Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence

John M. Butler, Bruce R. McCord, Janet M. Jung, Mark R. Wilson, Bruce Budowle, Ralph O. Allen

Research output: Contribution to journalArticle

71 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)271-280
Number of pages10
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume658
Issue number2
DOIs
StatePublished - 19 Aug 1994

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