Biomedical subjects

M J Doktycz

Publications and source records attributed to M J Doktycz.

At least 19 recordsLinked to original sources

PubMed2001

Towards environmental toxicogenomics -- development of a flow-through, high-density DNA hybridization array and its application to ecotoxicity assessment.

Assessment of the environmental hazard posed by soils/sediments containing low to moderate levels of contaminants using standard analytical chemical methods is uncertain due (in part) to a lack of information on contaminant bioavailability, the unknown interactive effects of contaminant mixtures, our inability to determine the species of a metal in an environmental matrix, and the relative sensitivity of bioassay species. Regulatory agencies compensate for this uncertainty by lowering cleanup goals, but in this process they effectively exclude otherwise attractive cleanup options (i.e. bioremediation). Direct evaluations of soil and sediment toxicity preclude uncertainty from most of these sources. However, the time and cost of chronic toxicity tests limits their general application to higher levels of tiered toxicity assessments. Transcriptional level (mRNA) toxicity assessments offer great advantages in terms of speed, cost and sample throughput. These advantages are currently offset by questions about the environmental relevance of molecular level responses. To this end a flow-through, high-density DNA hybridization array (genosensor) system specifically designed for environmental risk assessment was developed. The genosensor is based on highly regular microchannel glass wafers to which gene probes are covalently bound at discrete (200-microm diameter spot) and addressable (250-microm spot pitch) locations. The flow-through design enables hybridization and washing times to be reduced from approximately 18 h to 20 min. The genosensor was configured so that DNA from 28 environmental samples can be simultaneously hybridized with up to 64 different gene probes. The standard microscopic slide format facilitates data capture with most automated array readers and, thus high sample throughput (> 350 sample/h). In conclusion, hardware development for molecular analysis is enabling very tractable means for analyzing RNA and DNA. These developments have underscored the need for further developmental work in probe design software, and the need to relate transcriptional level data to whole-organism toxicity indicators.

Animals↗

PubMed2001

Improving spot homogeneity by using polymer substrates in matrix-assisted laser desorption/ionization mass spectrometry of oligonucleotides.

We describe a method for improving the homogeneity of MALDI samples prepared for analysis of small, single-stranded oligonucleotides using the widely used DNA matrix system, 3-hydroxypicolinic acid/picolinic acid/ ammonium citrate. This matrix system typically produces large crystals around the rim of the dried sample and requires tedious searching of this rim with the laser. However, when a substrate is prepared using both Nafion and a hydrophilic, high-molecular-weight polymer, such as linear polyacrylamide, linear poly(ethylene oxide), or methyl cellulose, oligonucleotide-doped matrix crystals tend to be smaller and more uniformly distributed across the entire spot, thus decreasing the time that is required for locating a usable signal. In addition to MALDI characterization of the spatial distribution of "sweet spots," fluorescence microscopy allows for imaging dye-labeled DNA in dried MALDI spots. The mechanism of enhanced uniformity may involve increased viscosity in the MALDI sample droplet due to partial solubilization of the substrate by the MALDI sample solvent as well as partitioning of the matrix or DNA between the solvent and the undissolved portion of the polymer substrate.

Base Sequence↗

PubMed2001

Spin-column isolation of DNA-protein interactions from complex protein mixtures for AFM imaging.

Applications of atomic force microscopy (AFM) to investigate structural-functional interactions between DNA and proteins, at the molecular level, should prove valuable for gaining a better understanding of gene expression. Specific genomic DNA-protein interactions occur within a sea of intracellular proteins. Successful AFM imaging requires isolating the specific DNA-protein complex free of background protein contamination. Using spin-column chromatography, we report the successful isolation and AFM imaging of transcription factor DNA complexes from DNA molecules incubated with crude cell lysates. This method should be applicable for the isolation and imaging of other specific DNA-protein complexes pertinent to functional genomic research.

Chromatography, Gel↗

PubMed2001

Modification of an automated liquid-handling system for reagent-jet, nanoliter-level dispensing.

Reducing the scale of biochemical reactions is becoming commonplace. Examples include the screening of large libraries of chemical compounds or gene sequences. These applications demand the ability to transfer sub-microliter volumes of fluid. To this end, we have modified a Hamilton MICROLAB 2200 with high-speed solenoids and a liquid pressurization system to modulate volume delivery down to the nanoliter level. Additional modifications include the use of sapphire-tipped dispensing nozzles and a high-resolution substage to assist in the construction of DNA microarrays. Techniques for characterizing the dispensed volume are presented.

Aluminum Oxide↗

PubMed2000

Identifying sequence similarities between DNA molecules.

