Automatic scanning of interphase FISH for prenatal diagnosis in uncultured amniocytes

Fluorescence in situ hybridization (FISH) of uncultured amniocytes using chromosome-specific DNA probes offers the opportunity for rapid aneuploidy screening. Between 80 and 95% of all chromosomal disorders expected in the second trimester of pregnancy can be discovered within 24 hr if DNA probes specific for chromosomes 21, 18, 13, X, and Y are used. Rapid results are crucial for clinical decision-making and are helpful in decreasing the anxiety level in most patients. One of the major factors that have been preventing the rapid FISH test from being broadly incorporated into the clinical setting is the limited staff in the cytogenetics laboratories. The present study demonstrates the use of an automated scanning system (Duet, BioView Ltd. Rehovot, Israel) for analyzing FISH in uncultured amniocytes. Fifty-six amniotic fluid samples were evaluated in parallel by karyotyping, manual FISH analysis, and automatic FISH scanning. Automatic scanning provided accurate results compared to both manual FISH scoring and karyotype analysis. The correlation between automatic and manual FISH scanning was found to be very high (r = 0.9, p < 0.0001). The availability of automation for aneuploidy screening in amniotic fluid samples will enable offering this test to a broader patient population while providing fast and reliable results.     

Video enhanced laser scanning microscopic analysis of DNA comet formation.

Laser Scanning Microscopy is a sensitive tool that provides a unique method of analyzing biological systems. Coupled with the Single Cell Gel assay, it allows for accurate and reproducible detection of DNA strand breaks. An understanding of the theory of DNA comet formation is lacking. Using dexamethasone induced apoptosis in murine thymocytes as a model for double strand breaks, we used video enhanced laser scanning microscopy to evaluate the leading edge of DNA migration in the single cell gel assay. In this system, comet length increases significantly within the first thirty seconds of electrophoresis, the greatest increase in length is completed within the first minute, and the first two minutes are important in significant increases in DNA migration during DNA comet formation.     

Electrochemical DNA biosensors based on thin gold films sputtered on capacitive nanoporous niobium oxide

Electrochemical DNA biosensors based on a thin gold film sputtered on anodic porous niobium oxide (Au@Nb(2)O(5)) are studied in detail here. We found that the novel DNA biosensor based on Au@Nb(2)O(5) is superior to those based on the bulk gold electrode or niobium oxide electrode. For example, the novel method does not require any time-consuming cleaning step in order to obtain reproducible results. The adhesion of gold films on the substrate is very stable during electrochemical biosensing, when the thin gold films are deposited on anodically prepared nanoporous niobium oxide. In particular, the novel biosensor shows enhanced biosensing performance with a 2.4 times higher resolution and a three times higher sensitivity. The signal enhancement is in part attributed to capacitive interface between gold films and nanoporous niobium oxide, where charges are accumulated during the anodic and cathodic scanning, and is in part ascribed to the structural stability of DNA immobilized at the sputtered gold films. The method allows for the detection of single-base mismatch DNA as well as for the discrimination of mismatch positions.     

Bayesian hierarchically weighted finite mixture models for samples of distributions

Finite mixtures of Gaussian distributions are known to provide an accurate approximation to any unknown density. Motivated by DNA repair studies in which data are collected for samples of cells from different individuals, we propose a class of hierarchically weighted finite mixture models. The modeling framework incorporates a collection of k Gaussian basis distributions, with the individual-specific response densities expressed as mixtures of these bases. To allow heterogeneity among individuals and predictor effects, we model the mixture weights, while treating the basis distributions as unknown but common to all distributions. This results in a flexible hierarchical model for samples of distributions. We consider analysis of variance-type structures and a parsimonious latent factor representation, which leads to simplified inferences on non-Gaussian covariance structures. Methods for posterior computation are developed, and the model is used to select genetic predictors of baseline DNA damage, susceptibility to induced damage, and rate of repair.     

