Rapid quantitation and monitoring of plasmid DNA using an ultrasensitive DNA-binding dye.
A sensitive fluorescence-based method for monitoring plasmid DNA during production was investigated. This simple method of assaying for plasmid DNA allows rapid monitoring of plasmid yields from a recombinant Escherichia coli fed-batch fermentation. The assay has several advantages over traditional methods of plasmid DNA measurement. The fluorescent dye is highly specific and can measure total plasmid DNA concentration in about 5 min. The assay is sensitive over a wide range of plasmid concentrations of between 15 and 280 ng/mL, even in the presence of impurities that occur within alkaline lysate preparations. The technique can also be applied to monitoring fermentation and downstream purification steps.
Noites
I S
IS
Centro de Engenharia Biologica e Quimica, Instituto Superior Tecnico, Lisbon, Portugal.
O'Kennedy
R D
RD
Levy
M S
MS
Abidi
N
N
Keshavarz-Moore
E
E
D001709:Biotechnology; D004269:DNA, Bacterial / Q000032:analysis; D004277:DNA, Single-Stranded / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D010957:Plasmids / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen