Sheared DNA fragment sizing: comparison of techniques.

DNA fragmented by conventional French press shearing procedures (30,000 lbs/in2) has a number-average fragment size of 230 base pairs. This is considerably smaller than the 450 base pairs typically reported for DNA sheared by this method. Comparison of 5 sizing techniques indicates that sheared DNA fragment size is overestimated by either measurement of velocity sedimentation or Kleinschmidt Electron Microscopic visualization. Both adsorption grid electron microscopic visualization and gel electrophoresis yield the most reliable estimates of the mean size of small DNA fragment populations. In addition, the assessment of fragment size distribution (not possible from sedimentation analysis) potentially allows more critical evaluation of DNA hybridization and reassociation kinetic and measurement parameters.     

Application of automated DNA sizing technology for genotyping microsatellite loci.

Highly polymorphic microsatellite loci offer great promise for gene mapping studies, but fulfillment of this potential will require substantial improvements in methods for accurate and efficient genotyping. Here, we report a genotyping method based on fluorescently labeled PCR primers and size characterization of PCR products using an automated DNA fragment analyzer. We capitalize on the availability of three distinct fluorescent dyes to label uniquely loci that overlap in size, and this innovation increases by threefold the number of loci that can be analyzed simultaneously. We label size standards with a fourth dye and combine these with the microsatellite PCR products in each gel lane. Computer programs provide very rapid and accurate sizing of microsatellite alleles and efficient data management. In addition, fluorescence signals are linear over a much greater range of intensity than conventional autoradiography. This facilitates multiplexing of loci (since signal intensities often vary greatly) and helps distinguish major peaks from artifacts, thereby improving genotyping accuracy.     

An automated method for counting and sizing fish eggs

This paper describes an automated method for counting and sizing fish eggs in the diameter range 200–2500 μm. Eggs, suspended in water, are pumped at a controlled rate through an electronic sensor which produces a voltage pulse which is proportional in amplitude to particle size. The sensor is connected to a HIAC Criterion PC-320 particle analyser which is in turn connected to a Tracor Northern pulse height analyser. This equipment separates the voltage pulses into 398 recording channels. An ACT 1 Sirius microcomputer is used to store the data, transform it to egg size and then produce a histogram of egg size distribution in 20-μm class intervals.
The method has a counting accuracy of 99.5 ± 0.5%. It has been compared with two manual methods of estimating egg numbers.     

Dideoxy linear PCR on a commercial fluorescent automated DNA sequencer.

The use of automated fluorescent DNA sequencer systems and PCR-based DNA sequencing methods play an important role in the actual effort to improve the efficiency of large-scale DNA analysis. Here we show the application of the linear PCR using a single fluorescent primer and dideoxynucleotide terminators in four separate sequencing reactions on the EMBL/Pharmacia's fluorescent automated DNA sequencer. We have used dideoxy/deoxynucleoside triphosphate ratios and linear amplification cycle conditions to obtain an accurate sequencing response of up to, and over, 500 bases from just 400 ng of double-stranded DNA template without chemical denaturation. The sequencing protocol described in this paper is effectively suited for enhancement of sensitivity and performance of the automated DNA sequencing system.     

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis.

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.     

Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.     

Energy-transfer cassette labeling for capillary array electrophoresis short tandem repeat DNA fragment sizing.

Energy-transfer (ET) dye-labeled primers significantly improve fluorescent DNA detection because they permit excitation at a single common wavelength and they produce well separated and intense acceptor dye emission. Recently, a new ET cassette technology was developed [Berti, L. et al. (2001) Anal. Biochem. 292, 188-197] that can be used to label any PCR, sequencing, or other primer of interest. In this report we examine the utility of this ET cassette technology by labeling seven different short tandem repeat (STR) specific primers with each of the four ET cassettes and analyzing the PCR products generated on a MegaBACE-1000 capillary array electrophoresis system. More than 60 amplicons were generated and successfully analyzed with the ET cassette-labeled primers. Both forward and reverse primers were labeled for multiplex PCR amplification and analysis. Single base pair resolution was achieved with all four ET cassettes. This ET cassette-primer labeling procedure is ideally suited for creating four-color fluorescent ET primers for STR and other DNA assays where large numbers of different loci are analyzed including sequencing, genetic identification, gene mapping, loss of heterozygosity testing, and linkage analysis.     

An analytical system based on a compact flow cytometer for DNA fragment sizing and single-molecule detection.