Detecting Isolation by Distance Using Phylogenies of Genes

We introduce a method for analyzing phylogenies of genes sampled from a geographically structured population. A parsimony method can be used to compute s, the minimum number of migration events between pairs of populations sampled, and the value of s can be used to estimate the effective migration rate M, the value of Nm in an island model with local populations of size N and a migration rate m that would yield the same value of s. Extensive simulations show that there is a simple relationship between M and the geographic distance between pairs of samples in one- and two-dimensional models of isolation by distance. Both stepping-stone and lattice models were simulated. If two demes k steps apart are sampled, then, s, the average value of s, is a function only of k/(Nm) in a one-dimensional model and is a function only of k/(Nm)2 in a two-dimensional model. Furthermore, log(M) is approximately a linear function of log(k). In a one-dimensional model, the regression coefficient is approximately -1 and in a two-dimensional model the regression coefficient is approximately -0.5. Using data from several locations, the regression of log(M) on log(distance) may indicate whether there is isolation by distance in a population at equilibrium and may allow an estimate of the effective migration rate between adjacent sampling locations. Alternative methods for analyzing DNA sequence data from a geographically structured population are discussed. An application of our method to the data of R. L. Cann, M. Stoneking and A. C. Wilson on human mitochondrial DNA is presented.     

DNA complexes, formed on aqueous phase surfaces: new planar polymeric and composite nanostructures

The formation of DNA complexes with Langmuir monolayers of the cationic lipid octadecylamine (ODA) and the new amphiphilic polycation poly-4-vinylpyridine with 16% of cetylpyridinium groups (PVP-16) on the surface of an aqueous solution of native DNA of low ionic strength was studied. Topographic images of Langmuir-Blodgett films of DNA/ODA and DNA/PVP-16 complexes applied to micaceous substrates were investigated by the method of atomic force microscopy. It was found that films of the amphiphilic polycation have an ordered planar polycrystalline structure. The morphology of planar DNA complexes with the amphiphilic cation substantially depended on the incubation time and the phase state of the monolayer on the surface of the aqueous DNA solution. Complex structures and individual DNA molecules were observed on the surface of the amphiphilic monolayer. Along with quasi-linear individual bound DNA molecules, characteristic extended net-like structures and quasi-circular toroidal condensed conformations of planar DNA complexes were detected. Mono- and multilayer films of DNA/PVP-16 complexes were used as templates and nanoreactors for the synthesis of inorganic nanostructures via the binding of metal cations from the solution and subsequent generation of the inorganic phase. As a result, ultrathin polymeric composite films with integrated DNA building blocks and quasi-linear arrays of inorganic semiconductor (CdS) and iron oxide nanoparticles and nanowires were obtained. The nanostructures obtained were characterized by scanning probe microscopy and transmission electron microscopy techniques. The methods developed are promising for investigating the mechanisms of structural organization and transformation in DNA and polyelectrolyte complexes at the gas-liquid interface and for the design of new extremely thin highly ordered planar polymeric and composite materials, films, and coatings with controlled ultrastructure for applications in nanoelectronics and nanobiotechnology.     

Some Results on the Tukey-Mclaughlin and Yuen Methods for Trimmed Means when Distributions are Skewed

In applied work, distributions are often highly skewed with heavy tails, and this can have disastrous consequences in terms of power when comparing groups based on means. One solution to this problem in the one-sample case is to use the TUKEY and MCLAUGHLIN (1963) method for trimmed means, while in the two-group case YUEN's (1974) method can be used. Published simulations indicate that they yield accurate confidence intervals when distributions are symmetric. Using a Cornish-Fisher expansion, this paper extends these results by describing general circumstances under which methods based on trimmed means can be expected to give more accurate confidence intervals than those based on means. The results cover both symmetric and asymmetric distributions. Simulations are also used to illustrate the accuracy of confidence intervals using trimmed means versus means.     

Analyzing ion distributions around DNA

We present a new method for analyzing ion, or molecule, distributions around helical nucleic acids and illustrate the approach by analyzing data derived from molecular dynamics simulations. The analysis is based on the use of curvilinear helicoidal coordinates and leads to highly localized ion densities compared to those obtained by simply superposing molecular dynamics snapshots in Cartesian space. The results identify highly populated and sequence-dependent regions where ions strongly interact with the nucleic and are coupled to its conformational fluctuations. The data from this approach is presented as ion populations or ion densities (in units of molarity) and can be analyzed in radial, angular and longitudinal coordinates using 1D or 2D graphics. It is also possible to regenerate 3D densities in Cartesian space. This approach makes it easy to understand and compare ion distributions and also allows the calculation of average ion populations in any desired zone surrounding a nucleic acid without requiring references to its constituent atoms. The method is illustrated using microsecond molecular dynamics simulations for two different DNA oligomers in the presence of 0.15 M potassium chloride. We discuss the results in terms of convergence, sequence-specific ion binding and coupling with DNA conformation.     

