Abstract
Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis.
Full text
PDF



Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bartholdi M. F., Sinclair D. C., Cram L. S. Chromosome analysis by high illumination flow cytometry. Cytometry. 1983 May;3(6):395–401. doi: 10.1002/cyto.990030602. [DOI] [PubMed] [Google Scholar]
- Brettell T. A., Saferstein R. Forensic science. Anal Chem. 1991 Jun 15;63(12):148R–164R. doi: 10.1021/ac00012a010. [DOI] [PubMed] [Google Scholar]
- Kostichka A. J., Marchbanks M. L., Brumley R. L., Jr, Drossman H., Smith L. M. High speed automated DNA sequencing in ultrathin slab gels. Biotechnology (N Y) 1992 Jan;10(1):78–81. doi: 10.1038/nbt0192-78. [DOI] [PubMed] [Google Scholar]
- Peck K., Stryer L., Glazer A. N., Mathies R. A. Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin. Proc Natl Acad Sci U S A. 1989 Jun;86(11):4087–4091. doi: 10.1073/pnas.86.11.4087. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Strathmann M., Hamilton B. A., Mayeda C. A., Simon M. I., Meyerowitz E. M., Palazzolo M. J. Transposon-facilitated DNA sequencing. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1247–1250. doi: 10.1073/pnas.88.4.1247. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Studier F. W. A strategy for high-volume sequencing of cosmid DNAs: random and directed priming with a library of oligonucleotides. Proc Natl Acad Sci U S A. 1989 Sep;86(18):6917–6921. doi: 10.1073/pnas.86.18.6917. [DOI] [PMC free article] [PubMed] [Google Scholar]