Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).     

Label-free quantitative DNA detection using the liquid core optical ring resonator

We demonstrated quantitative real-time label-free detection of DNA sequences using the liquid core optical ring resonator (LCORR) sensor. The LCORR is a recently developed sensing platform that integrates microfluidics and photonic sensing technology with low detection limit and sub-nanoliter detection volume. We analyzed experimentally and theoretically the LCORR response to a variety of DNA samples that had different strand lengths (25-100 bases), number of base- mismatches (1-5), and concentrations (10 pM to 10 microM) to evaluate the LCORR sequence detection capability. In particular, we established the linear correlation between the LCORR sensing signal and the molecule density, which allows us to accurately calculate the molecule density on the surface. It is found that the probe surface coverage was 26-51% and the extent of hybridization was 40-50%. The titration curve for 25-base probe and 25-base target DNA yields a dissociation constant of 2.9 nM. With a 37.1 nm/RIU LCORR, detection of 10 pM bulk DNA concentration was demonstrated. The mass detection limit was estimated to be 4 pg/mm(2), corresponding to a density of 10(10) molecules/cm(2) on the surface. We also showed that the LCORR was sensitive enough to differentiate DNA with only a few base-mismatches based on the raw sensing signal and kinetic analysis. Our work will provide important insight into the light-DNA interaction at the ring resonator surface and lay a foundation for future LCORR-based DNA label-free microarray development.     

Multimerization-cyclization of DNA fragments as a method of conformational analysis

Ligation of short DNA fragments results in the formation of linear and circular multimers of various lengths. The distribution of products in such a reaction is often used to evaluate fragment bending caused by specific chemical modification, by bound ligands or by the presence of irregular structural elements. We have developed a more rigorous quantitative approach to the analysis of such experimental data based on determination of j-factors for different multimers from the distribution of the reaction products. j-Factors define the effective concentration of one end of a linear chain in the vicinity of the other end. To extract j-factors we assumed that kinetics of the reaction is described by a system of differential equations where j-factors appear as coefficients. The assumption was confirmed by comparison with experimental data obtained here for DNA fragments containing A-tracts. At the second step of the analysis j-factors are used to determine conformational parameters of DNA fragments: the equilibrium bend angle, the bending rigidity of the fragment axis, and the total twist of the fragments. This procedure is based on empirical equations that connect the conformational parameters with the set of j-factors. To obtain the equations, we computed j-factors for a large array of conformational parameters that describe model fragments. The approach was tested on both simulated and actual experimental data for DNA fragments containing A-tracts. A-tract DNA bend angle determined here is in good agreement with previously published data. We have established a set of experimental conditions necessary for the data analysis to be successful.     

Exonuclease I hydrolyzes DNA with a distribution of rates

We report heterogeneity in the time necessary for Exonuclease I to hydrolyze identical DNA fragments. A real-time fluorescence method measured the time required by molecules of Exonuclease I to hydrolyze single-stranded DNA that was synthesized to have two fluorescently labeled nucleotides. One fluorescently labeled nucleotide was located near the 3′ end of the DNA and the other near the 5′ end. Heterogeneity in the hydrolysis rate of the exonuclease population was inferred from the distribution of times necessary to cleave these DNA fragments. In particular, we found simple first-order kinetics, using a single hydrolysis rate, did not result in a good fit to the data. Better fits to the data were obtained if one assumed a distribution of hydrolysis rates for the exonuclease population. Under our experimental conditions, this broad distribution of rates was centered near 100 nt/s.     

Sulfur signaling: is the agent sulfide or sulfane?

Triplex-forming oligonucleotides (TFOs) are sequence-dependent DNA binders that may be useful for DNA targeting and detection. A sensitive and convenient method to monitor triplex formation by a TFO and its target DNA duplex is required for the application of TFO probes. Here we describe a novel design by which triplex formation can be monitored homogeneously without prelabeling the target duplex. The design uses a TFO probe tagged with a fluorophore that undergoes fluorescence resonance energy transfer with fluorescent dyes that intercalate into the target duplex. Through color compensation analysis, the specific emission of the TFO probe reveals the status of the triple helices. We used this method to show that triple helix formation with TFOs is magnesium dependent. We also demonstrated that the TFO probe can be used for detection of sequence variation in melting analysis and for DNA quantitation in real-time polymerase chain reaction.     

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication

Background

Intrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease.

Results

In this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data.

Conclusions

We conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.     

Temperature-Regulated Surface Plasmon Resonance Imaging System for Bioaffinity Sensing

We describe a temperature-regulated surface plasmon resonance (SPR) imaging biosensor in this article. The sample temperature can be regulated for specific requirements of the bioaffinity sensing, and stabilized to suppress the measurement noise caused by temperature fluctuations. The water thermo optic coefficient is measured to test the temperature regulation performance. The protein interaction is monitored to demonstrate the feasibility of this system for real-time biomolecular interaction analysis. This temperature-regulated SPR imaging biosensor can be readily implemented by adding the common water path and peristaltic pump to the conventional SPR imaging system, which may provide an economical and convenient scheme to improve the analysis accuracy and quality of bioaffinity sensing using SPR sensing platform.     

