Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry.

Abstract

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.