An atomic force microscope (AFM) imaging technique is described to compare sequences between two different DNA molecules and precisely locate nonhomologies in DNA strands. Sequence comparisons are made by forming heteroduplexes between the two molecules and, by AFM imaging the intact molecules formed, identifying both homologous and nonhomologous regions. By forming heteroduplexes between linearized wildtype pSV-beta-galactosidase plasmid (6821 bp) and a series of deletion mutants we have identified nonhomologies (deletions) as small as 22 bp and as large as 418 bp. Furthermore, by incorporating our technique for AFM-mediated restriction mapping of DNA these mutations can be positioned relative to EcoRI restriction sites. These results suggest AFM can be useful in identifying molecular level similarities and differences in DNA.

Cloning, Molecular↗

PubMed1998

Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs.

The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention. Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing. Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared. A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data. A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides. DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done. The modification data support a very similar intracellular structure. When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo. A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model. With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Base Sequence↗

PubMed1998

MALDI-TOF analysis of polymerase chain reaction products from methanotrophic bacteria.

Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.

Biodegradation, Environmental↗

PubMed1997

Mapping individual cosmid DNAs by direct AFM imaging.

Individual cosmid clones have been restriction mapped by directly imaging, with the atomic force microscope (AFM), a mutant EcoRI endonuclease site-specifically bound to DNA. Images and data are presented that locate six restriction sites, predicted from gel electrophoresis, on a 35-kb cosmid isolated from mouse chromosome 7. Measured distances between endonuclease molecules bound to lambda DNA, when compared to known values, demonstrate the accuracy of AFM mapping to better than 1%. These results may be extended to identify other important site-specific protein-DNA interactions, such as transcription factor and mismatch repair enzyme binding, difficult to resolve by current techniques.

Animals↗

PubMed1996

Direct atomic force microscope imaging of EcoRI endonuclease site specifically bound to plasmid DNA molecules.

Direct imaging with the atomic force microscope has been used to identify specific nucleotide sequences in plasmid DNA molecules. This was accomplished using EcoRI (Gln-111), a mutant of the restriction enzyme that has a 1000-fold greater binding affinity than the wild-type enzyme but with cleavage rate constants reduced by a factor of 10(4). ScaI-linearized plasmids with single (pBS+) and double (pGEM-luc and pSV-beta-galactosidase) EcoRI recognition sites were imaged, and the bound enzyme was localized to a 50- to 100-nt resolution. The high affinity for the EcoRI binding site exhibited by this mutant endonuclease, coupled with an observed low level of nonspecific binding, should prove valuable for physically mapping large DNA clones by direct atomic force microscope imaging.

Binding Sites↗

PubMed1996

Detection of bacterial DNA polymerase chain reaction products by matrix-assisted laser desorption/ionization mass spectrometry.

Accurate monitoring and identification of Legionella species, the causative agents of Legionnaires' and other diseases, in environmental water sources is an important public health issue. Traditional culture methods often lack the sensitivity and specificity that can be attained using the polymerase chain reaction (PCR) to amplify targeted regions of the bacterial genome. Matrix-assisted laser desorption/ionization combined with time-of-flight (MALDI-TOF) mass spectrometry is shown to be useful for detection of 108- and 168-base PCR products specific to Legionella. A rapid purification aimed at removal of salts and unreacted primers is demonstrated. The addition of a synthetic DNA 20-mer to the MALDI sample facilitates aiming the laser at a favorable spot on the sample probe from which the PCR products can be detected.

Bacteria↗

PubMed1996

Discontinuous electrophoresis revisited: a review of the process.

The use and development of discontinuous buffer systems for the separation of proteins and nucleic acids are reviewed, and the advantages over a continuous buffer system are presented. Emphasis is given to the more recent applications in DNA separations where discontinuous systems have aided in the resolution and mobility tailoring of DNA fragments. Various DNA size ranges can be separated in short time periods by simple choice of buffer components rather than gel alterations. Guidelines for choosing appropriate buffer systems are offered as well as the application of borate complexes to modify the mobility of DNA. This article reviews the historical development of discontinuous buffers in the electrophoresis of proteins and nucleic acids with emphasis on the characteristics and applicability to nucleic acid separations.

Boric Acids↗

PubMed1995

Analysis of polymerase chain reaction-amplified DNA products by mass spectrometry using matrix-assisted laser desorption and electrospray: current status.

