Detection of single lambda DNA fragments by flow cytometry
- Los Alamos National Lab., NM (United States)
The authors have demonstrated flow cytometric detection and sizing of single pieces of fluorescently stained lambda DNA (48.5 kb) and individual Kpn I restriction fragments of lambda DNA at 17.05 kb and 29.95 kb. DNA fragments were stained stoichiometrically with an intercalating dye such that the fluorescence from each fragment was directly proportional to fragment length. Laser powers range from 10 to 100 mW and transit times through the focused laser beam were several milliseconds. Measurements were made using time-resolved single photon counting of the detected fluorescence emission from individual stained DNA fragments. Samples were analyzed at rates of about 50 fragments per second. The measured fluorescence intensities are linearly correlated with DNA fragment length over the range measured. Detection sensitivity and resolution needed for analysis of small pieces of DNA are discussed and a comparison of single photon counting measurements of DNA fragments to measurements using more conventional flow cytometers is made. Applications of this methodology to DNA sizing and DNA fingerprinting are discussed.
- OSTI ID:
- 6574734
- Report Number(s):
- CONF-9303114-; CODEN: CYTODQ
- Journal Information:
- Cytometry (Baltimore); (United States), Vol. 6; Conference: 16. congress of the International Society for Analytical Cytology, Colorado Springs, CO (United States), 21-26 Mar 1993; ISSN 0196-4763
- Country of Publication:
- United States
- Language:
- English
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