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

Huang, Zhengping; Petty, Jeffrey T.; O'Quinn, Brian; Longmire, Jonathan L.; Brown, Nancy; Jett, James H.; Keller, Richard A.

doi:10.1093/nar/24.21.4202

Cited by 48 publications

(71 citation statements)

References 23 publications

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“…A typical background rate for the data presented below was three photoelectrons (pe) per MCS bin, and a typical threshold was chosen to be 4 pe per bin. The criterion for choosing the proper threshold has been discussed elsewhere in detail (9)(10)(11). The areas of the bursts were integrated (LabVIEW) and histogrammed to produce a burst size distribution using Sigmaplot software (Jandel Scientific Inc., San Rafael, CA).…”

Section: Discussionmentioning

confidence: 99%

“…The experimental apparatus is similar to that described previously (11). Briefly, the pulled acid-etched capillary was introduced into the center of a 250-ϫ 250-µm square-bore flow cuvette, with the detection side fabricated to a 100-µm coverslip thickness.…”

Section: Flow Cytometric Measurementsmentioning

confidence: 99%

“…In addition to high sensitivity and speed, our approach is quantitative, i.e., individual fragments are counted. The analysis is conformation independent; linearization of circular and supercoiled DNA before analysis is not required (11). Application of this technology to the sizing of a restriction digest of the bacterial genome S. aureus has been reported recently (12).…”

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confidence: 99%

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Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

et al. 1999

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Section: Discussionmentioning

confidence: 99%

Section: Flow Cytometric Measurementsmentioning

confidence: 99%

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confidence: 99%

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Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

et al. 1999

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“…This method in principle allows the measurement of extremely long DNA molecules because the signal increases with the length of the molecule. This technique has been used with traditional methods of flow cytometry to measure length distributions of DNA molecules (3,4). Other groups have imaged restriction enzymes digesting extended single DNA molecules for ''optical mapping'' (5,6).…”

mentioning

confidence: 99%

A microfabricated device for sizing and sorting DNA molecules

Chou

Spence²,

Scherer³

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

281

196

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We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.Many assays in biology require measurement of the length distribution of DNA molecules in a heterogeneous solution. This measurement is commonly done with gel electrophoresis; the molecules are separated by mobility, from which the lengths are inferred. This method is powerful, yet has some drawbacks. For medium to large DNA molecules the resolution is limited to approximately 10%. Gel electrophoresis is time consuming. It generally takes at least an hour to run the gel, not including the setup time to cast the gel. Furthermore, for large molecules the procedure fails. This problem has been alleviated to some extent by the development of pulsed-field gel electrophoresis (1), but running times can be days.With the development of high affinity intercalating DNA stains (2), it has become possible to directly measure the length of single molecules by quantitating fluorescence. The amount of intercalated dye is proportional to the length of the molecule, so measuring the total fluorescent intensity from a single molecule gives a direct measurement of its length. This method in principle allows the measurement of extremely long DNA molecules because the signal increases with the length of the molecule. This technique has been used with traditional methods of flow cytometry to measure length distributions of DNA molecules (3, 4). Other groups have imaged restriction enzymes digesting extended single DNA molecules for ''optical mapping'' (5, 6).We have developed microfabricated devices to size and sort microscopic objects based on measurement of fluorescent properties. The devices have a network of microfluidic channels and are fabricated from a silicone elastomer by using a replica technique (7). Master molds are made from silicon wafers by using standard micromachining techniques. Because the molds can be reused indefinitely, this method of fabrication allows economical mass production of the devices. The devices were patterned as shown in Fig. 1. This fabrication technique is one of a new set of technologies known as soft lithography. Previous work has demonstrated that elastomers can replicate gratings and other test patterns with high (Ϸ50 nm) resolution and fidelity (8, 9). Although some groups have made large (Ϸ30 m) elastomer structures for capillary electrophoresis (10), there is only one other example of a micron scale fluidic network with the elastomer (11). MATERIALS AND METHODSDevice Fabrication. Negative master devices were fabricated in silicon and use...

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“…Flow cytometric fragment sizing characterizes individual DNA fragments as they transit a focused laser beam. FCM has been shown to have significant advantages over traditional methods of fragment sizing, including pulsed-field gel electrophoresis (PFGE) (40). Demonstrated applications of single-molecule FCM measurements include: characterization of artificial chromosomes for DNA sequencing, analysis of PCR fragments, and bacterial identification.…”

Section: Application To Flow Cytometry Sizing Of Dna Fragmentsmentioning

confidence: 99%

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

Ferris¹,

Habbersett²,

Wolinsky³

et al. 2004

Cytometry Pt A

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Background:The measurement of physical properties from single molecules has been demonstrated. However, the majority of single-molecule studies report values based on relatively large data sets (e.g., N Ͼ 50). While there are studies that report physical quantities based on small sample sets, there has not been a detailed statistical analysis relating sample size to the reliability of derived parameters. Methods: Monte Carlo simulations and multinomial analysis, dependent on quantifiable experimental parameters, were used to determine the minimum number of singlemolecule measurements required to produce an accurate estimate of a population mean. Simulation results were applied to the fluorescence-based sizing of DNA fragments by ultrasensitive flow cytometry (FCM). Results: Our simulations show, for an analytical technique with a 10% CV, that the average of as few as five

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

Cited by 48 publications

References 23 publications

Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

Bacterial fingerprinting by flow cytometry: Bacterial species discrimination

A microfabricated device for sizing and sorting DNA molecules

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

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