Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence.
In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.
Butler
J M
JM
Department of Chemistry, University of Virginia, Charlottesville 22903.
McCord
B R
BR
Jung
J M
JM
Wilson
M R
MR
Budowle
B
B
Allen
R O
RO
D004279:DNA, Viral / Q000032:analysis; D004586:Electrophoresis; D017403:In Situ Hybridization; D007834:Lasers; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D012015:Reference Standards; D013050:Spectrometry, Fluorescence
D004279:DNA, Viral