Rapid separation, quantitation and purification of products of polymerase chain reaction by liquid chromatography.
The polymerase chain reaction (PCR), a new, powerful method for rapid enzymatic amplification of specific DNA fragments, has gained tremendous popularity in molecular biology. This paper describes the successful application of liquid chromatography to the analysis of products of the PCR. Efficient separation of both DNA restriction fragments and amplified PCR products were achieved in 10-12 min on a new ion-exchange column, DEAE-NPR, packed with 2.5-microns non-porous particles. The PCR products were quantitated with a reproducibility within 10%. Use of liquid chromatography was demonstrated for separation and quantitation of PCR products in amounts below those required for direct analysis by ethidium bromide gel electrophoresis or a Hoechst 33258 dye-based fluorescence assay. Liquid chromatography was also demonstrated to be effective for quick optimization of PCR procedures.
Katz
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Perkin-Elmer, Norwalk, CT 06859.
Haff
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Eksteen
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D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D002853:Chromatography, Liquid; D004247:DNA / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D016133:Polymerase Chain Reaction; D013056:Spectrophotometry, Ultraviolet
D007202:Indicators and Reagents; D004247:DNA