Subject(s)
DNA/chemistry , Nucleosides/analysis , Nucleotides/analysis , Oligonucleotides/analysis , Purines/analysis , Pyrimidines/analysis , Capillary Action , Cytarabine/blood , Drug Monitoring/methods , Electrophoresis/methods , Humans , Leukemia/metabolism , Lymphocytes/metabolism , Nucleosides/isolation & purification , Nucleotides/blood , Nucleotides/isolation & purification , Oligonucleotides/isolation & purification , Purines/isolation & purification , Pyrimidines/isolation & purification , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, UltravioletABSTRACT
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.
Subject(s)
Poliovirus/chemistry , Polymerase Chain Reaction , RNA-Directed DNA Polymerase/metabolism , Viral Proteins/analysis , Viral Proteins/isolation & purification , Electrophoresis , Humans , Poliovirus Vaccine, Inactivated/analysis , RNA, Viral/isolation & purification , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
Capillary electrophoresis (CE) is a versatile microanalytical technique that has gained much attention, particularly from those working with biologically active molecules. Its appealing characteristics include unprecedented sensitivity and the ability for automating the rapid electrophoretic separation of a number of low-volume samples in a reproducible manner, with relatively short analysis times. The picomole-femtomole (10(-12)-10(-15) mol) sensitivity of UV-CE has been enhanced tremendously by the interfacing of detection systems such as laser-induced fluorescence, which has extended the sensitivity into the attomole-zeptomole (10(-18)-10(-21) mol) range. Fluorescence detection has shown great potential for the CE analysis of a wide range of biomolecules including peptides, proteins and DNA. CE research and development has taken on directions focused primarily on improving detection, understanding and exploiting the basic chemistry of CE and devising new applications.
Subject(s)
Electrophoresis/methods , Biotechnology , Electrophoresis/instrumentation , Evaluation Studies as Topic , Oligonucleotides/isolation & purification , Peptides/isolation & purification , Proteins/isolation & purificationABSTRACT
Capillary electrophoresis (CE) with a sieving buffer containing ethidium bromide was applied to the detection of PCR-amplified RFLP samples. With CE, in contrast to agarose gel electrophoresis, run times are short, i.e., typically less than 30 min, the capillary can be re-used, and full automation is feasible. The addition of ethidium bromide to the buffer system in conjunction with a field amplification injection technique led to increased sample detectability and resolution. Migration time precision was better than 0.2% RSD with a approximately 12-bp resolution for the DNA fragment sizes of interest. RFLP samples were analyzed for homo- or heterozygosity based on the presence of 500- and/or 520-bp DNA fragments. Special software was used to correct for run-to-run migration time variations, thus facilitating genotype assignment.
