Large DNA fragment sizing by flow cytometry: application to the characterization of P1 artificial chromosome (PAC) clones.

A flow cytometry-based, ultrasensitive fluorescence detection technique is used to size individual DNA fragments up to 167 kb in length. Application of this technology to the sizing of P1 artificial chromosomes (PACs) in both linear and supercoiled forms is described. It is demonstrated that this method is well suited to characterizing PAC/BAC clones and will be very useful for the analysis of large insert libraries. Fluorescence bursts are recorded as individual, dye stained DNA fragments pass through a low power, focused, continuous laser beam. The magnitudes of the fluorescence bursts are linearly proportional to the lengths of the DNA fragments. The histograms of the burst sizes are generated in <3 min with <1 pg of DNA. Results on linear fragments are consistent with those obtained by pulsed-field gel electrophoresis. In comparison with pulsed-field gel electrophoresis, sizing of large DNA fragments by this approach is more accurate, much faster, requires much less DNA, and is independent of the DNA conformation.     

Bacteria genome fingerprinting by flow cytometry.

BACKGROUND: A flow cytometry-based, ultrasensitive fluorescence detection technique has been developed that demonstrates unique advantages in the analysis of large DNA fragments over the currently most widely used technology, pulsed-field gel electrophoresis (PFGE). The technique described herein is used to characterize the restriction fingerprints of the bacteria genome Staphylococcus aureus in this study. METHODS: The isolation of the bacterial genomic DNA and the subsequent complete digestion by a restriction endonuclease were performed inside an agarose plug. Electroelution was used to move the DNA fragments out-of the agarose plug into a solution containing low concentrations of spermine and spermidine, added to stabilize the large DNA fragments. DNA was stained with the bisintercalating dye thiazole orange homodimer (TOTO-1) and subsequently introduced into our ultrasensitive flow cytometer from a capillary. RESULTS: Individual DNA fragments up to 351 kbp were successfully handled and sized. The histograms of the burst sizes were generated from signals associated with individual fragments in <7 min with <2 pg of DNA. The sizing accuracy was better than 98%. In contrast, standard PFGE takes approximately 20 h and requires approximately 1 microg of DNA with a sizing accuracy of approximately 90%. CONCLUSIONS: With the demonstrated success and advantages, our approach has the potential of being applied to fast, accurate bacteria species and strain identification.     

Bacterial fingerprinting by flow cytometry: bacterial species discrimination.

BACKGROUND: A flow cytometric measurement (FCM) technique has been developed to size DNA fragments. Individual fragments of a restriction digest of genomic DNA, stained with an intercalating dye, are passed through an ultrasensitive cytometer. The measured fluorescence intensity from each fragment is proportional to the fragment length. METHODS: The isolation of bacterial genomic DNA and digestion by restriction enzymes were performed inside an agarose plug. Rare cutting enzymes were employed to produce a manageable number of DNA fragments. Electroelution was used to move the DNA fragments from the agarose plug into a solution containing polyamines to protect the DNA from shear-induced breakage. The DNA was stained with the bisintercalating dye thiazole orange homodimer and introduced into our ultrasensitive flow cytometer. A histogram of the fluorescence intensities (fingerprint) was constructed. RESULTS: Gram-positive Bacillus globigii and gram-negative bacteria Escherichia coli and Erwinia herbicola were distinguished by the fingerprint pattern of restriction fragments of their genomic DNA. DNA sizes determined by FCM are in good agreement with pulsed-field gel electrophoresis (PFGE) analysis. Flow cytometry requires only picogram quantities of purified DNA and takes less than 10 min for data collection and analysis. When the total sample preparation time is included, the analysis times for PFGE and FCM are similar ( approximately 3 days). CONCLUSIONS: FCM is an attractive technique for the identification of bacterial species. It is more sensitive and potentially much faster than PFGE.     

Construction of a micro-library enriched with genomic replication origins of carrot somatic embryos by laser microdissection.

In this paper, we describe an effective method for constructing a micro-library enriched with chromosomal DNA replication origins. Carrot (Daucus carota L.) somatic embryos at early globular stage were incubated for 15 min in the presence of bromodeoxyuridine (BrdU) to pulse label newly synthesized DNA strands. Nuclei were isolated from the cells, and the DNA was extracted on microscopic slides. DNA fibers spread on slides were visualized using anti-BrdU and FITC-conjugated secondary antibodies. DNA regions where BrdU was incorporated were clearly visualized under a fluorescent microscope as dots on DNA fibers. Regions of DNA fiber containing many fluorescent dots should contain replicons in them. DNA fibers showing many fluorescence dots, or replicons were easily cut and collected using a laser microdissection system equipped with a pulse laser beam. DNA fragments containing many replicons were able to be collected with an efficiency of 20-30 DNA fragments per 1 h. Using degenerate oligonucleotide primed PCR, fragments were randomly amplified from the microdissected fragments, and subcloned to construct a micro-library. This is the first report of the application of a laser microdissection technique for constructing a micro-library enriched with replication origins of chromosomal DNA, although there were some reports on laser microdissection of chromosomes. The simple procedure established here should open up a new application of laser optics.     

A moving spot high resolution scanning microspectrometer using focused laser illumination: illustrations with acriflavin stained sephadex beads and human fibroblast nuclei.

We describe a high resolution moving spot scanning microspectrometer, capable of absorption or fluorescence detection, using focused laser illumination which is moved over the sample by rotating the laser beam direction prior to focusing. This rotation is achieved by reflecting the beam from mirrors mounted on bending mode piezoelectric transducers which, when bent by an applied voltage, cause the mirrors to rotate. The images of optically thin samples are analyzed by considering the convolution of the focused spot intensity distribution with the absorbance of a uniformly stained spherical particle. This analysis is verified experimentally with data from acriflavin stained Sephadex beads. Data from acriflavin-Feulgen stained human fibroblasts indicate that the efficiency of this type of nuclear staining is about 2 to 3 dye molecules incorporated per 100 nucleotide pairs. Quantitative data on fading of acriflavin fluorescence in stained fibroblasts indicate that fading is negligible in the time required to record the microscope images.     

Cylindrical illumination confocal spectroscopy: rectifying the limitations of confocal single molecule spectroscopy through one-dimensional beam shaping

Cylindrical illumination confocal spectroscopy (CICS) is a new implementation of single molecule detection that can be generically incorporated into any microfluidic system and allows highly quantitative and accurate analysis of single fluorescent molecules. Through theoretical modeling of confocal optics and Monte Carlo simulations, one-dimensional beam shaping is used to create a highly uniform sheet-like observation volume that enables the detection of digital fluorescence bursts while retaining single fluorophore sensitivity. First, we theoretically show that when used to detect single molecules in a microchannel, CICS can be optimized to obtain near 100% mass detection efficiency, <10% relative SD in burst heights, and a high signal/noise ratio. As a result, CICS is far less sensitive to thresholding artifacts than traditional single molecule detection and significantly more accurate at determining both burst rate and burst parameters. CICS is then experimentally implemented, optically characterized, and integrated into separate two microfluidic devices for the analysis of fluorescently stained plasmid DNA and single Cy5 labeled oligonucleotides. CICS rectifies the limitations of traditional confocal spectroscopy-based single molecule detection without the significant operational complications of competing technologies.     

Visualization of intracellular trafficking of exogenous DNA delivered by cationic liposomes

To visualize the intracellular trafficking of exogenous DNAs delivered by cationic liposomes, rhodamine-labeled DNAs were transfected into NIH3T3 cells and observed by confocal laser microscopy. After 0.5- to 1-h incubations, the DNAs reached the nucleus with a much higher frequency than that expected from the cell division rate. This result suggests that DNAs can enter the nucleus in the presence of the nuclear membrane. Interestingly, some DNAs appeared to extend through the nuclear membrane in the aggregated form which were much larger than the nuclear pore complex. The DNAs which have passed through the nuclear membrane were stained with SYTO 24, a DNA labeling reagent. The stained part may be "naked" DNA that is free of lipids or proteins. This observation indicates that a complex containing DNA fuses with the nuclear membrane and then naked DNA is released into the nucleus.     

Optical studies of complexes of quinacrine with DNA and chromatin: implications for the fluorescence of cytological chromosome preparations

