Preparation and evaluation of packed capillary columns for the separation of nucleic acids by ion-pair reversed-phase high-performance liquid chromatography

Oligonucleotides and double stranded DNA fragments were separated in 200 microm I.D. capillary columns packed with micropellicular, octadecylated, 2.1 microm poly(styrene-divinylbenzene) particles by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC). Both the length and the diameter of the connecting capillaries (150 x 0.020 mm I.D.) as well as the detection volume (3 nl) had to be kept to a minimum in order to maintain the high efficiency of this chromatographic separation system with peak widths at half height in the range of a few seconds. Three different types of frits, namely sintered silica particles, sintered octadecylsilica particles, and monolithic poly(styrene-divinylbenzene) (PS-DVB) frits were evaluated with respect to their influence on chromatographic performance. Best performance for the separation of oligonucleotides and long DNA fragments was observed with the PS-DVB frits, whereas the short DNA fragments were optimally resolved in columns terminated by octadecylsilica frits. The maximum loading capacity of 60 x 0.20 mm I.D. columns ranged from 20 fmol (7.7 ng) for a 587 base pair DNA fragment to 500 fmol (2.4 ng) for a 16-mer oligonucleotide. Lower mass- and concentration detection limits in the low femtomol and low nanomol per liter range, respectively, make capillary IP-RP-HPLC with UV absorbance detection highly attractive for the separation and characterization of minute amounts of synthetic oligonucleotides, DNA restriction fragments, and short tandem repeat sequences amplified by polymerase chain reaction.     

Exciton transport in disordered crystals: velocity correlation functions

The two-particle coherent-potential approximation is used to calculate Frenkel-exciton group-velocity correlation functions for substitutionally disordered crystals. Most of the results can be described by a relaxation-time approximation, provided that k-dependent and complex relaxation times are allowed; however, some evidence for long-time tails, associated with the finite frequency range of the scattering potentials, is found. The probable accuracy of the approximation and its relationships with localization and kinetic theory are discussed, as is its relevance to experimental systems (triplet excitons in isotopic mixed naphthalene and anthracene crystals).     

Direct molecular haplotyping of multiple polymorphisms within exon 4 of the human catechol-O-methyltransferase gene by liquid chromatography–electrospray ionization time-of-flight mass spectrometry

The applicability of ion-pair reversed-phase high-performance liquid chromatography hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS) for the haplotyping of five SNPs (rs769223, rs4818, rs4986871, rs8192488, rs4680) located within exon 4 of the human catechol-O-methyltransferase (COMT, EC 2.1.1.6) gene is demonstrated. Two differently sized products of polymerase chain reaction—a 71-bp amplicon partially covering the sequence of a 124-bp amplicon—were used to determine unequivocally the allelic states of the single nucleotide polymorphisms linked on both chromosomes. The two amplicons were co-loaded onto the chromatographic column and simultaneously analyzed within a single gradient run. Using the described strategy, 101 individuals representing an Austrian population sample were typed. The obtained haplotype frequencies will serve as reference values in future association studies to examine the impact of the COMT gene on neuropsychiatric disorders. Additionally, two newly discovered polymorphic sites within the sequence of the COMT gene are described (a synonymous C>T mutation at the third position of the amino acid codon 99 in the soluble COMT protein or 149 in the membrane-bound COMT protein; a non-synonymous G>A substitution at the second position of the amino acid codon 95 in the soluble COMT protein or 145 in the membrane-bound-COMT protein).     

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.     

Applicability of tandem mass spectrometry to the automated comparative sequencing of long-chain oligonucleotides

An algorithm for the comparative sequencing (COMPAS) of oligonucleotides is shown to be suitable for the sequence verification of nucleic acids ranging in length from a few to 80 nucleotides. The algorithm is based on the matching of a fragment ion spectrum generated by collision-induced dissociation to m/z values predicted from a known reference sequence employing established fragmentation pathways. Prior to mass spectrometric investigation, the oligonucleotides were on-line purified by ion-pair reversed-phase high-performance liquid chromatography using monolithic separation columns. This study evaluated the potential and the limits of COMPAS regarding the length and the charge state of oligonucleotides, the selected collision energy, and the analyzed amount of sample using a quadrupole ion trap mass spectrometer. The results revealed that oligonucleotides could be very reliably re-sequenced up to 60-mers, although the algorithm was successfully used to even verify sequences up to 80-mers. The relative collision energy was typically in the range between 13 and 20%, which allowed in most cases a verification of the reference sequence in a window of at least three consecutive collision energies. To select a proper charge state for fragmentation, a compromise had to be found between high signal intensity and low charge state. Furthermore, by reducing the eluent flow rate during elution of the oligonucleotide, the sequence of a 50-mer was successfully verified from the analysis of 295 fmol of the raw product. COMPAS was proven to be reproducible and was applied to the genotyping of the polymorphic, Y-chromosomal locus M9 contained in a 62-base pair polymerase chain reaction product.     

Photodissociation dynamics of IBr(-)(CO(2))(n), n<15

We report the ionic photoproducts produced following photoexcitation of mass selected IBr(-)(CO(2))(n), n=0-14, cluster ions at 790 and 355 nm. These wavelengths provide single state excitation to two dissociative states, corresponding to the A(') (2)Pi(1/2) and B 2 (2)Sigma(1/2) (+) states of the IBr(-) chromophore. Excitation of these states in IBr(-) leads to production of I(-)+Br and Br(-)+I( *), respectively. Potential energy curves for the six lowest electronic states of IBr(-) are calculated, together with structures for IBr(-)(CO(2))(n), n=1-14. Translational energy release measurements on photodissociated IBr(-) determine the I-Br(-) bond strength to be 1.10+/-0.04 eV; related measurements characterize the A(') (2)Pi(1/2)<--X (2)Sigma(1/2) (+) absorption band. Photodissociation product distributions are measured as a function of cluster size following excitation to the A(') (2)Pi(1/2) and B 2 (2)Sigma(1/2) (+) states. The solvent is shown to drive processes such as spin-orbit relaxation, charge transfer, recombination, and vibrational relaxation on the ground electronic state. Following excitation to the A(') (2)Pi(1/2) electronic state, IBr(-)(CO(2))(n) exhibits size-dependent cage fractions remarkably similar to those observed for I(2) (-)(CO(2))(n). In contrast, excitation to the B 2 (2)Sigma(1/2) (+) state shows extensive trapping in excited states that dominates the recombination behavior for all cluster sizes we investigated. Finally, a pump-probe experiment on IBr(-)(CO(2))(8) determines the time required for recombination on the ground state following excitation to the A(') state. While the photofragmentation experiments establish 100% recombination in the ground electronic state for this and larger IBr(-) cluster ions, the time required for recombination is found to be approximately 5 ns, some three orders of magnitude longer than observed for the analogous I(2) (-) cluster ion. Comparisons are made with similar experiments carried out on I(2) (-)(CO(2))(n) and ICl(-)(CO(2))(n) cluster ions.     

Two-particle continuous-time random walks and binary reactions in disordered media

The relation between unary-(trappihg) and binary (mutual annihilation) reactions in disordered systems is studied in the framework of the continuous-time random walk. It is found that if the waiting-time distribution of the walk has infinite moments, a time-independent binary rate constant may exist even though a unary one does not.     

Potential anticonvulsants. III. The condensation of isatin with cyclic ketones

Isatin has been condensed with a series of cyclic ketones to give 3-substituted-3-hydroxyoxindoles as potential anticonvulsant agents.