James H. Jett

Detection of single molecules of phycoerythrin in hydrodynamically focused flows by laser-induced fluorescence

Single molecules of B-phycoerythrin were detected by laser-induced fluorescence in hydrodynamically focused flows as each molecule transited a focused laser beam. Phycoerythrin is a large protein molecule containing the equivalent of 25 rhodamine-6G chromophores. Single molecule detection is documented by the following: (1 )the number of counts per molecules is in agreement with the expected number, (2) the number of molecules per second is in agreement with the concentration and the flow rate, and (3) the time interval distribution between detected molecules is In agreement with the concentration and the flow rate. The molecular transit time through the 1.1-pL probe volume was 180 micros.

Single-Molecule Fluorescence Analysis in Solution

Over the past five years, several groups have developed the capability to detect and identify single fluorescent molecules in solution as the molecules flow through a focused laser beam. The history of the approach to single-molecule detection in fluid solution is shown in Fig. 1. Approximately one dozen molecular species have been detected at this level of sensitivity. Fluorescence-based, single-molecule detection techniques are expected to have a significant impact in fields where fluorescence detection and quantification are broadly applied, e.g., analytical chemistry, biology, and medicine. Single-molecule detection is a new way of doing analytical chemistry, and new applications will arise. In this article, we describe our approach to single-molecule detection and explore assays that can be done at the single-species level that would be difficult or impossible with bulk measurements.

High-speed DNA sequencing: An approach based upon fluorescence detection of single molecules

We are developing a laser based technique for the rapid sequencing of large fragments (approximately 40 kb) of DNA based upon the detection of single, fluorescently tagged nucleotides cleaved from a single DNA fragment. We have demonstrated significant progress on several of the important steps of this technique. The projected rate of sequencing is several hundred bases per second which is orders of magnitude faster than existing methods. Once developed, this technology could be utilized by investigators for rapid sequencing of genetic material from virtually any source.

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.

Rapid DNA sequencing based upon single molecule detection

We are developing a laser-based technique for the rapid sequencing of 40-kb or larger fragments of DNA at a rate of 100 to 1000 bases per second. The approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA fragment into a flowing sample stream, and detection of individual fluorescently labeled bases as they are cleaved from the DNA fragment by an exonuclease. The ability to sequence large fragments of DNA will significantly reduce the amount of subcloning and the number of overlapping sequences required to assemble megabase segments of sequence information.

Application of Single Molecule Detection to Dna Sequencing

Abstract A flow cytometric, single molecule approach to DNA sequencing is described. A single, fluorescently labeled DNA fragment is suspended in a flow stream. An exonuclease is added to sequentially cleave the end base into the flow stream where it is detected and identified by laser-induced fluorescence.

Single particle high resolution spectral analysis flow cytometry

While conventional multiparameter flow cytometers have proven highly successful, there are several types of analytical measurements that would benefit from a more comprehensive and flexible approach to spectral analysis including, but certainly not limited to spectral deconvolution of overlapping emission spectra, fluorescence resonance energy transfer measurements, metachromic dye analysis, free versus bound dye resolution, and Raman spectroscopy.

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

Previous reports have demonstrated accurate DNA fragment sizing of linear DNA fragments, from 564 to ≈4 × 105 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.

Progress towards single-molecule DNA sequencing: a one color demonstration

Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.

The characterization and isolation of plant heterokaryons by flow cytometry

SummaryPlant heterokaryons were identified and isolated from a population of protoplasts following fusion. Endogenous chlorophyll autofluorescence and exogenously supplied fluorochromes were utilized to differentiate between parental and heterokaryon populations. Flow cytometric analysis detected heterokaryons based upon the simultaneous presence of both chlorophyll and an exogenously supplied fluorochrome within one cell. These parameters were utilized to sort large numbers of heterokaryons from the fusion mixture using modified flow instrumentation. Modifications to the instrumentation which allowed this sorting are discussed.