Hostname: page-component-586b7cd67f-2plfb Total loading time: 0 Render date: 2024-11-28T17:52:13.286Z Has data issue: false hasContentIssue false
  • English
  • Français

Sizing Dna Fragments by Ultrasensitive Flow Cytometry

Published online by Cambridge University Press:  02 July 2020

James H. Jett ,
Robert C. Habbersett ,
Xiaomei Yan ,
Thomas M. Yoshida ,
Babetta L. Marrone  and
Richard A. Keller
James H. Jett
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Robert C. Habbersett
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Xiaomei Yan
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Thomas M. Yoshida
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Babetta L. Marrone
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Richard A. Keller
Affiliation:
Biosceince Division, Los Alamos National Laboratory, Los Alamos, NM , 87545
Get access

Abstract

As originally developed in the 1960's, flow cytometry was primarily a technique for the analysis of mammalian cells. Analysis of cellular constituents such as DNA or cell surface antigens made fluorescent by a variety of reagents has been the main stay of flow cytometric applications. Over the years, flow cytometric analysis techniques have been developed that range from multicellular spheroids containing a million or more cells down to single molecule detection. An outgrowth of single molecule detection capability is DNA fragment size analysis.

DNA fragment size analysis starts with a sample of naked DNA that can be derived from a variety of sources including PCR products, double stranded viral genomes, BAC/PAC clones, and bacterial genomes. For genomic or cloned DNA, restriction enzyme digests are analyzed to produce a fingerprint pattern. The fingerprint, i. e., the distribution of fragment sizes produced by the restriction enzyme digestion, is characteristic of the source of DNA and forms the basis for identifying the source.

Type
Correlative Fluorescent Microscopy and Flow Cytometry Techniques (Organized by R. Smith)
Information
Microscopy and Microanalysis , Volume 7 , Issue S2: Proceedings: Microscopy & Microanalysis 2001, Microscopy Society of America 59th Annual Meeting, Microbeam Analysis Society 35th Annual Meeting, Long Beach, California August 5-9, 2001 , August 2001 , pp. 612 - 613
Copyright
Copyright © Microscopy Society of America 2001

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

1.Shapiro, H. M., Practical Flow Cytometry - Third Edition, Wiley-Liss, Inc, New York, NY, (1995).Google Scholar
2.Ambrose, W. P., Goodwin, P. M., Jett, J. H., Van Orden, A., Werner, J. H., and Keller, R. A.; “Single Molecule Fluorescence Spectroscopy at Ambient Temperature”, Chemical Reviews, 99:2929–2956 (1999).Google ScholarPubMed
3.Yan, X., Habbersett, R. C., Nolan, J. P., Yoshida, T. M., Jett, J.H., and Marrone, B. L.; “Development of a Mechanism-Based, DNA Staining Protocol using SYTOX Orange Nucleic Acid Stain and DNA Fragment Sizing Flow Cytometry”, Analytical Biochemistry 286:138–148 (2000).CrossRefGoogle Scholar
4.Habbersett, R.C., Jett, J.H., and Keller, R.A.. “Single DNA Fragment Detection by Flow Cytometry” in: Emerging Tools for Cell Analysis: Advances in Optical Measurement Technologies, Durack, G. and Robinson, J. P., Eds., Wiley-Liss: New York, Pgs 115–138, (2000).Google Scholar
5.Larson, E. J., Penttila, J. R., Cai, H., Jett, J. H., Burde, S., and Keller, R. A.; “Rapid DNA Fingerprinting of Pathogens by Flow Cytometry”, Cytometry, 41:203 (2000).Google ScholarPubMed