Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Ferris, Matthew M.; Habbersett, Robbert C.; Wolinsky, Murray; Jett, James H.; Yoshida, Thomas M.; Keller, Richard A.

doi:10.1002/cyto.a.20000

Cited by 19 publications

(14 citation statements)

References 51 publications

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“…In particular, we have addressed the importance of these topics as they relate to collection and interpretation of results as has been pointed out previously for single molecule measurements (12).…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we have examined the impact of several empirical variables on the variance of ''bead array'' data and suggested methods to obviate this impact. In particular, we have addressed the importance of these topics as they relate to collection and interpretation of results as has been pointed out previously for single molecule measurements (12).…”

Section: Discussionmentioning

confidence: 99%

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Analysis of individual data from bead‐based assays (“bead arrays”)

2006

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Introduction: Typically, bead‐based assays (“bead arrays”) use the mean or median value of a population of measurements to judge ligand binding or other activity, which results in a change in fluorescence intensity. Individual bead measurements are used here to calculate population parameters integral to the measurement of a bead array. Methods: Using a commercially‐available instrument designed for bead array measurements, a set of beads were labeled with biotin and then titrated with PE‐Streptavidin. Data were collected under normal machine conditions as well as variations. Results: The “sensitivity” of the measurements was determined using parametric and nonparametric statistical methods as well as regression analysis over a limited range of the titration (concentration vs. response profile). Conclusions: Results at low ligand concentrations suggest that precise measurements with bead array systems require a large number of individual bead measurements to be acquired. Individual bead measurements should be used to determine the mean and confidence intervals for the calculated measurements. These results also apply to regression analysis of concentration‐response profiles. Furthermore, features of the instrument can be manipulated to achieve increased sensitivity and detection of lower amounts of ligand bound to the bead populations. © 2006 International Society for Analytical Cytology

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Analysis of individual data from bead‐based assays (“bead arrays”)

2006

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“…728 While this may appear to be a relatively slow rate of analysis, simulations suggest that one could obtain a result within one standard deviation of the mean with only 5 measurements, although in practice one needs closer to 100 measurements to obtain an accurate value. 729 The requisite throughput is thus quite small; in principle, a fingerprint containing 40 restriction fragments could be sized in less than a minute. In practice the analysis time is somewhat longer (e.g., 30 minutes 100 ), which is still much faster than pulsed field gel electrophoresis if one skips the electroelution step 723 and instead dissolves the gel using GELase.…”

Section: Fluorescence Burst Analysismentioning

confidence: 99%

Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

et al. 2012

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“…For molecules, this concentration, which corresponds to 1.25 × 10 −12 M, is readily attainable. Moreover, fragment size distributions of restriction digests of microbial DNA can be accurately characterized by measuring only a few hundred fragments (14), allowing analyses to be done in less than a minute.…”

Section: “Personal” Cytometers—what and Why?mentioning

confidence: 99%

Personal cytometers: Slow flow or no flow?

Shapiro¹,

Perlmutter²

2006

Cytometry Pt A

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Background: Although some manufacturers have optimistically described instruments with prices in the US$40,000 range as “personal cytometers”, analogy with the personal computer suggests that the target price for a true “personal” cytometer should be under $5,000. Since such an apparatus could find a wide range of applications in cytomics in both developing and developed countries, it seemed desirable to consider its technical and economic feasibility. Methods: Using resolution targets and a variety of fluorescent bead standards immobilized on filters and/or slides, we evaluated high‐intensity LEDs as fluorescence excitation sources, relatively inexpensive CCD cameras as detectors, and 35 mm camera lenses and plastic low‐power microscope optics for light collection in a simple, inexpensive low‐resolution imaging cytometer. Results: The components tested could be combined toproduce an instrument capable of detecting fewer than 10,000 molecules of cell‐associated fluorescent label, and thus applicable to a broad range of cytometric tasks. Conclusions: Given the requirements for light sources, detectors, optics, mechanics, electronics and data analysis hardware and software, and the components presently available, it should be easier to reach the desired $5,000 price point with an image cytometer than with a flow cytometer. © 2006 International Society for Analytical Cytology

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Statistics of single‐molecule measurements: Applications in flow‐cytometry sizing of DNA fragments

Cited by 19 publications

References 51 publications

Analysis of individual data from bead‐based assays (“bead arrays”)

Analysis of individual data from bead‐based assays (“bead arrays”)

Beyond Gel Electrophoresis: Microfluidic Separations, Fluorescence Burst Analysis, and DNA Stretching

Personal cytometers: Slow flow or no flow?

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