Restriction fragment length polymorphism: molecular weight analysis and calculation with a scanner-based computer system.

Abstract

For an exact and reproducible molecular weight calculation, an essential requirement in all applications of DNA profiling, we present a new system, called RFLP-MAC (restriction fragment length polymorphism-molecular weight analysis and calculation). The system is composed of a sensitive digital gray-scale scanner unit and an Apple Macintosh computer supported by a powerful combination of a shareware and self-developed software package. Molecular weights are calculated by profiling the optical density and band particle processing parameters to identify the unknown DNA fragments. Mathematical models were used to interpolate the transverse and horizontal migration directions of the molecular weight standards, insuring consistent results from gel to gel. All data are stored in a relational multi-user database system. Descriptive statistics are performed and allele frequencies are analyzed. Procedures for image-analysis, molecular weight calculation, statistical evaluations and database management were developed and run within the multitasking environment of 4th Dimension. Two sets of data, intragel and intergel measurements, are investigated in order to check the precision of the RFLP-MAC system. All these data are subjected to a standard analysis of variance resulting in a maximal 3 SD value of 0.005 (0.288%) for the intragel and of 0.091 (1.654%) for the intergel variation.