The fluorescence and circular dichroism of quinacrine complexed with nucleic acids and chromatin were measured to estimate the relative magnitudes of factors influencing the fluorescence banding patterns of chromosomes stained with quinacrine or quinacrine mustard. DNA base composition can influence quinacrine fluorescence in at least two ways. The major effect, evident at low ratios of quinacrine to DNA, is a quenching of dye fluorescence, correlating with G-C composition. This may occur largely prior to relaxation of excited dye molecules. At higher dye/DNA saturations, which might exist in cytological chromosome preparations stained with high concentrations of quinacrine, energy transfer between dye molecules converts dyes bound near G-C base pairs into energy sinks. In contrast to its influence on quinacrine fluorescence, DNA base composition has very little effect on either quinacrine binding affinity or the circular dichroism of bound quinacrine molecules. The synthetic polynucleotides poly(dA-dT) and poly(dA)-poly(dT) have a similar effect on quinacrine fluorescence, but differ markedly in their affinity for quinacrine and in the circular dichroism changes associated with quinacrine binding. Quinacrine fluorescence intensity and lifetime are slightly less when bound to calf thymus chromatin than when bound to calf thymus DNA, and minor differences in circular dichroism between these complexes are observed. Chromosomal proteins probably affect the fluorescence of chromosomes stained with quinacrine, although this effect appears to be much less than that due to variations in DNA base composition. The fluorescence of cytological chromosome preparations may also be influenced by fixation effects and macroscopic variations in chromosome coiling.     

Flow microfluorometric and light-scatter measurement of nuclear and cytoplasmic size in mammalian cells.

A technique for rapid measurement of nuclear and cytoplasmic size relationships in mammalian cell populations has been developed. Based on fluorescence staining of either the nucleus alone or in combination with the cytoplasm using two-color fluorescence methods, this technique permits the simultaneous determination of nuclear and cytoplasmic diameters from fluorescence and light-scatter measurements. Cells stained in liquid suspension pass through a flow chamber at a constant velocity, intersecting a laser beam which excites cell fluorescence and causes light scatter. Depending upon which analysis procedure is used, optical sensors measure nuclear fluorescence and light scatter (whole cell size) or two-color nuclear and cytoplasmic fluorescence from individual cells crossing the laser beam. The time durations of signals generated by the nucleus and cytoplasm are converted electronically into signals proportional to the respective diameters and are displayed as frequency distribution hitograms. Illustrative examples of measurements on uniform microspheres, cultured mammalian cells and human exfoliated gynecologic cells are presented.     

Quantifying Double-Strand Breaks and Clustered Damages in DNA by Single-Molecule Laser Fluorescence Sizing

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.     

Development and use of metaphase chromosome flow-sorting methodology to obtain recombinant phage libraries enriched for parts of the human X chromosome

Metaphase chromosomes isolated from human lymphoblastoid cell lines containing structurally abnormal X chromosomes have been stained with the bisbenzimidazole dye Hoechst 33258 and analyzed on a FACS II flow system equipped with a 5-W all-lines argon ion laser. The chromosomal fluorescence has been highly resolved at flow rates of 1,000-3,000 chromosomes per second. With the goal of obtaining recombinant DNA libraries from parts of the human X chromosome, fluorescence populations enriched for a dicentric X (Xpter- greater than Xq24::Xq24-greater than Xpter) chromosome and an isochromosome of the long arm of the X [i(Xq)] have been identified. The dicentric X chromosome has been resolved as a discrete peak in the fluorescence flow histogram. In contrast, the fluorescence intensity of the isochromosome is indistinguishable from that of chromosomes 3 and 4. Recombinant DNA libraries from the flow-sorted chromosomes have been constructed in the lambda phage, Charon 21A, and consist of 1.6 X 10(5) and 0.7 X 10(5) plaque-forming units in the case of the dicentric X and the isochromosome, respectively. Ninety percent of the phage in both recombinant libraries contain inserts which hybridize to highly repetitive human DNA sequences. The recombinant phage library from the flow-sorted dicentric X chromosome, which could be assigned to a discrete fluorescence peak, has been further characterized and shows at least a tenfold enrichment for X chromosome-specific DNA sequences as determined by Southern blot hybridization of cloned fragments.     

Slit-scan flow cytometry of mammalian chromosomes.

A flow cytometer has been constructed which measures total fluorescence and the distribution of fluorescence along isolated, stained mammalian chromosomes. In this device, chromosomes flow lengthwise at 4 m/sec through a 1-micrometer thick laser beam. The fluorescence from each chromosome is recorded at 10 nsec intervals; the sequence of recorded values represents the distribution of fluorescence along the chromosome and is stored in the memory of a waveform recorder. The total fluorescence of each chromosome is also measured and recorded. Preliminary studies show that doublets of 1.83 micrometers diameter microspheres flow with their long axes parallel to the direction of flow and that the two microspheres are resolved in the slit-scan profile. Ethidium bromide stained Muntjac and Chinese hamster chromosomes have also been slit-scanned. Centromeres were resolved in many of the Nos. 1 and 2 Chinese hamster chromosomes and the Nos. 1 and X + 3 Muntjac chromosomes.     

