A comparison of electrophoretic resolution for snapshot and finish-line imaging

John C. Sutherland, David J. Fisk, Denise C. Monteleone, John G. Trunk

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Finish-line imaging, in which DNA or other macromolecules are detected after electrophoresis for a constant distance, usually improves resolution compared to snapshot imaging, in which molecules are electrophoresed for a constant time in an apparatus of comparable dimensions. Resolving power, which is an objective measure of the ability of different separatory methods to detect closely spaced molecular species, can be used to compare directly the performance of systems employing both snapshot and finish-line imaging [E. A. Ribeiro and J. C. Sutherland, Anal. Biochem. 210, 378-388 (1993)]. Experimentally determined values of resolving power are influenced both by the method of imaging (snapshot vs finish-line) and by instrument-specific factors that affect resolution. Previous comparisons of the resolving power obtained with finish-line and snapshot imaging involved data sets acquired by different instruments with different instrumental resolutions. To reduce the influence of instrumental effects, we constructed a scanning laser fluorometer that can measure both snapshot and finish-line images of fluorochrome-labeled DNA. Snapshot and finish-line images of a DNA sample containing HaeII restriction fragments of the DNA from bacteriophage T7, which range in length from 474 to 6514 base pairs, were obtained under otherwise identical electrophoretic conditions. Snapshot and finish-line imaging give similar resolving powers for DNA molecules up to about 1.5 kbp long. For both imaging modes, maximum resolving power was achieved for DNA molecules between 2 and 3 kbp in length. For larger DNA molecules, finish- line imaging provided higher resolving power. The ratio of the resolving power of finishline images to that of snapshot images increased monotonically as a function of DNA length. For the longest restriction fragments studied (6514 bp), the resolving power for finish.line images exceeded that of snapshot images by about 50%.

Original languageEnglish (US)
Pages (from-to)136-144
Number of pages9
JournalAnalytical Biochemistry
Volume239
Issue number2
DOIs
StatePublished - Aug 1 1996
Externally publishedYes

Fingerprint

Optical resolving power
Imaging techniques
DNA
Molecules
Bacteriophage T7
Fluorometers
Bacteriophages
Fluorescent Dyes
Base Pairing
Electrophoresis
Macromolecules
Lasers
Scanning

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

A comparison of electrophoretic resolution for snapshot and finish-line imaging. / Sutherland, John C.; Fisk, David J.; Monteleone, Denise C.; Trunk, John G.

In: Analytical Biochemistry, Vol. 239, No. 2, 01.08.1996, p. 136-144.

Research output: Contribution to journalArticle

Sutherland, John C. ; Fisk, David J. ; Monteleone, Denise C. ; Trunk, John G. / A comparison of electrophoretic resolution for snapshot and finish-line imaging. In: Analytical Biochemistry. 1996 ; Vol. 239, No. 2. pp. 136-144.
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