COMPUTERIZED ESTIMATION OF SIZE OF NUCLEIC-ACID FRAGMENTS USING THE 4-PARAMETER LOGISTIC MODEL

We have developed improved methods for statistical estimation of the size of linear duplex DNA after continuous- or pulsed-field electrophoresis in agarose and polyacrylamide gels. We employ the four-parameter logistic model to describe the smooth, symmetrical, sigmoidal relationship between electrophoretic mobility (distance migrated) and log of molecular size (kb): distance = a-d 1+( size c)b+d The four parameters (a, b, c, d) are estimated by nonlinear least-squares curve fitting using the Marquardt-Levenberg algorithm, where a represents an upper plateau, b a slope factor, c the midpoint, and d the lower plateau. Estimates of size for unknown species are accompanied by estimates of the standard error (SE) or coefficient of variation (%CV). A plot of SE and %CV versus size results in a "precision profile" which objectively defines the useful range of the calibration curve. This logistic relationship is more general than the rectangular hyperbola or linear methods, provides excellent goodness of fit, and can be used as a "global" method for the entire calibration curve, rather than as a "local" method for small segments of the curve. This computerized, automated method has been applied successfully to a wide range of DNA standards and gel concentrations, including: (i) 20 runs of a mixture of λ HindIII and φX HaeIII fragments (0.3-23 kb, on a 0.7% agarose gel); (ii) extensive data of N. C. Stellwagen addressing the effect of systematic variation of gel concentration (0.2 - 1.5% agarose) and field strength (0.64 - 3.8 V/cm); (iii) pBR322 HinfI and HaeIII fragments (0.05 - 1.6 kb, on a 10% polyacrylamide gel); and (iv) data of Birren et al. for pulsed-field gel electrophoresis of 1- to 50-kb DNA fragments using various switching intervals, and other systems for pulsed-field gel electrophoresis extending to the megabase range. © 1990.