A novel standardization method for two-dimensional DNA fingerprints.
Two-dimensional (2-D) DNA fingerprinting is a technique that allows for parallel genome analysis through the simultaneous detection of up to 500 mini- or microsatellite loci on a 2-D gel. Separation is performed according to size and melting temperature in the gel. In the application of this technique in genome analysis, a standardized method for the identification of individual spots is required. However, due to the polymorphic nature of up to 80% of the spots, existing standardization methods that have been primarily developed for 2-D protein patterns are not suitable for this task. We developed a robust method that standardizes 2-D DNA fingerprint spots on the basis of melting temperature - or denaturing gradient position - and fragment size. An external marker was used as a basis for standardization. A normalization surface was calculated over the gel dimensions by adapting an established numerical iteration technique previously used in physics termed "relaxation method". The relaxation method works robustly with the irregularly spaced marker spots. The evaluation of the method for a spot of preknown position derived from the TP53 gene revealed a median observed error 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 different individuals. We proved experimentally that alleles of a single minisatellite locus cloned from a 2-D gel cluster on an isothermal line can be reliably identified using the presented standardization method. In conclusion, a standardization tool for a broader application of 2-D DNA fingerprinting in both tumor analysis and possibly parallel mutation screening is now available.
Hampe
J
J
Institute of Medical Genetics, Charité University Hospital, Berlin, Germany. hampe@rz.charite.hu-berlin.de
Mrowka
R
R
Marczinek
K
K
Nürnberg
P
P
D000483:Alleles; D016172:DNA Fingerprinting; D015180:Electrophoresis, Gel, Two-Dimensional; D005819:Genetic Markers; D015894:Genome, Human; D006801:Humans; D018598:Minisatellite Repeats; D009691:Nucleic Acid Denaturation; D012150:Polymorphism, Restriction Fragment Length; D012015:Reference Standards; D015203:Reproducibility of Results; D013696:Temperature
D005819:Genetic Markers