BACKGROUND: Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to approximately 4 x 10(5) bp, in a flow system. B-phycoerythrin (B-PE), commonly used in conventional cytometric applications that require high-sensitivity, was the first fluorophore detected in flow at the single-molecule level. METHODS: Dilute solutions of stained DNA fragments or B-PE were analyzed in a simplified, compact flow system, with enhanced performance and lower cost, utilizing a solid-state laser and a single-photon sensing avalanche photodiode detector (SSAPD). Extensive data processing and display software, developed specifically for the photon-counting data stream, extracts correlated height, width, and area features from bursts of photons due to discrete molecules passing through the sensing region in the flow channel. RESULTS: DNA fragment sizing in flow has now been demonstrated for SYTOX-orange-stained fragments ranging in size over 3.4 orders of magnitude, from 125 to 5 x 10(5) bp. For Lambda bacteriophage DNA (lambda DNA; 48.5 kbp) a CV of 1.2 % has been achieved. Analysis of a femtomolar B-PE solution demonstrates that the bursts of photons from individual molecules can be baseline-resolved with 0.5 mW of laser power at a signal to noise ratio (SNR) of approximately 30, with approximately 100 photons detected from each molecule. CONCLUSIONS: A compact, low-power, high-sensitivity system detects DNA fragments as small as 125 bp or individual B-PE molecules in a flowing liquid stream. Demonstrated linearity, sensitivity, and resolution indicate that <1.0 mW of laser power is optimal, permitting further miniaturization of the system and additional cost reduction. Comprehensive analytical software exploits the standard cytometric paradigm of multiple 2D graphs and gating to extract features from classes of individually analyzed biomolecules. This complete system is thus poised to engage high-sensitivity applications not amenable to conventional flow cytometric instrumentation.     

Comparison of a prototype magnetoresistive biosensor to standard fluorescent DNA detection

We present a comparative analysis of a magnetoresistive biosensor to standard fluorescent DNA detection. The biosensor consists of giant magnetoresistive (GMR) type Cu/Ni(80)Fe(20) multilayers in the second antiferromagnetic coupling maximum. Each of the 206 elements of the magnetoresistive biosensor is patterned into a spiral-shaped line that can cover the area of a typical DNA spot (70 microm diameter). The probe DNA is assembled on top of the sensor elements in different concentrations ranging from 16 pg/microl to 10 ng/microl. Complementary biotin-labeled analyte DNA is hybridized to the probe DNA at a concentration of 10 ng/microl. A number of different commercially available magnetic microspheres are investigated to determine the most appropriate markers. The experimental comparison shows that the relative sensitivity of the magnetoresistive biosensor is superior to the fluorescent detection at low probe DNA concentrations.     

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.     

Development of an automated procedure for fluorescent DNA sequencing

We describe here the development of a procedure for complete automation of the dideoxynucleotide DNA sequencing chemistry using fluorescent dye-labeled oligonucleotide primers. This procedure combines rapid preparation of template DNA using a modification of the polymerase chain reaction, automation of the DNA sequencing reactions using a robotic laboratory workstation, and subsequent analysis of the fluorescent-labeled reaction products on a commercial automated fluorescent sequencer. Using this procedure, we were able to produce sufficient quantities of template DNA directly from bacterial colonies or bacteriophage plaques, perform the DNA sequencing reactions on these templates, and load the reaction products on the fluorescent DNA sequencer in a single work day. This scheme for automation of the fluorescent DNA sequencing method allows the fluorescent sequencer to be run at its full capacity every day and eliminates much of the labor required to obtain a high level of data output. Currently, we are able to perform and analyze 16 fluorescent-labeled reactions every day, with an average output of over 7000 bp per sequencer run.     

Universal fluorescent labeling (UFL) method for automated microsatellite analysis.

We have devised a novel method for automated microsatellite analysis using "universal" fluorescent labeling. This system is based on polymerase chain reactions driven by sequence-specific primers and a reporter primer labeled with a fluorescent dye at its 5' end. The forward sequence-specific primer is designed with a tag region bearing no homology to any human genomic sequence. Complementary tag sequences act as templates for the 6-carboxyfluorescein-labeled reporter primer, and those products can be analyzed with an autosequencer. The results we achieved with this assay system were consistent with the results of conventional assays using radioisotope-labeled primers, and diagnosis required less time. Furthermore, the fluorescent-labeled reporter primer is "universal" in that it can be used with different sequence-specific primers designed to carry the appropriate tag sequence at their 5'-ends. Our observations suggest that the "universal" fluorescent labeling method is an efficient tool for analyzing sequence variations in human DNA.     