Denaturation and renaturation of DNA. I. Equilibrium statistics of copolymeric DNA

The one-dimensional Ising model, with nearest neighbor correlation only, suitably modified, is used to explain the observed linear dependence of melting temperature of copolymeric DNA with GC content. Transition curves are plotted for regular, random, and Markoff distribution of base pairs for various values of a correlation parameter U between nearest neighbor bonds. Exact analytic formulas are given for fraction of bonds intact at a particular temperature for various regular distributions for all U and approximate ones for random and Markoff distributions for small U. A scheme is indicated for further improvement. The model, in principle, makes it possible to estimate the statistical distribution of base pairs from the detailed shape of the transition curve.     

Preselection of alarms in a hybrid system for screening of cervical cancer.

In this report, a preselection of alarms in a system for automated screening of cervical cancer based on depositing the cell sample linearly as a "cell trace" on a tape and analyzing it at different decision levels with increasing complexity, and preliminary results on analyzing cervical material with this system are discussed. The "cell trace" is analyzed with the slit-scan technique. Six parameters are computed: 1) cellular diameter; 2) nuclear diameter; 3) nuclear fluorescence (acriflavin-Feulgen) as nuclear DNA; 4) cellular fluorescence; 5) nuclear to cytoplasm ratio (N/C ratio); and 6) nuclear density. At present, only nuclear fluorescence is used to define a decision boundary between normal and potentially atypical cells. Under this criteria the slit-scan analysis leaves 5% of the events in a sample that must be rechecked at a second decision level in normal cell samples. A further reduction is expected when several slit-scan parameters are used at the first decision step. All events declared suspicious will be investigated in more detail by a two dimensional image analyzing system where the fluorescence image is generated by a laser scanning system. Results obtained in preliminary experiments are discussed in this paper.     

Spatial distribution of radiation-induced double-strand breaks in plasmid DNA as resolved by atomic force microscopy

Atomic force microscopy (AFM) has been used to directly visualize, size and compare the DNA fragments resulting from exposure to low- and high-LET radiation. Double-stranded pUC-19 plasmid ("naked") DNA samples were irradiated by electron-beam or reactor neutron fluxes with doses ranging from 0.9 to 10 kGy. AFM scanning in the tapping mode was used to image and measure the DNA fragment lengths (ranging from a few bp up to 2864 bp long). Double-strand break (DSB) distributions resulting from high-LET neutron and lower-LET electron irradiation revealed a distinct difference between the effects of these two types of radiation: Low-LET radiation-induced DSBs are distributed more uniformly along the DNA, whereas a much larger proportion of neutron-induced DSBs are distributed locally and densely. Furthermore, comparisons with predictions of a random DSB model of radiation damage show that neutron-induced DSBs deviate more from the model than do electron-induced DSBs. In summary, our high-resolution AFM measurements of radiation-induced DNA fragment-length distributions reveal an increased number of very short fragments and hence clustering of DSBs induced by the high-LET neutron radiation compared with low-LET electron radiation and a random DSB model prediction.     

GelJ – a tool for analyzing DNA fingerprint gel images

Background

DNA fingerprinting is a technique for comparing DNA patterns that has applications in a wide variety of contexts. Several commercial and freely-available tools can be used to analyze DNA fingerprint gel images; however, commercial tools are expensive and usually difficult to use; and, free tools support the basic functionality for DNA fingerprint analysis, but lack some instrumental features to obtain accurate results.

Results

In this paper, we present GelJ, a feather-weight, user-friendly, platform-independent, open-source and free tool for analyzing DNA fingerprint gel images. Some of the outstanding features of GelJ are mechanisms for accurate lane- and band-detection, several options for computing migration models, a number of band- and curve-based similarity methods, different techniques for generating dendrograms, comparison of banding patterns from different experiments, and database support.

Conclusions

GelJ is an easy to use tool for analyzing DNA fingerprint gel images. It combines the best characteristics of both free and commercial tools: GelJ is light and simple to use (as free programs), but it also includes the necessary features to obtain precise results (as commercial programs). In addition, GelJ incorporates new functionality that is not supported by any other tool.