Recent advances in molecular biology are making it possible to diagnose genetic diseases and identify pathogens through the analysis of DNA. As clinical applications for molecular diagnosis increase, rapid, reliable methods for determination of DNA size will be needed. Mass spectrometry offers the potential of analyzing amplified DNA quickly and reliably, without the need for gel-based separation and sample labeling steps that are conventionally employed. Both electrospray ionization and matrix-assisted laser desorption/ionization have been evaluated for the size analysis of DNA using both synthetic oligonucleotides and PCR-amplified samples corresponding to bases 1626 to 1701 of the cystic fibrosis transmembrane conductance regulator gene. Both technologies have been demonstrated to have mass range and sensitivity required for the analysis of PCR-amplified DNA in this size range using minimal sample preparation. Steps required to incorporate either ionization technique into a reliable analytical scheme for the rapid, routine analysis of DNA are outlined.

Base Sequence↗

PubMed1995

Optical melting of 128 octamer DNA duplexes. Effects of base pair location and nearest neighbors on thermal stability.

The use of short oligonucleotide probes is finding increased application in DNA sequencing and genome characterization techniques, but a lack of knowledge of the hybridization properties of short duplexes hinders their use. Melting data were acquired on 128 DNA duplexes based on the length proposed in sequencing by hybridization procedures and formed from the general sequences 5'-XYZTGGAC-3',5'-GTCCAXYZ-3',5'-GCXYZGAC-3', and 5'-GTCXYZGC-3' where X, Y, and Z are either A, T, G, or C. These molecules were designed to elucidate the effects of location and nearest-neighbor stacking on the stability of base pairing in short DNA duplexes. The type of base pairs present had a major effect on stability, but was insufficient to predict stability without the inclusion of nearest-neighbor terms. Furthermore, the addition of information on position, or distance from the end, of the nearest-neighbor doublets led to statistically better fitting of the melting data. However, the positionally dependent stabilization differences are small compared with the contributions of base pairing and stacking.

Base Composition↗

PubMed1994

Accumulation and storage of ionized duplex DNA molecules in a quadrupole ion trap.

Evidence for the accumulation and storage of ionized duplex DNA molecules in a quadrupole ion trap is presented. Aqueous solutions of complementary single-strand molecules of DNA were annealed to form duplexes in solution and subjected to electrospray ionization. The ions liberated in this process were transported through an atmosphere/vacuum interface and injected into a quadrupole ion trap operated with a bath gas present at a pressure of 1 mTorr. Despite the roughly 2 order of magnitude poorer signal levels noted for electrospray of aqueous solutions relative to those observed for single-strand oligonucleotides in methanol solutions, aqueous solutions were used to avoid denaturing the duplexes. Ion trap mass spectra are reported here for duplexes consisting of two complementary 20-mer single strands and two complementary 10-mers. Tandem mass spectrometry results are also reported for the 10-mer duplex. These results are significant in that they indicate that the ions are kinetically stable under the ion injection, storage, and mass analysis conditions of the quadrupole ion trap operated with a relatively high pressure of bath gas. The tools of ion trap mass spectrometry can therefore be applied to this important class of compounds.

Base Sequence↗

PubMed1993

Sequence dependence of the free energy of B-Z junction formation in deoxyoligonucleotides.

The NaCl-induced transition from the right-handed B form to a hybrid form containing both left and right-handed DNA, joined by a B-Z junction, was investigated. Transition curves were constructed from circular dichroism spectra collected as a function of NaCl concentration for a series of 16 bp deoxyoligonucleotides. The sequence of the series (one strand of the duplex) was: 5'CGCGCGCGAMNGACTG, where C indicates m5dC, and -MN- was varied to include the possible Py:Py stacks: -CC-, -CT-, -TC- and -TT-. Transition curves for the conversion of all deoxyoligonucleotides were found to be biphasic. Singular value decomposition was used to analyze the experimental circular dichroism spectra obtained as a function of NaCl, and showed that the transition was not a simple two-state process, but rather required at least three species to account for the experimental data. A sequential three-state model, B<-->I<-->BZ, was derived and applied to analyze experimental transition curves. Non-linear least-squares analysis was used to evaluate the salt-dependent equilibrium constants for each step in the sequential reaction model. The results indicate that the free energy change for B-Z junction formation (delta Gj) depends on the dinucleotide sequence near the junction. At a Na activity of 5, delta Gj values ranging from +1.2 to +1.7 kcal mol-1 were determined, depending on the sequence near the junction. delta Gj was found to be strongly dependent on salt concentration, with its magnitude decreasing with increasing Na activity. In addition to studies on linear duplex molecules, the B to BZ transition was also investigated in "dumbbell" forms of selected sequences. In these molecules, the ends were covalently linked by a single-stranded T4 segment. These studies show that junction formation is energetically more costly in dumbbells than in their linear counterparts. A striking correlation was found between delta Gj and two independent conformational properties of the dinucleotide steps that were introduced into the linear duplex molecules. These are the free energy of unstacking and the estimated free energy change for the conversion of the dinucleotide from the B to the A conformation. The more stable against unstacking, or the more resistant to the B to A conversion, the sequence near the junction site is, the more costly is B-Z junction formation. These correlations reveal that the DNA sequence near the junction apparently must be pliable in order to accommodate the unusual structure of the junction.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Composition↗

PubMed1993

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base-pair sequence 5'G-T-A-T-C-C-C-T-C-T-G-G-A-T-A-C3' linked on the ends by dodecyl chains.

