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS.
Ion-pair reversed-phase high-performance liquid chromatography online hyphenated to electrospray ionization mass spectrometry (ICEMS) represents an efficient method for the characterization of nucleic acids amplified by polymerase chain reaction (PCR). Since sample preparation is limited to PCR, the optimization of its solution conditions is of utmost importance for efficient mass spectrometric detection. The compatibility of a number of different commercially available PCR components including DNA polymerases, deoxynucleotide triphosphates, bovine serum albumin, enhancer, and ionic buffers was evaluated. These experiments revealed that higher concentration of enhancer and detergents such as Tween-20 or Nonidet P-40 impairs the mass spectrometric detection of nucleic acids and should be avoided within the PCR mixture. The optimized analytical platform was applied to the characterization of PCR products covering parts of the first hypervariable region of the noncoding mitochondrial control region. Truncated amplicons were detected attributable to the use of low quality primers. Furthermore, due to the proofreading activity of the applied polymerase system, mismatches between the primer and the target sequence located at the last or the second last base at the 3'-end of primers were corrected and detected within the corresponding amplicons.
Oberacher
Herbert
H
Institute of Legal Medicine, Innsbruck Medical University, Innsbruck, Austria. herbert.oberacher@uibk.ac.at
Niederstätter
Harald
H
Casetta
Bruno
B
Parson
Walther
W
D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D004272:DNA, Mitochondrial / Q000737:chemistry / Q000235:genetics; D006801:Humans; D007202:Indicators and Reagents; D009841:Oligonucleotides / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization
D004272:DNA, Mitochondrial; D007202:Indicators and Reagents; D009841:Oligonucleotides; D004247:DNA