Capillary electrophoresis: separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus.
In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.
Rossomando
E F
EF
Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06030.
White
L
L
Ulfelder
K J
KJ
D004586:Electrophoresis; D006801:Humans; D017955:Poliovirus / Q000737:chemistry*; D011054:Poliovirus Vaccine, Inactivated / Q000032:analysis; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014764:Viral Proteins / Q000032:analysis* / Q000302:isolation & purification*
D011054:Poliovirus Vaccine, Inactivated; D012367:RNA, Viral; D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase