Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry

Goodwin, Peter M.; Johnson, Mitchell E.; Martin, John; Ambrose, W. P.; Marrone, Babetta L.; Jett, James H.; Keller, Richard A.

doi:10.1093/nar/21.4.803

Cited by 131 publications

(109 citation statements)

References 6 publications

Supporting

Mentioning

104

Contrasting

Unclassified

Order By: Relevance

“…The mean RSD values associated with FCM and PFGE were 1.2% Ϯ 0.8% and 3% Ϯ 2%, respectively. These values are again in agreement with previously reported literature values (11,20,23,29,31,33). While the mean numerical precision of FCM was slightly better than that of PFGE, the two values were equivalent, within 1 standard deviation.…”

Section: Vol 42 2004 Assessment Of Flow Cytometry and Gel Electrophsupporting

confidence: 92%

“…The largest observed error, for either method, was the underestimation of the 216.623-kb fragment by FCM (13% error). This discrepancy is noteworthy given the relatively accurate sizing of this fragment by PFGE (4.6% error) and the linear response of our FCM method in previous studies (20,29,31,33,39). In an attempt to understand this inconsistency, we examined the sequence of this 216-kb restriction fragment more closely.…”

Section: Vol 42 2004 Assessment Of Flow Cytometry and Gel Electrophmentioning

confidence: 92%

“…This method utilizes fluorescence-based, single-molecule detection technology to size individual DNA fragments in flowing streams (1,7,8,15,20,23,43). Through the use of intercalating dyes that selectively bind to DNA, the fragment length can be determined from its fluorescence intensity (50).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

et al. 2004

Self Cite

View full text Add to dashboard Cite

The sizing of restriction fragments is the chief analytical technique utilized in the production of DNA fingerprints. Few techniques have been able to compete with pulsed-field gel electrophoresis (PFGE), which is capable of discriminating among bacteria at species and strain levels by resolving restriction fragments. However, an ultrasensitive flow cytometer (FCM) developed in our lab has also demonstrated the ability to discriminate bacteria at species and strain levels. The abilities of FCM warrant a quantitative parallel comparison with PFGE to assess and evaluate the accuracy and precision of DNA fragment sizing by both techniques. Replicate samples of Staphylococcus aureus Mu50 were analyzed along with two clinical S. aureus isolates. The absolute fragment sizing accuracy was determined for PFGE (5% ؎ 2%) and FCM (4% ؎ 4%), with sequence-predicted Mu50 SmaI fragment sizes used as a reference. Precision was determined by simple arithmetic methods (relative standard deviation for PFGE [RSD PFGE ] ‫؍‬ 3% ؎ 2% and RSD FCM ‫؍‬ 1.2% ؎ 0.8%) as well as by the use of dendrograms derived from Dice coefficient-unweighted pair group method with arithmetic averages (UPGMA) and Pearson-UPGMA analyses. All quantitative measures of PFGE and FCM precision were equivalent, within error. The precision of both methods was not limited by any single sample preparation or analysis step that was tracked in this study. Additionally, we determined that the curve-based clustering of fingerprint data provided a more informative and useful assessment than did traditional bandbased methods.DNA fragment sizing is arguably the most widely used analytical method in molecular biology, biochemistry, and microbiology. Specific applications of DNA fragment sizing include microbe identification and discrimination, genotyping, and sequencing. Traditionally, DNA fragments are characterized via size-dependent separation methods such as gel electrophoresis.Conventional gel electrophoresis, using a solid polymer (e.g., agarose or polyacrylamide) and a static electric field, is ubiquitous in today's molecular biology and biochemistry labs. Such methods are routinely used to separate and size DNA fragments of Յ20 kb (4).Pulsed-field gel electrophoresis (PFGE), developed in 1984 by Schwartz et al. (44), extended the fragment sizing range to over 1 Mb (4,6,9,19,44). For PFGE, large DNA fragments (i.e., Ͼ10 kb) are separated in an agarose gel by a pulsed electric field. A popular application of PFGE has been strainlevel bacterial fingerprinting through the sizing of DNA fragments resulting from the digestion of whole genomic DNAs with rare-cutting restriction endonucleases (5,11,28,35,47,49). Macrorestriction-based bacterial fingerprinting has found applications primarily in the public health and food safety industries. For example, the Centers for Disease Control and Prevention (CDC) have formed PulseNet, the National Molecular Subtyping Network for Foodborne Disease Surveillance (46; http://www.cdc.gov/pulsenet). PulseNet utilizes macrorestriction fi...

show abstract

Section: Vol 42 2004 Assessment Of Flow Cytometry and Gel Electrophsupporting

confidence: 92%

Section: Vol 42 2004 Assessment Of Flow Cytometry and Gel Electrophmentioning

confidence: 92%

See 1 more Smart Citation

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

et al. 2004

Self Cite

View full text Add to dashboard Cite

show abstract

“…Currently, two strategies are feasible. In a flow system, a dilute solution of DNA is made to sequentially stream pass the detector [Goodwin et al, 1993]. The DNA dilution is such that most DNA molecules do not travel with any other DNA molecule and the time interval between two DNA molecules as they arrive at the detector is not too long.…”

Section: Physical Methods For Dna Separationmentioning

confidence: 99%

Single-molecule analysis for molecular haplotyping

Kwok

Xiao

2004

Hum. Mutat.

