James E. Girard

Electrophoretic separations of polymerase chain reaction-amplified DNA fragments in DNA typing using a capillary electrophoresis- laser induced fluorescence system

Abstract Analysis of polymerase chain reaction (PCR)-amplified DNA fragments for human identification requires high-resolution separation and efficient detection of amplified alleles. Capillary electrophoresis (CE) with its speed, automation, high resolution and efficiency shows promise for analysing the amplified DNA fragments. CE with UV detection, however, suffers from lack of detector sensitivity owing to the limited detection path length of the capillary. By the use of intercalating dyes (TOTO and YOYO) a laser-induced fluorescence (LIF) detection system can provide much greater sensitivity for detecting DNA fragments. Femtogram quantities of dsDNA (φX174 HaeIII restriction digest mixture) per nanoliter of injected volume have been detected. Application of CE-LIF to analysis of PCR-amplified DNA fragments from three different genetic loci (apolipoprotein B, VNTR locus D1S80, mitochondrial DNA) is shown here. Further, the resolving power of a polymer-network capillary separation system is compared to that of a capillary-gel separation system.

A comparison of coulometric detectors for catecholamine analysis by LC-EC

An ion-pairing chromatographic method which uses a controlled potential coulometric detector is described. Two coulometric detectors with different electrolytic cell designs have been investigated. The resulting sensitivity can be comparable to the conventional amperometric detector. This technique has been applied to the analysis of catecholamines.

Isolation and identification of a sex attractant of a mushroom-infesting sciarid fly

A sciarid fly,Lycoriella mali (Fitch), is one of the most destructive pests to the commercial mushroom,Agaricus bisporus (Lange) Singer. Sanitation procedures and pesticide applications were ineffectual in controlling this pest. A supplemental pest control method employing sex pheromones was suggested since previous work indicated the presence of a sex attractant in the female. Diagnostic reactions performed on female extracts indicated saturated hydrocarbons as active components. The biologically most active fractions eluted from the gas chromatograph between standard hydrocarbons C15 and C22. Hydrocarbons C15, C16, C17, and C18 were isolated and identified via TLC, GC, and GC-MS. Male response to standard hydrocarbons C15 to C26 indicated that C17 is a major attractant. Male response to varying amounts of C17 indicated a preference for levels 10−6g and 10−9g.

Characterization of three Aroclor mixtures using a new cyanobiphenyl stationary phase

Abstract The retention characteristics of all 209 polychlorinated biphenyl (PCB) congeners were determined on a new p,p-cyanobiphenyl stationary phase using gas chromatography with electron capture detection (GC-ECD). Response factors were determined relative to decachlorobiphenyl, PCB 209. Several congeners that coelute on the phases routinely used for PCB analysis are separated on this phase, including the hexachlorobiphenyls 138, 163, and 164. The p,p-cyanobiphenyl stationary phase exhibits altered retention for planar congeners, such that the toxic coplanar PCBs 77, 126, and 169 are eluted free from interference. Of the 209 congeners, 61 were separated using the p,p-cyanobiphenyl phase in conjunction with GC-ECD. When analyzed by gas chromatography with mass selective detection (GC-MSD), the number of congeners determined increased to 133. Therefore GC-MSD was used with the p,p-cyanobiphenyl phase to characterize three PCB mixtures: Aroclor 1242, Aroclor 1254, and Aroclor 1260.

Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection

DNA analysis using capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection requires that polymerase chain reaction products either be prepared using primers with fluorescent molecules covalently bonded to them, or stained with a fluorescent intercalation dye following amplification. The intercalation technique has the advantage of allowing fluorescence detection of any double-stranded DNA (dsDNA) product regardless of the amplification primers used. The increased sensitivity of LIF detection is sometimes compromised by the intercalation dye changing the mass to charge ratio of the DNA. The purpose of this study was to evaluate the changes of migration rate, resolution and fluorescent intensity of dye-DNA complexes during electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1, F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and YOPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes, Eugene, OR, USA). Alleles intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The results of this experimentation were then applied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THOI locus were separated and typed correctly with a resolution of 0.49 in less than 20 min, and the only sample preparation necessary after amplification was a dilution step.

Evaluation of phenylthiourea derivatives as inhibitors of transglutaminase-catalyzed reaction in Chinese hamster ovary cells

1-(5-Aminopentyl)-3-phenylthiourea (PPTU), a recently developed inhibitor of the transglutaminase-catalyzed reaction (K.N. Lee, L. Fesus, S.T. Yancey, J.E. Girard, and S.I. Chung, (1985) J. Biol. Chem. 260, 14689-14694) was evaluated as a possible probe to examine the physiological role of transglutaminase (EC 2.3.2.13) in Chinese hamster ovary (CHO) cells. The [14C]PPTU in cell culture was readily taken up by CHO cells and was found to be covalently attached to high-molecular-weight proteins which are associated with the particulate fractions. Incubating cell homogenates, in the presence of Ca2+, incorporated the labeled PPTU exclusively into high-molecular-weight proteins that were also undergoing polymerization. PPTU at 0.1 mM, a concentration close to the Ki value reported for inhibition of tissue transglutaminase-catalyzed amine incorporation into the B chain of oxidized insulin, decreased high-molecular-weight protein polymerization, whereas PPTU at the same concentrations showed no effect on CHO cell proliferation or on in vitro calmodulin activation of cyclic nucleotide phosphodiesterase. These results suggest that transglutaminase may not be a constitutive enzyme in cell proliferation.

Pheromone of the male flesh fly,Sarcophaga bullata

Hexanal, isolated from a whole animal extract of the flesh fly,Sarcophaga bullata, attracts over 65% of the females tested with no apparent effect on males.

High-performance liquid chromatography separation methods for the analysis of peptide nucleic acids

An analytical analysis of peptide nucleic acids (PNAs) was carried out by reversed-phase HPLC using a solvent system comprised of aqueous trifluoroacetic acid and acetonitrile. A regression equation was obtained which represents the relationship of the molecular mass, sequence composition and retention time. This equation can be used to estimate the retention time of a known PNA under certain HPLC conditions. In addition to this equation, new HPLC conditions were also optimized which can be used for separation of pure PNAs.

Carbohydrate-borate eluents for anion chromatography

Abstract The chromatographic efficiencies of four different carbohydrate-borate eluents at pH values between 8.0 and 9.5 were compared. The carbohydrates studied were gluconate, mannonic acid, glucose and mannitol. The mannonic acid-borate eluent was a efficient as the original gluconate-borate eluent but the glucose-borate and mannitol-borate eluents gave poor results. For each eluent several carbohydrate borate complexes were responsible for the elution of anions.

Multivariate mixture analysis using reduced-mass-resolution membrane introduction mass spectrometry and variable selection

The feasibility of analyzing mixtures of spectrally similar analytes with low resolution mass spectrometers coupled with membrane introduction was studied. The performance of the multivariate calibration of an isometric ethyl benzene and p-xylene mixture remained essentially unchanged as the mass resolution degraded. The calibration performance also improved slightly as the data used for calibration decreased from the full mass spectra to only 12 or 18 fragment ions judiciously chosen by variable selection. The multivariate calibration and prediction for a more complex mixture of benzene and toluene only degraded slightly as the resolution decreased, while the result for the two isomers ethyl benzene and p-xylene got progressively worse. Depending on the variable selection algorithm and the number of fragment ions used, using only a select few ions for multivariate calibration and prediction gave results that were either similar to or slightly worse than results using the entire mass spectra. This paper demonstrates that mixture analysis performed with membrane introduction coupled to future miniature, low resolution mass spectrometers is possible.