A NOVEL STANDARDIZATION METHOD FOR 2-DIMENSIONAL DNA FINGERPRINTS

Two-dimensional (2-D) DNA fingerprinting is a technique that allows fo r parallel genome analysis through the simultaneous detection of up to 500 mini- or microsatellite loci on a 2-D gel. Separation is performe d according to size and melting temperature in the gel. In the applica tion of this technique in genome analysis, a standardized method for t he identification of individual spots is required. However, due to the polymorphic nature of up to 80% of the spots, existing standardizatio n methods that have been primarily developed for 2-D protein patterns are not suitable for this task. We developed a robust method that stan dardizes 2-D DNA fingerprint spots on the basis of melting temperature - or denaturing gradient position - and fragment size. An external ma rker was used as a basis for standardization. A normalization surface was calculated over the gel dimensions by adapting an established nume rical iteration technique previously used in physics termed ''relaxati on method''. The relaxation method works robustly with the irregularly spaced marker spots. The evaluation of the method for a spot of prekn own position derived from the TP53 gene revealed a median observed err or below 1% for fragment length and denaturing gradient position. The search for candidate minisatellite loci in genomic difference analysis depends on the reliable identification of alleles of this locus in di fferent individuals. We proved experimentally that alleles of a single minisatellite locus cloned from a 2-D gel cluster on an isothermal li ne can be reliably identified using the presented standardization meth od. In conclusion, a standardization tool for a broader application of 2-D DNA fingerprinting in both tumor analysis and possibly parallel m utation screening is now available.