Simplex maximization of the correlation coefficient for DNA sizing analysis by capillary electrophoresis.
The separation of DNA molecules in polymeric solution by capillary electrophoresis involves the optimization of several variables, such as polymer solution concentration, electric field separation, temperature, etc. The optimization of each variable individually usually is a time-consuming process and the results may reach a false optimum point. Chemometric methods are suitable to be applied in such cases in which a number of variables can be optimized simultaneously. The simplex is a chemometric method that can perform such a task easily and efficiently. In this study, a simplex method was carried out to maximize the correlation coefficient (r(2)) of a logarithmic plot of mobility (mu) vs. base pair (bp), which was obtained from the separation of DNA fragments of size between 75 and 4072 bp. The simplex showed three vertexes with r(2) > 0.98 and the vertex 21 showing the highest resolution. For the fragments between 201 and 2036 bp, the r(2) increased to 0.992 with and relative standard deviation (RSD) lower than 0.2% (inter- and intra-day variation). The precision of the method in determining the size of a PCR DNA fragment was carried out using a 1 kbp DNA ladder. With the addition of an internal standard to the sample, the precision could be further improved.
Catai
Jonatan R
JR
Instituto de Química de São Carlos, Universidade de São Paulo, Av. Trabalhador Sãocarlense 400, 13560-970 São Carlos-São Paulo, Brazil.
Formenton-Catai
Ana P
AP
Carrilho
Emanuel
E
D005591:Chemical Fractionation; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary
D004247:DNA