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C12H24 (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T4 loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na+ and revealed, regardless of [Na+], the duplex linked by dodecyl loops is more stable by at least 6 degrees C than the same duplex linked by T4 loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T4 end loops are essentially two state in 25 and 55 mM Na+. In contrast, significant deviations from two-state behavior were observed in 85 mM Na+. From our previously published melting data of DNA dumbbells with Tn end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881-892] and the dumbbell with T4 end loops of this study, a plot of d(Tm)/d ln [Na+] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (Tm)/d ln [Na+] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue.

Base Sequence↗

PubMed1993

Studies of DNA dumbbells. V. A DNA triplex formed between a 28 base-pair DNA dumbbell substrate and a 16 base linear single strand.

CD spectra and melting curves were collected for a 28 base-pair DNA fragment in the form of a DNA dumbbell (linked on both ends by T4 single-strand loops) and the same DNA sequence in the linear form (without end loops). The central 16 base pairs (bp) of the 28-bp duplex region is the poly(pu) sequence: 5'-AGGAAGGAGGAAAGAG-3'. Mixtures of the dumbbell and linear DNAs with the 16-base single-strand sequence 5'-TCCTTCCTCCTTTCTC-3' were also prepared and studied. At 22 degrees C, CD measurements of the mixtures in 950 mM NaCl, 10 mM sodium acetate, 1 mM EDTA, pH 5.5, at a duplex concentration of 1.8 microM, provided evidence for triplex formation. Spectroscopic features of the triplexes formed with either a dumbbell or linear substrate were quite similar. Melting curves of the duplex molecules alone and in mixtures with the third strand were collected as a function of duplex concentration from 0.16 to 2.15 microM. Melting curves of the dumbbell alone and mixtures with the third strand were entirely independent of DNA concentration. In contrast, melting curves of the linear duplex alone or mixed with the third strand were concentration dependent. At identical duplex concentrations, the dumbbell alone melts approximately 20 degrees C higher than the linear duplex. The curve of the linear duplex displayed a significant pretransition probably due to end fraying. On melting curves of mixtures of the dumbbell or linear duplex with the third strand, a low temperature transition with much lower relative hyperchromicity change (approximately 5%) was observed. This transition was attributed to the melting of a new molecular species, e.g., the triplex formed between the duplex and single-strand DNA molecules. In the case of the dumbbell/single-strand mixture, these melting transitions of the triplex and the dumbbell were entirely resolvable. In contrast, the melting transitions of the linear duplex and the triplex overlapped, thereby preventing their clear distinction. To analyze the data, a three-state equilibrium model is presented. The analysis utilizes differences in relative absorbance vs temperature curves of dumbbells (or linear molecules) alone and in mixtures with the third strand.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1993

Discontinuous electrophoresis of DNA: adjusting DNA mobility by trailing ion net mobility.

The use of a discontinuous buffer system, where a moving boundary separates ions of like charge but different ionic mobilities, for DNA electrophoresis may hold advantages over continuous zone electrophoresis in terms of resolution and electrophoresis time. Discontinuous buffer systems with calculated trailing ion net mobility were used to evaluate DNA mobility on gels of a constant pore size. Standard double-stranded DNA ladders and dideoxy sequencing ladders were electrophoresed on open-faced gels and standard sequencing gels, respectively. Trailing ion net mobility was systematically varied, while the leading ion mobility and concentration were kept constant. Decreasing trailing ion net mobility from 2.17 x 10(-4) to 0.59 x 10(-4) cm2 V-1s-1 generally led to increased DNA migration on both native and denaturing gels, allowing resolution of higher molecular weight DNAs with decreased electrophoresis time. However, on native open-faced gels, net trailing ion mobilities between 1.38 x 10(-4) and 1.76 x 10(-4) cm2 V-1s-1 had no differential effects for a 10-cm separation and kept DNAs smaller than approximately 75 bp stacked in the moving boundary and clearly resolved DNA between 100 and 600 bp. These results indicate that various DNA size ranges can be separated in short time periods by adjusting the net mobility of the trailing ion.

Amino Acids↗