Single-molecule sorting reveals how ubiquitylation affects substrate recognition and activities of FBH1 helicase

DNA repair helicases function in the cell to separate DNA duplexes or remodel nucleoprotein complexes. These functions are influenced by sensing and signaling; the cellular pool of a DNA helicase may contain subpopulations of enzymes carrying different post-translational modifications and performing distinct biochemical functions. Here, we report a novel experimental strategy, single-molecule sorting, which overcomes difficulties associated with comprehensive analysis of heterologously modified pool of proteins. This methodology was applied to visualize human DNA helicase F-box–containing DNA helicase (FBH1) acting on the DNA structures resembling a stalled or collapsed replication fork and its interactions with RAD51 nucleoprotein filament. Individual helicase molecules isolated from human cells with their native post-translational modifications were analyzed using total internal reflection fluorescence microscopy. Separation of the activity trajectories originated from ubiquitylated and non-ubiquitylated FBH1 molecules revealed that ubiquitylation affects FBH1 interaction with the RAD51 nucleoprotein filament, but not its translocase and helicase activities.     

Processing of gene expression data generated by quantitative real-time RT-PCR

Quantitative real-time PCR represents a highly sensitive and powerful technique for the quantitation of nucleic acids. It has a tremendous potential for the high-throughput analysis of gene expression in research and routine diagnostics. However, the major hurdle is not the practical performance of the experiments themselves but rather the efficient evaluation and the mathematical and statistical analysis of the enormous amount of data gained by this technology, as these functions are not included in the software provided by the manufacturers of the detection systems. In this work, we focus on the mathematical evaluation and analysis of the data generated by quantitative real-time PCR, the calculation of the final results, the propagation of experimental variation of the measured values to the final results, and the statistical analysis. We developed a Microsoft Excel-based software application coded in Visual Basic for Applications, called Q-Gene, which addresses these points. Q-Gene manages and expedites the planning, performance, and evaluation of quantitative real-time PCR experiments, as well as the mathematical and statistical analysis, storage, and graphical presentation of the data. The Q-Gene software application is a tool to cope with complex quantitative real-time PCR experiments at a high-throughput scale and considerably expedites and rationalizes the experimental setup, data analysis, and data management while ensuring highest reproducibility.     

Self-assembled pearling structure of long duplex DNA with histone H1.

We report that complexes of giant DNA molecules with histone H1 proteins form a pearl necklace-like structure when the complexes are prepared by natural dilution from a high-salt solution (2 M NaCl) to a low-salt solution (0.2 M and 50 mM NaCl). We performed real-time observations on the conformational changes of individual T4 phage DNA (166 kb) molecules in bulk solution by fluorescence microscopy. To identify H1-binding regions on individual DNA molecules, we also performed immunofluorescence microscopic observations on the DNA-H1 complex spread on a glass surface. It was found that histone H1 binds DNA in a highly co-operative manner and is accompanied by local folding of the DNA. On the basis of the experimental observations and a theoretical simulation, we propose a self-assembling mechanism for the pearling structure.     

Nondetectability of restriction fragments and independence of DNA fragment sizes within and between loci in RFLP typing of DNA.

We provide experimental evidence showing that, during the restriction-enzyme digestion of DNA samples, some of the HaeIII-digested DNA fragments are small enough to prevent their reliable sizing on a Southern gel. As a result of such nondetectability of DNA fragments, individuals who show a single-band DNA profile at a VNTR locus may not necessarily be true homozygotes. In a population database, when the presence of such nondetectable alleles is ignored, we show that a pseudodependence of alleles within as well as across loci may occur. Using a known statistical method, under the hypothesis of independence of alleles within loci, we derive an efficient estimate of null allele frequency, which may be subsequently used for testing allelic independence within and across loci. The estimates of null allele frequencies, thus derived, are shown to agree with direct experimental data on the frequencies of HaeIII-null alleles. Incorporation of null alleles into the analysis of the forensic VNTR database suggests that the assumptions of allelic independence within and between loci are appropriate. In contrast, a failure to incorporate the occurrence of null alleles would provide a wrong inference regarding the independence of alleles within and between loci.     

Fabrication and characterization of a radiation sensor based on bacteriorhodopsin

Available techniques of X-ray detection have been under development due to specific shortcomings such as finite lifetime, low sensitivity, and post-processing requirements. Here we report on the fabrication of an X-ray sensor based on bacteriorhodopsin (BR) with a radius of r=3mm as the sensing area on a flexible substrate. The flexible X-ray detector can be placed on the targeted area for real-time monitoring of radiation dosage. We show that BR sensor is a potential candidate for such a powerful sensing device. For this purpose, we measure the electrical current generated by the BR sensor under different radiation dosages, energies and dose rates. This averaged current is in the range of nanoampere and is proportional to the dose rate of the received X-ray. The current also increases with the increase of radiation energy. BR radiation sensor can be readily miniaturized and is relatively easy to fabricate. The capability for real-time data collection and reusability are other advantages of this radiation sensor.     