Fingerprinting of single viral genomes

We demonstrate the use of technology developed for optical mapping to acquire DNA fingerprints from single genomes for the purpose of discrimination and identification of bacteria and viruses. Single genome fingerprinting (SGF) provides not only the size but also the order of the restriction fragments, which adds another dimension to the information that can be used for discrimination. Analysis of single organisms may eliminate the need to culture cells and thereby significantly reduce analysis time. In addition, samples containing mixtures of several organisms can be analyzed. For analysis, cells are embedded in an agarose matrix, lysed, and processed to yield intact DNA. The DNA is then deposited on a derivatized glass substrate. The elongated genome is digested with a restriction enzyme and stained with the intercalating dye YOYO-1. DNA is then quantitatively imaged with a fluorescence microscope and the fragments are sized to an accuracy >or=90% by their fluorescence intensity and contour length. Single genome fingerprints were obtained from pure samples of adenovirus, from bacteriophages lambda and T4 GT7, and from a mixture of the three viral genomes. SGF will enable the fingerprinting of uncultured and unamplified samples and allow rapid identification of microorganisms with applications in forensics, medicine, public health, and environmental microbiology.     

Unmethylated domains in vertebrate DNA.

We have detected a fraction that is rich in unmethylated HpaII and HhaI sites by end-labelling HpaII fragments of chicken DNA. The fraction is not obvious when DNA fragments are stained with ethidium bromide as it amounts to less than 2% of the genome. The average frequency of sites for HpaII is over thirteen times greater in the unmethylated fraction than in total DNA. Partial digests indicate that the unmethylated sites are clustered in the genome. Similar unmethylated fractions were detected in six other vertebrates in both somatic and germ line DNA.     

Caffeine dissociates complexes between DNA and intercalating dyes: application for bleaching fluorochrome-stained cells for their subsequent restaining and analysis by laser scanning cytometry

BACKGROUND: Removal of the nucleic acid-bound fluorochrome is desirable when stained cells have to be reanalyzed using other fluorochromes. It is also often desirable to remove DNA-bound antitumor drugs from drug-treated cells, to improve cell staining. We have previously observed that in aqueous solutions, the methylxanthine caffeine (CFN) decreases interactions between planar aromatic molecules such as intercalating dyes or antitumor drugs and nucleic acids. The aim of this study was to explore whether this property of CFN can be utilized to remove the DNA-bound intercalating dyes propidium iodide (PI) or 7-aminoactinomycin D (7-AAD) from the cells and whether the bleached cells can be restained and reanalyzed. METHODS: HL-60 cells were fixed in 70% ethanol and their DNA was stained with PI or 7-AAD. The cells were then rinsed with a 0.05 M solution of CFN in phosphate-buffered saline (PBS) or with PBS alone. The decrease in intensity of cell fluorescence during rinsing was measured by laser scanning cytometry (LSC) to obtain the bleaching kinetics of individual cells. The bleached cells were then restained with PI, 7-AAD, or the protein-specific fluorochrome sulforhodamine 101(S101). Their fluorescence was measured again by LSC. In addition, free DNA was subjected to gel electrophoresis, DNA bands in the gels were stained with ethidium bromide (EB), and the gels were rinsed with a solution of CFN or PBS to bleach the DNA band's fluorescence. RESULTS: Rinsing the PI or 7-AAD-stained cells with solutions of CFN led to nearly complete removal of PI and a more than 75% decrease in 7-AAD fluorescence after 10 min. The rinse with PBS decreased the PI cell fluorescence intensity by less than 30% and the 7-AAD fluorescence by about 50%. The differences in kinetics of PI or 7-AAD removal by CFN from G2/M versus G1 cells suggest that these intercalators bind more strongly to DNA in chromatin of G2/M than G1 cells. The CFN-bleached cells were then successfully stained with S101 and again with PI or 7-AAD. The bivariate analysis of the LSC merged files of the cells sequentially stained with PI and S101 revealed typical DNA/protein distributions. The fluorescence of EB-stained DNA bands in gels was also nearly completely removed by rinsing gels in 0.05 M CFN; PBS alone had a distinctly lesser effect. CONCLUSION: Solutions of CFN can dissociate the DNA-bound PI, 7-AAD, EB, and possibly other intercalating fluorochromes. The bleached cells can be restained and reanalyzed by LSC. This approach can also be used to remove such fluorochromes from nucleic acids immobilized in gels and perhaps in other solid matrices. Analysis of the kinetics of fluorochrome removal from cells can possibly be used to study their binding affinities to nucleic acids in situ.     