     

Loading/release behavior of (chitosan/DNA)n layer-by-layer films toward negatively charged anthraquinone and its application in electrochemical detection of natural DNA damage

In the present work, positively charged chitosan (CS) and negatively charged DNA were alternately adsorbed on the surface of pyrolytic graphite (PG) electrodes, forming (CS/DNA)(n) layer-by-layer films. Cyclic voltammetry (CV) results showed that negatively charged electroactive probe, 9,10-anthraquinone-2,6-disulfonate (AQDS), could be loaded into the (CS/DNA)(n) films from its solution (1 mM at pH 7.0, containing 0.1 M NaCl), designated as (CS/DNA)(n)-AQDS, and then released from the films in blank buffers. The loading/release behavior of (CS/DNA)(n) films toward AQDS was found to be obviously different between double-stranded (dsDNA) and single-stranded DNA (ssDNA). The release rate of AQDS from (CS/dsDNA)(n) films was much slower than that from the ssDNA counterparts mainly because AQDS could be intercalated into the double helix structure of dsDNA despite the repulsion between likely charged AQDS and DNA. The loading/release behavior of (CS/DNA)(n) films toward AQDS in recognition of dsDNA and ssDNA was then successfully applied to electrochemically detect the damage of natural DNA caused by Fenton reaction. To further understand the essence of the interactions involved in the AQDS loading/release process for (CS/DNA)(n) films, comparison experiments were performed, in which either positively charged intercalator brilliant cresyl blue (BCB) was used to replace AQDS as the redox probe, or poly(diallyldimethylammonium) (PDDA) with relatively high positive charge density was used to replace CS as the constituent of layer-by-layer films with DNA. The loading/release behavior of DNA films toward electroactive intercalator may open new possibilities for dsDNA/ssDNA recognition and of DNA damage detection by electrochemistry.     

Accurate Determination of Membrane Dynamics with Line-Scan FCS

Here we present an efficient implementation of line-scan fluorescence correlation spectroscopy (i.e., one-dimensional spatio-temporal image correlation spectroscopy) using a commercial laser scanning microscope, which allows the accurate measurement of diffusion coefficients and concentrations in biological lipid membranes within seconds. Line-scan fluorescence correlation spectroscopy is a calibration-free technique. Therefore, it is insensitive to optical artifacts, saturation, or incorrect positioning of the laser focus. In addition, it is virtually unaffected by photobleaching. Correction schemes for residual inhomogeneities and depletion of fluorophores due to photobleaching extend the applicability of line-scan fluorescence correlation spectroscopy to more demanding systems. This technique enabled us to measure accurate diffusion coefficients and partition coefficients of fluorescent lipids in phase-separating supported bilayers of three commonly used raft-mimicking compositions. Furthermore, we probed the temperature dependence of the diffusion coefficient in several model membranes, and in human embryonic kidney cell membranes not affected by temperature-induced optical aberrations.     

Transition between two regimes describing internal fluctuation of DNA in a nanochannel

We measure the thermal fluctuation of the internal segments of a piece of DNA confined in a nanochannel about 50-100 nm wide. This local thermodynamic property is key to accurate measurement of distances in genomic analysis. For DNA in ~100 nm channels, we observe a critical length scale ~10 m for the mean extension of internal segments, below which the de Gennes' theory describes the fluctuations with no fitting parameters, and above which the fluctuation data falls into Odijk's deflection theory regime. By analyzing the probability distributions of the extensions of the internal segments, we infer that folded structures of length 150-250 nm, separated by ~10 m exist in the confined DNA during the transition between the two regimes. For ~50 nm channels we find that the fluctuation is significantly reduced since the Odijk regime appears earlier. This is critical for genomic analysis. We further propose a more detailed theory based on small fluctuations and incorporating the effects of confinement to explicitly calculate the statistical properties of the internal fluctuations. Our theory is applicable to polymers with heterogeneous mechanical properties confined in non-uniform channels. We show that existing theories for the end-to-end extension/fluctuation of polymers can be used to study the internal fluctuations only when the contour length of the polymer is many times larger than its persistence length. Finally, our results suggest that introducing nicks in the DNA will not change its fluctuation behavior when the nick density is below 1 nick per kbp DNA.     

