Influence of the electrokinetic injection conditions on the separation of DNA fragments in capillary electrophoresis

Abstract

Em análises genéticas por eletroforese capilar com soluções poliméricas existem muitas variáveis que afetam a separação dos fragmentos de DNA. Uma das mais críticas é o processo da injeção da amostra, a qual pode afetar consideravelmente a eficiência e a resolução dos picos. Neste trabalho, estudou-se a influência da composição da amostra e as condições da injeção eletrocinética na separação dos fragmentos de DNA em eletroforese capilar com solução polimérica renovável. Os estudos foram realizados injetando-se eletrocineticamente as amostras do DNA sob uma variedade de condições tais como, a força iônica da amostra e sua capacidade tamponante e a qualidade da matriz de separação. Em todos os experimentos, os fragmentos de DNA foram corados com brometo de etídio para detecção por fluorescência induzida a laser e separados sob um campo elétrico de 200 V cm -1 em uma coluna capilar revestida com polialcoolvinílico e preenchida com uma solução 0,5% de hidroxietilcelulose como matriz de separação. Sob estas condições de análise, a composição da amostra deve conter entre 1 e 5 mmol L -1 de tampão de separação para a obtenção de injeções reprodutíveis com um coeficiente de variação menor que 4% tanto para o tempo de migração quanto para a quantidade de material injetado.In genetic analysis by capillary electrophoresis with polymer solutions there are many variables that affect separation of the DNA fragments. A very critical one is the sample injection process, which can considerably affect the peak efficiency and the resolution. In this work, we have studied the influence of the DNA sample composition and the electrokinetic injection conditions in the separation of DNA fragments by capillary electrophoresis using replaceable polymer solutions. The studies were carried out by electrokinetically injecting the DNA samples under a variety of conditions, such as sample ionic strength, buffering capacity and the quality of the separation matrix. In all experiments DNA was stained with ethidium bromide for laser-induced fluorescence (LIF) detection and separated in a poly(vinylalcohol) coated capillary column filled with 0.5% hydroxyethylcellulose solution under a 200 V cm -1 electric field. Under such conditions, samples prepared with 1 to 5 mmol L -1 of running buffer have been shown to produce reproducible injections with RSD < 4% for both migration time and peak area.

Suppression of Apparent Fluid Flow in Capillary Isotachophoresis without Recourse to Capillary Coating

“…Due to the fact that, in EKI, if the EOF is in the opposite direction to the μ ep of the analytes, the analyte injection is less effective, it becomes interesting to suppress the EOF during the EKI of the Aβ 1-40. Moreover, under suppressed EOF conditions, the injected sample amount depends on the transference number of the analyte (which is a function of the ionic mobility, concentration, and charge of the injected ion relative to all other competing ions) . Thus, the optimal injection of the Aβ 1-40 was anticipated to be at pH around 11.…”

“…Moreover, under suppressed EOF conditions, the injected sample amount depends on the transference number of the analyte (which is a function of the ionic mobility, concentration, and charge of the injected ion relative to all other competing ions). 33 Thus, the optimal injection of the Aβ 1-40 was anticipated to be at pH around 11. However, covalently or noncovalently coatings are often easily removed at extreme pHs.…”

Evaluation of capillaries with different inner coatings for DNA analysis using dilute polymer solutions by capillary electrophoresis

“…This column was used as a reference, since it has already provided good results for DNA separations with HEC. 25 As an alternative capillary coating, we used another covalently bound coating of PDMA, a polymer with hydrophobic characteristics. 26 Based on successful results acquired with uncoated capillaries, described by Barron et al, 1 we also used this kind of capillary in our experiments for comparison.…”

Simplex maximization of the correlation coefficient for DNA sizing analysis by capillary electrophoresis

“…In order to define the variables to be optimized, we have taken into account the variables that affect not only the separation mechanism but also the separation performance (resolution). It has been demonstrated elsewhere [15,16] that the performance of a DNA separation is greatly influenced by the injection conditions. Besides, depending on these conditions, changes in the DNA conformation can occur leading to different separation mechanisms [17].…”