Evaluation of automated threshold selection methods for accurately sizing microscopic fluorescent cells by image analysis

The accurate measurement of bacterial and protistan cell biomass is necessary for understanding their population and trophic dynamics in nature. Direct measurement of fluorescently stained cells is often the method of choice. The tedium of making such measurements visually on the large numbers of cells required has prompted the use of automatic image analysis for this purpose. Accurate measurements by image analysis require an accurate, reliable method of segmenting the image, that is, distinguishing the brightly fluorescing cells from a dark background. This is commonly done by visually choosing a threshold intensity value which most closely coincides with the outline of the cells as perceived by the operator. Ideally, an automated method based on the cell image characteristics should be used. Since the optical nature of edges in images of light-emitting, microscopic fluorescent objects is different from that of images generated by transmitted or reflected light, it seemed that automatic segmentation of such images may require special considerations. We tested nine automated threshold selection methods using standard fluorescent microspheres ranging in size and fluorescence intensity and fluorochrome-stained samples of cells from cultures of cyanobacteria, flagellates, and ciliates. The methods included several variations based on the maximum intensity gradient of the sphere profile (first derivative), the minimum in the second derivative of the sphere profile, the minimum of the image histogram, and the midpoint intensity. Our results indicated that thresholds determined visually and by first-derivative methods tended to overestimate the threshold, causing an underestimation of microsphere size. The method based on the minimum of the second derivative of the profile yielded the most accurate area estimates for spheres of different sizes and brightnesses and for four of the five cell types tested. A simple model of the optical properties of fluorescing objects and the video acquisition system is described which explains how the second derivative best approximates the position of the edge.     

A solvothermal method to synthesize fluorescent carbon nanoparticles and application to photocatalysis and electrocatalysis

A novel solvothermal approach to synthesize fluorescent carbon nanoparticles (CNPs) was developed using glucose and ammonium oxalate as the carbon source, and glycol as the solvent. The solution of as‐prepared CNPs emitted blue‐green fluorescence under ultraviolet (UV) light at 365 nm, and the carbon nanoparticle formation was investigated through XRD, TEM, DLS, FT‐IR, UV, PL, XPS. The particle was well dispersed with an average diameter of about 10–30 nm. In contrast with previous methods, in this synthesis process neither strong acid treatment nor further surface modification was necessary. The solution of as‐prepared CNPs were applied to photocatalytic degradation of mountain green in the present H₂O₂, and the decolorization rate was over 97% when the reaction time was more than 7 h under visible light. The as‐prepared CNPs were also applied to electrocatalysis and showed excellent electrocatalytic activity. Copyright © 2014 John Wiley & Sons, Ltd.     

A quick,reliable, and automated method for fat cell sizing

Mean diameters of fat cells from abdominal tissues from 31 volunteers were determined by three methods based on fat cell isolation after collagenase digestion and methylene blue staining. The three methods were direct microscopy (Micro), manual measurement of diameters from digital images by using the public domain NIH Image program (Scion), and automated measurement of diameters from digital images using a customized program developed by Biomedical Imaging Resource at Mayo Clinic (AdCount). There was excellent agreement between the methods' measurement of mean abdominal fat cell diameter (concordance correlation coefficient >0.84). The Scion method gave slightly but systematically lower mean abdominal fat cell diameters than did either AdCount or Micro. The AdCount approach produced results that are comparable to those from Micro. Comparison of AdCount and Micro in measuring diameters of fat cells from thigh confirmed the good comparability between the two methods independent of fat depot. AdCount is very reliable, and the quickest and most objective of the three methods in measuring fat cell diameters from various depots.     

A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing

A cost-effective, reliable, and reproducible method has been developed to produce good-quality, double-stranded plasmid DNA for automated sequence analysis. The method incorporates modifications to a previously described plasmid-purification protocol used in manual sequencing. The quality of the DNA produced from the present protocol is suitable for automated fluorescent sequencing. Using a dye-terminator sequencing protocol, most runs using plasmid DNA prepared using this protocol produced over 700 bases with greater than 99% base-calling accuracy.     

Dual fluorescence self‐assembled multilayers bearing a fluorescent internal standard for interfacial sensing

A novel dual fluorescence film Quartz/PSS/AO/PSS/CS/CdTe sensor was constructed based on layer‐layer electrostatic attraction using CdTe as a fluorescence probe. Acridine orange is shielded effectively by sodium poly(4‐styrenesulfonate) and chitosan, thus its fluorescence intensity does not change in the presence of the analyte, thereby acting as a fluorescent internal standard providing a reference signal. The ratio of dual fluorescence intensities of the film sensor is not affected by the fluctuation of the external environment factors such as excitation intensity, detector voltage of photo multiplier tube and sensor position in the cell holder. Assays based on this dual fluorescence self‐assembled multilayers sensor afforded an extremely high sensitivity for DNA with greatly accuracy. Copyright © 2010 John Wiley & Sons, Ltd.