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.     

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.     

Detection and discrimination of individual viruses by flow cytometry.

A new flow cytometer with a very small observation volume has been developed to detect individual viruses with good resolution, and has been used to discriminate between two types of viral particles based on differences in their light scattering. Measurements of light scattering and fluorescence made with such an instrument can provide a basis for quantitative analysis and sorting of viruses and other particles in the micron and submicron size range.     

DNA analysis by flow cytometry

Accurate quantification of DNA from cells of several species is possible with flow cytometry. When one species is used as a reference, cytometric readings from two or more different species can be compared to obtain relative percent DNA or DNA indices. Differences in DNA from the male and female of the same species also can be measured. The method allows rapid screening of chromosomal abnormalities among large clinical populations, and evaluation of errors of sex determination such as XY sex reversal.     

Rapid assessment of bacterial viability by flow cytometry

The ability of a flow cytometer to rapidly assess microbial viability was investigated using three vital stains: rhodamine 123 (Rh123); 3,3'-dihexyloxacarbocyanine iodide [DiOC₆(3)] and fluorescein diacetate (FDA). Rh123 was found to clearly differentiate viable from non-viable bacteria. The methodology for staining bacteria with this dye was optimised. Rh123 was shown to stain and discriminate several different species of viable bacteria although this was not universal. Viable cells of Bacillus subtilis were found to stain better with FDAthan with Rh123. The results demonstrate the ability of flow cytometry to rapidly detect and estimate the viability of bacterial populations.Correspondence to: J. P. Diaper     

Multiparameter DNA flow cytometry of keratoacanthoma.

Keratoacanthomas (KAs) are rapidly growing cutaneous lesions that frequently look much like well-differentiated squamous cell carcinomas (SCCs) but spontaneously regress. It is uncertain whether KA is a reactive hyperplastic lesion that mimics a neoplasm or a true (but defective) neoplasm that cannot sustain progressive growth. To address this question, we performed DNA flow cytometric analysis on 14 KAs and 10 cutaneous SCCs for comparison. By multiparameter DNA flow cytometry using forward scatter and orthogonal scatter, 10 KAs and 4 SCCs had peridiploid DNA aneuploid populations (DNA indices of 1.03-1.14), and 2 SCCs had grossly aneuploid populations (DNA index, 1.69 and 2.33). Our data thus support aneuploidy in KAs. It is argued that KA is a true neoplasm.     

Rapid titration of retroviral vectors encoding intracellular antigens by flow cytometry.

Flow cytometry was used to detect cells infected with retroviral vectors encoding both simian virus 40 large T antigen and G418 resistance after indirect immunofluorescence staining using a T-antigen-specific monoclonal antibody and a fluorescein-conjugated secondary antibody. Titers of viral stocks determined by flow cytometry were equivalent to those determined by quantitation of G418-resistant colonies.     

Rapid identification of sex in birds by flow cytometry

A rapid method to identify sex in birds is described. The method requires microliter volumes of blood, and, under appropriate conditions, results can be available within an hour of sample collection. Samples can be stored at 4 degrees C or -20 degrees C without sacrificing the ability to discriminate sex differences in DNA content. The assay will find utility in laboratory, field, and applied studies, in other classes of vertebrates, and in studies on the dynamics of genome size within and among populations.     

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.     

Fluorescence spectra of cells stained with a DNA-specific dye, measured by flow cytometry

Fluorescence spectra of ethanol-fixed rat thymocytes stained with the DNA-specific dye Hoechst 33258 have been measured in an arc lamp-based flow cytometer including a grating monochromator in front of the fluorescence detector. Spectral resolution was 5-10 nm. Increasing dye concentration was found to yield an increasing shift of the fluorescence spectrum toward longer wavelengths, thus supporting previous work on soluble DNA that indicated several different binding modes of this dye. The results show that similar data may be obtained for all commonly used DNA-specific dyes. It appears that this type of spectral information may be used to probe the structure of cell chromatin.     

Rapid detection of mutagen induced micronucleated erythrocytes by flow cytometry

Summary The micronucleus test is a cytogenetic method for the screening of mutagens and carcinogens which exhibit clastogenic mechanisms of action. After application of clastogenic agents chromosomal fragments or even whole chromsomes can remain as conspicuous structures (micronuclei) in a small fraction of anucleated polychromatic erythrocytes which can be visually scored using a microscope following staining with May-Grünwald-Giemsa solutions. These time-consuming, painstaking, and tedious manual evaluations are often sources of unreliability and uncertainty. Here, a fluorescence technique is presented which applies DNA and protein fluorochromes to discriminate normal anucleated erythrocytes from micronucleated erythrocytes using a fluorescence microscope. This method is particularly tailored to be applied to flow cytometric instrumentation. Data obtained manually and automatically in flow show a strong linear correlation with high significance (r=0.96) as far as the percentage of micronucleated erythrocytes as an indicator for the mutagenicity of a given drug is concerned. These results have been obtained by means of the established clastogens cyclophosphamide and mitomycin C.     

Rapid pyrazinamide susceptibility testing of Mycobacterium tuberculosis by flow cytometry

The resurgence of tuberculosis along with the increased resistance of Mycobacterium tuberculosis has emphasized the need for timely susceptibility testing for control of the disease. Previous studies have shown that rapid susceptibility testing can be accomplished for isoniazid, ethambutol, and rifampin using the flow cytometric assays. In this study we compared the flow cytometric susceptibility assay with the BACTEC TB 460 and BACTEC MGIT 960 for pyrazinamide (PZA). There was 93% agreement between the BACTEC MGIT 960 and the flow cytometric methods for 100 microg/mL of PZA. Additionally, there was a 95% and 86% agreement between the BACTEC TB 460 and flow cytometric methods for 50 microg/mL and 100 microg/mL of PZA, respectively. These findings show that susceptibility testing by the flow cytometric assay is accurate. Most importantly, susceptibility results by the flow cytometric assay were available 24 h after initiation of the testing procedure. The advantages of simplicity, speed and accuracy make the flow cytometric susceptibility assay an immediate impact technology to improve patient care.     

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.     

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.     

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.