RESUMEN
A capillary electrophoresis system has been developed which has the ability to rapidly analyse DNA restriction fragments, PCR products, oligonucleotides and complex deoxyribonucleoside tri-, di- and mono-phosphate mixtures in a single separating medium. Separations are performed in an internally coated capillary containing a solution of linear polymers. The separation of all DNA species is achieved through the novel use of acidic rather than alkaline pH. This has the added advantage of preserving the internal capillary coating. The use of the technique is described for rapid, high resolution separation of pBR322 and phi X174 DNA restriction digests, quality control of dNTP and oligonucleotide primer PCR components, detection of a PCR amplified region of lambda-phage DNA and detection of a PCR amplified human hypervariable region of forensic interest. The technique is termed "acidic non-gel capillary electrophoresis".
Asunto(s)
Dermatoglifia del ADN/métodos , ADN/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , ADN/genética , Cartilla de ADN/aislamiento & purificación , ADN Viral/genética , ADN Viral/aislamiento & purificación , Desoxirribonucleótidos/aislamiento & purificación , Electroforesis/métodos , Estudios de Evaluación como Asunto , Humanos , Concentración de Iones de HidrógenoRESUMEN
Heteroduplex DNA polymorphism analysis (HPA) makes use of conformational polymorphisms to alter electrophoretic mobility of fragments and can be used to detect non-restrictable loci. We have developed a novel application of entangled solution capillary electrophoresis (ESCE) to separate heteroduplex and homoduplex DNA molecules. The addition of ethidium bromide and glycerol to the free solution sieving buffer resulted in the improved peak resolution and good reproducibility. Reannealed polymerase chain reaction products could be used directly for mutation screening and with fully automated ESCE the entire HPA may be completed in less than 30 min including sample handling. This technology could provide a rapid and highly efficient way for screening rare mutations among large numbers of individuals.
Asunto(s)
ADN de Hongos/análisis , Ácidos Nucleicos Heterodúplex/análisis , Polimorfismo Genético , Secuencia de Bases , Tampones (Química) , Electroforesis , Etidio , Datos de Secuencia Molecular , Reacción en Cadena de la PolimerasaRESUMEN
Methods are described for the HPLC and genetic analysis of herbal Cannabis sativa. The latter method was applied to 17 plants grown simultaneously, at the same site. Sixteen of these samples were also compared using HPLC, which resulted in differentiation of the plants into 3 groups. Within two of these groups, the members could not be distinguished. By RAPD analysis, using certain combinations of primers and cladistic analysis, differentiation was possible between all but two of the plants. The use of the RAPD technique enables differentiation between samples that cannot be differentiated by HPLC analysis alone.