Temporal analysis of DNA restriction digests by capillary electrophoresis.
We demonstrate a facile means for temporal analysis of DNA restriction enzyme digests by capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection. phi X-174 DNA was digested with HaeIII restriction enzyme under conditions that allowed the monitoring of digestion as it proceeded toward completion. Separation by a polymer solution of methylcellulose in a polyacrylamide coated capillary allowed high resolution and a high degree of reproducibility between sequential runs. At pre-selected time intervals an injection of the digest, directly from the reaction mixture, was made. Sensitive detection was achieved by using ethidium bromide as an intercalation dye and allowing intercalation to occur on-column. It is demonstrated that the course of the digestion (i.e., the creation and diminishing of fragment peaks) can be followed using this methodology. Also demonstrated is the ability to use temporal analysis to determine ideal conditions for producing a single cut within a cloning and expression vector (pET3a-PAI-1) which contains 11 potential restriction endonuclease cleavage sites. This initial attempt to follow a restriction digest on-column not only provides meaningful information for the biochemical researcher, but also furthers the use of CE a diagnostic tool for the biochemical laboratory.
Stebbins
M A
MA
Department of Chemistry, University of Tennessee, Knoxville 37996-1600, USA.
Schar
C R
CR
Peterson
C B
CB
Sepaniak
M J
MJ
D010584:Bacteriophage phi X 174 / Q000235:genetics*; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D019075:Electrophoresis, Capillary; D010957:Plasmids; D013997:Time Factors
D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases