Some guidelines for the analysis of genomic DNA by PCR-LC-ESI-MS

Abstract

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. (J Am Soc Mass Spectrom 2006, 17, 124 -129)

Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry

“…Such limitations can be controlled through recent developments in instrument software, otherwise the valve for the C-trap inside the chassis must be tuned as described in the protocol section. As for sample preparation, use of detergent-free reagents is critical for the subsequent LC in terms of appropriate peak shape, sufficient peak intensity and stable retention time 31 .…”

“…A PS-DVB capillary monolith column 21,[24][25][26][27][28][29][30][31][32][33] , a C 18 reverse-phase particulate silica column 23,40,41 and a hydrophobic interaction chromatography (HILIC) column 42 have been used for the LC/ESI-MS analysis of oligonucleotides. Among them, the capillary monolith column is superior to the others in terms of separation capacity, however, the capillary monolith column used in past studies was made in-house and operated at a low flow rate (2 µl/min), which requires instrumentation dedicated to micro LC.…”

“…LC provides an on-line separation of the analytes from coexisting substances and does not require a laborious sample preparation prior to ionization 21,22 . Application of LC/ESI-MS genotyping for differentiation of pathogen species, single-nucleotide polymorphisms and short tandem repeats has been reported using a capillary polymer-monolith column that can separate longer oligonucleotides 1,21,[24][25][26][27][28][29][30][31][32][33] .…”

Genetics and the History of the Samaritans: Y-Chromosomal Microsatellites and Genetic Affinity between Samaritans and Cohanim

“…The choice of thermostable DNA polymerase is of utmost importance for efficient mass spectrometric sizing of PCR-amplified STRs (Oberacher et al 2006). DNA polymerases with intrinsic 3ʹ′>5ʹ′ exonuclease activity can proofread repeat deletion intermediates occurring due to enzyme slippage, thus lowering the frequency of deletion mutants by 2-to 10-fold (Kroutil and Kunkel 1999).…”

“…Aside from differences in PCR fidelity, provision of polymerases in storage buffers devoid of detergents eliminates the detrimental effect of detergents on performance of both reversed-phase high-performance liquid chromatography (Hecker 2003) and mass spectrometry (Oberacher et al 2006). …”