Rapid DNA fingerprinting of pathogens by flow cytometry

BACKGROUND: A new method for rapid discrimination among bacterial strains based on DNA fragment sizing by flow cytometry is presented. This revolutionary approach combines the reproducibility and reliability of restriction fragment length polymorphism (RFLP) analysis with the speed and sensitivity of flow cytometry. METHODS: Bacterial genomic DNA was isolated and digested with a rare-cutting restriction endonuclease. The resulting fragments were stained stoichiometrically with PicoGreen dye and introduced into an ultrasensitive flow cytometer. A histogram of burst sizes from the restriction fragments (linearly related to fragment length in base pairs) resulted in a DNA fingerprint that was used to distinguish among different bacterial strains. RESULTS: Five different strains of gram-negative Escherichia coli and six different strains of gram-positive Staphylococcus aureus were distinguished by analyzing their restriction fragments with DNA fragment sizing by flow cytometry. Fragment distribution analyses of extracted DNA were approximately 100 times faster and approximately 200,000 times more sensitive than pulsed-field gel electrophoresis (PFGE). When sample preparation time is included, the total DNA fragment analysis time was approximately 8 h by flow cytometry and approximately 24 h by PFGE. CONCLUSIONS: DNA fragment sizing by flow cytometry is a fast and reliable technique that can be applied to the discrimination among species and strains of human pathogens. Unlike some polymerase chain reaction (PCR)-based methods, sequence information about the bacterial strains is not required, allowing the detection of unknown, newly emerged, or unanticipated strains.     

Flow cytometry of DNA content using oxazine 750 or related laser dyes with 633 nm excitation

The laser dyes oxazine 750 (OX750), LD700, and rhodamine 800 (R800) can be used in an instrument employing a low-power helium-neon laser source for flow cytometry of DNA content in ethanol-fixed or detergent-permeabilized cells. Cells in near-isotonic medium are stained with 10-30 microM dye, and fluorescence excited at 633 nm is measured at wavelengths above 665 nm. The dyes do not appear to stain RNA, and the intensity of DNA staining is not changed when 2 microM Hoechst 33342 is added to cells simultaneously with a red-excited dye. The effects on fluorescence of addition of DNA to LD700 or R800 in aqueous solution are strongly influenced by the base composition of the DNA; binding mechanisms remain to be determined.     

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.     

Sizing of single fluorescently stained DNA fragments by scanning microscopy

We describe an approach to determine DNA fragment sizes based on the fluorescence detection of single adsorbed fragments on specifically coated glass cover slips. The brightness of single fragments stained with the DNA bisintercalation dye TOTO-1 is determined by scanning the surface with a confocal microscope. The brightness of adsorbed fragments is found to be proportional to the fragment length. The method needs only minute amount of DNA, beyond inexpensive and easily available surface coatings, like poly-L-lysine, 3-aminoproyltriethoxysilane and polyornithine, are utilizable. We performed DNA-sizing of fragment lengths between 2 and 14 kb. Further, we resolved the size distribution before and after an enzymatic restriction digest. At this a separation of buffers or enzymes was unnecessary. DNA sizes were determined within an uncertainty of 7-14%. The proposed method is straightforward and can be applied to standardized microtiter plates.     

The orientation of DNA fragments in the agarose gels

A microscopic method of measuring the orientation of nucleic acids in the agarose gels is described. A nucleic acid undergoing electrophoresis is stained with the dye ethidium bromide and is viewed under high magnification with a polarization microscope. A high-numerical-aperture microscope objective is used to illuminate and to collect the fluorescence signal, and therefore the orientation of the minute quantities of nucleic-acid can be measured: in a typical experiment we can detect the orientation of one-tenth of a picogram (10(13)g) of DNA. Polarization properties of the fluorescent light emitted by the separate bands corresponding to different molecular weights of the DNA are examined. A linear dichroism equation relates the measured fluorescence to the mean orientation of the absorption dipole of the ethidium bromide (and therefore DNA) and to the extent to which it is disorganized. As an example, we measured the orientation of phi X174 DNA RF/HaeIII fragments undergoing electrophoresis in a field of 10 V/cm. Ethidium bromide bound to the fragments with an angle of the absorption dipole largely perpendicular to the direction of the electrophoretic current. The dichroism declined as the molecular weight of the fragments decreased which is interpreted as an increase in the degree of disorder for shorter DNA.