View full text Add to dashboard Cite

In the genome era, there is great hope that genetic approaches such as linkage equilibrium mapping can be used to study common human disorders using a case-control population association study design. Ideally, the parental chromosomes are marked so that chromosomal regions in the form of haplotypes are compared in these studies to increase the power of association. Determining the haplotypes in a diploid individual is a major technical challenge in genetic studies of complex traits. A molecular approach to haplotyping is therefore highly desirable. Recent advances in DNA preparation, separation, labeling, and image analysis provide hope that a strategy of using a three-dye system coupled with DNA distance measurements between alleles will yield haplotype information of sufficiently high quality for genetic studies. In this work, we present the outline of the major challenges one must meet in developing a robust strategy for SNP detection and molecular haplotyping using single molecule analysis. Hum Mutat 23: [442][443][444][445][446] 2004. r 2004 Wiley-Liss, Inc.

show abstract

“…For small molecules and short exposure times (10 ms), we have found that can become comparable to w, and hence w does not serve as a good measure of the amount of DNA in a single restriction fragment. Therefore, we determined the relative lengths of restriction fragments by integrating the fitted function and using the proportionality between total fluorescence intensity and length (17). The absolute number of base pairs was determined by first calculating the relative lengths of the restriction fragments and then multiplying it by the known length of the DNA.…”

Section: [1]mentioning

confidence: 99%

Restriction mapping in nanofluidic devices

Riehn

Wang

et al. 2005

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

194

183

View full text Add to dashboard Cite

We have performed restriction mapping of DNA molecules using restriction endonucleases in nanochannels with diameters of 100 -200 nm. The location of the restriction reaction within the device is controlled by electrophoresis and diffusion of Mg 2؉ and EDTA. We have successfully used the restriction enzymes SmaI, SacI, and PacI, and have been able to measure the positions of restriction sites with a precision of Ϸ1.5 kbp in 1 min using single DNA molecules.R estriction mapping with endonucleases is a central method in molecular biology (1, 2). It is based on the measurement of fragment lengths after digestion, while possibly maintaining the respective order. We present here an approach to restriction mapping that is based on stretching DNA in nanofluidic channels, in which DNA is linearized to a length-independent fraction of its contour length (3, 4).To date, the most powerful method for constructing restriction maps of long DNA molecules (100 kbp and above) is the one developed by Schwartz and coworkers (5, 6). Their technique consists of stretching the DNA on a surface to establish a one-to-one mapping between spatial and genomic position, initiating the restriction by exposing the DNA to restriction enzymes and Mg 2ϩ , and optically observing the lengths of the resulting fragments. An elegant study of the same basic approach showing separation of specific binding and induced cutting was published by Taylor et al. (7).A fundamental principle in determining the error of a measurement, and a common strategy to reduce the experimental error, is to take multiple, statistically independent measurements of the same quantity. Note that the fixing of the molecule to the surface in Schwartz's approach prevents any fluctuations, and hence multiple molecules have to be observed to obtain statistically independent measurements of the same cut position. Moreover, genomic-length DNA molecules stretched on surfaces often exhibit inhomogeneous stretching, including breaks, and thus averaging over multiple molecules becomes imperative (6,8). In contrast, a molecule stretched inside a nanochannel is not subject to any forces other than those causing lateral confinement and thus is able to thermally relax and fluctuate around an equilibrium conformation. The evaluation of a single molecule is thus sufficient if complete digestion is achieved. A detailed description of the statistics and dynamics of DNA molecules in nanochannels is published in ref. 9.The main challenge in employing the concept of stretching and mapping DNA inside a closed fluidic system is to separate the steps of stretching and cutting. We have solved this problem by introducing the DNA electrophoretically into nanochannels and controlling the concentration of the enzyme cofactor Mg 2ϩ in the device shown in Fig. 1. It consists of a microfluidic ''loading'' channel containing the DNA to be analyzed, the restriction enzyme, and EDTA, and a microfluidic ''exit'' channel containing Mg 2ϩ and the restriction enzyme. The two microfluidic channels are linked by 10 n...

show abstract

Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry

Cited by 131 publications

References 6 publications

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

Performance Assessment of DNA Fragment Sizing by High-Sensitivity Flow Cytometry and Pulsed-Field Gel Electrophoresis

Single-molecule analysis for molecular haplotyping

Restriction mapping in nanofluidic devices

Contact Info

Product

Resources

About