Easy and rapid method of zygosity determination in transgenic mice by SYBR<Superscript>®</Superscript> Green real-time quantitative PCR with a simple data analysis

Establishment and maintenance of transgenic mouse strains require being able to distinguish homozygous from heterozygous animals. To date, the developed real-time quantitative PCR techniques are often complicated, time-consuming and expensive. Here, we propose a very easy and rapid method with a simple data analysis to determine zygosity in transgenic mice. We show that the real-time quantitative PCR using SYBR Green fluorescent dye can be applied to discriminate two-fold differences in copy numbers of the transgene. Our procedure has to fit only three simple requirements: (1) to design primers capable of detecting one Ct difference for two-fold differences in DNA amounts (2) to measure genomic DNA concentrations accurately and (3) to have a reference animal of known zygosity in each run. Then, if the Ct values for the control gene are similar in all samples, we are able to compare directly the Ct values for the transgene in every sample, and so, to deduce the zygosity status of each mouse relative to the reference animal. This method is really simple and reliable, and it may be valuable as a rapid screening tool for zygosity status in transgenic animals.     

The presence of Saccharomyces cerevisiae DNA in various media used to propagate yeasts and its removal by ethidium monoazide

Aims:  In this study we demonstrate the interference of yeast extract in enumeration of Saccharomyces cerevisiae using real-time PCR and develop a method for its removal from the media using ethidium monoazide (EMA).
Methods and Results:  Using real-time PCR and primers to S. cerevisiae we demonstrate the presence of yeast DNA in various media as well as the media impact on S. cerevisiae real-time PCR standard curves. By pretreatment with EMA, we were able to remove this interference.
Conclusions:  Saccharomyces cerevisiae DNA can be found in a number of common laboratory media and may impact the enumeration of this yeast by real-time PCR. However, pretreatment with EMA eliminates this concern.
Significance and Impact of the Study:  We have developed a method for removal of contaminating DNA in yeast growth media.     

A rational approach in probe design for nucleic acid-based biosensing

Development of nucleic acid-based sensing attracts the interest of many researchers in the field of both basic and applied research in chemistry. Major factors for the fabrication of a successful nucleic acid sensor include the design of probes for target sequence hybridization and their immobilization on the chip surface. Here we demonstrate that a rational choice of bioprobes has important impact on the sensor's analytical performances. Computational evaluations, by a simple and freely available program, successfully led to the design of the best probes for a given target, with direct application to nucleic acid-based sensing. We developed here an optimized and reproducible strategy for in silico probe design supported by optical transduction experiments. In particular Surface Plasmon Resonance imaging (SPRi), at the forefront of optical sensing, was used here as proof of principle. Five probes were selected, immobilized on gold chip surfaces by widely consolidated thiol chemistry and tested to validate the computational model. Using SPRi as the transducting component, real-time and label free analysis was performed, taking the Homo sapiens actin beta (ACTB) gene fragment as model system in nucleic acid detection. The experimental sensor behavior was further studied by evaluating the strength of the secondary structure of probes using melting experiments. Dedicated software was also used to evaluate probes' folding, to support our criteria. The SPRi experimental results fully validate the computational evaluations, revealing this approach highly promising as a useful tool to design biosensor probes with optimized performances.     

Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis

Real-time PCR is being used increasingly as the method of choice for mRNA quantification, allowing rapid analysis of gene expression from low quantities of starting template. Despite a wide range of approaches, the same principles underlie all data analysis, with standard approaches broadly classified as either absolute or relative. In this study we use a variety of absolute and relative approaches of data analysis to investigate nocturnal c-fos expression in wild-type and retinally degenerate mice. In addition, we apply a simple algorithm to calculate the amplification efficiency of every sample from its amplification profile. We confirm that nocturnal c-fos expression in the rodent eye originates from the photoreceptor layer, with around a 5-fold reduction in nocturnal c-fos expression in mice lacking rods and cones. Furthermore, we illustrate that differences in the results obtained from absolute and relative approaches are underpinned by differences in the calculated PCR efficiency. By calculating the amplification efficiency from the samples under analysis, comparable results may be obtained without the need for standard curves. We have automated this method to provide a means of streamlining the real-time PCR process, enabling analysis of experimental samples based upon their own reaction kinetics rather than those of artificial standards.     

Application of method of moments analysis to fluorescence decay lifetime distributions

In fluorescence decay work, distributions of exponential decay lifetimes are anticipated where complex systems are examined. We describe here methods of gaining information on such distributions using the method of moments analysis approach. The information obtained may be as simple as the average and deviation of the lifetime distribution, quantities which we show may be estimated directly from the results of a multiexponential analysis. An approximation to the actual distribution shape may also be obtained using a procedure we call the variable filter analysis (VFA) method without making any assumptions about the shape of the distribution. Tests of VFA using both simulated and experimental data are described. Limitations of this method and of distribution analysis methods in general are discussed. Results of analyses on experimental decays for ethidium intercalated in core particles and in free DNA are reported.