Analysis of TPOX short tandem repeat locus with matrix-associated laser desorption/ionization time-of-flight-based restriction fragment mass polymorphism assay

Short tandem repeat (STR) loci are routinely analyzed by capillary electrophoresis. However, this method has several disadvantages, including long operational time, low throughput, and inaccuracy. As a result of the introduction of matrix-associated laser desorption/ionization time-of-flight (MALDI–TOF) and electrospray ionization (ESI), mass spectrometry has become an alternative method for genotyping polymorphic STR loci. Here we established a restriction fragment mass polymorphism (RFMP) assay for genotyping STR locus, TPOX, by typeIIS restriction endonuclease cleavage of polymerase chain reaction (PCR) amplicon followed by MALDI–TOF mass spectrometry. The resulting TPOX genotypes from this assay were in good agreement with the results from direct DNA sequencing and GeneScan assays. Our results showed that the RFMP assay is an accurate and high-throughput method for analyzing long DNA fragments such as STR markers. Further research with multiple STR loci may allow this assay to be used for diverse applications such as forensics, paternity tests, and detection of genetic disorders.     

Correlated cleavage of damaged DNA by bacterial and human 8-oxoguanine-DNA glycosylases

Many enzymes acting on specific rare lesions in DNA are suggested to search for their targets by facilitated one-dimensional diffusion. We have used a recently developed correlated cleavage assay to investigate whether this mechanism operates for Fpg and OGG1, two structurally unrelated DNA glycosylases that excise an important oxidative lesion, 7,8-dihydro-8-oxoguanine (8-oxoG), from DNA. Similar to a number of other DNA glycosylases or restriction endonucleases, Fpg and OGG1 processively excised 8-oxoG from pairs with cytosine at low salt concentrations, indicating that the lesion search likely proceeds by one-dimensional diffusion. At high salt concentrations, both enzymes switched to a distributive mode of lesion search. Correlated cleavage of abasic site-containing substrates proceeded in the same manner as cleavage of 8-oxoG. Interestingly, both Fpg and especially OGG1 demonstrated higher processivity if the substrate contained 8-oxoG.A pairs, against which these enzyme discriminate. Introduction of a nick into the substrate DNA did not decrease the extent of correlated cleavage, suggesting that the search probably involves hopping between adjacent positions on DNA rather than sliding along DNA. This was further supported by the observation that mutant forms of Fpg (Fpg-F110A and Fpg-F110W) with different sizes of the side chain of the amino acid residue inserted into DNA during scanning were both less processive than the wild-type enzyme. In conclusion, processive cleavage by Fpg and OGG1 does not correlate with their substrate specificity and under nearly physiological salt conditions may be replaced with the distributive mode of action.     

On numerical uncertainty in evaluation of pest population size

Obtaining reliable estimates of pest insect species abundance is an essential part of ecological monitoring programs. It is often the case that data available for obtaining such estimates are sparse which in turn makes achieving an accurate evaluation difficult. This is especially true for strongly heterogeneous pest population density distributions. In our paper we discuss the accuracy of a mean density estimate when a certain class of high aggregation density distributions is considered and a standard statistical method is employed to handle sparse sampled data. It will be shown in the paper that conventional conclusions about the accuracy of the pest population size evaluation do not work when the data are sparse and a new approach is required. Namely, if the number of traps is small, an estimate of the mean density becomes a random variable with an error of high magnitude and we have to compute the probability of an accurate estimate rather than computing the estimate itself. We have obtained a probability of an accurate estimate based on the assumption that only one trap falls within a sub-domain where the pest population density is different from zero. The probability has been calculated for the one-dimensional and the two-dimensional problem.     

The measurement of exonuclease activities by atomic force microscopy

We have applied atomic force microscopy (AFM) to the measurement of BAL 31 nuclease activities. BAL 31 nuclease, a species of exonuclease, is used to remove unwanted sequences from the termini of DNA before cloning. For cutting out only the appropriate sequences, it is important to know the nuclease properties, such as digestion speed and the distribution of the lengths of the digested DNA. AFM was used to obtain accurate measurements on the lengths of DNA fragments before and after BAL 31 nuclease digestion. We analyzed 4 DNAs with known number of base pairs (288, 778, 1818, and 3162 base pairs) for correlating the contour length measured by AFM with the number of base pairs under the deposition conditions used. We used this calibration for analyzing DNA degradation by BAL 31 nuclease from the AFM measurement of contour lengths of digested DNAs. In addition, the distribution of digested DNA could be analyzed in more detail by AFM than by electrophoresis, because digested DNA were measured as a population by electrophoresis, but were measured individually by AFM. These results show that AFM will be a useful new technique for measuring nuclease activities. Received: 8 August 1997 / Accepted: 10 September 1997