Marker pattern instabilities as a major cause of reproducibility problems in two-dimensional DNA fingerprinting.
Two-dimensional (2-D) DNA fingerprinting is a promising technique for multilocus analysis of eukaryotic genomes. It has been successfully applied to the detection of DNA variation in tumors, to linkage analyses and to genomic comparisons of inbred mouse strains. However, there are still problems with inter-gel comparisons of 2-D DNA typing patterns as documented by the inter-gel reproducibility rates reported in the literature, which range from 84 to 98%. The basis for standardization in almost all of these studies has been a set of lambda fragments (digested separately with the restriction enzymes HaeIII, RsaI, Bg/I) that produces a spot pattern scattered across the gel. These spots are used as markers for gel comparisons. Since we noticed considerable variations in the marker spot patterns, we evaluated the properties of the lambda marker using both computer simulation and an empirical analysis of forty independent consecutive gels from our laboratory. We explain the instabilities of the spot pattern on the basis of the melting properties of the individual lambda fragments. A subset of spots is presented that has been stable in all our experiments. Only this set of spots should be used for gel standardization purposes until a new, completely reproducible marker becomes available. Finally, suggestions for an improved marker system are made.
Hampe
J
J
Institut für Medizinische Genetik, Universitätsklinikum Charité, Berlin, Germany. joe@genetik.charite.hu-berlin.de
Marczinek
K
K
Preuss
A
A
Nürnberg
P
P
D010582:Bacteriophage lambda / Q000235:genetics*; D001483:Base Sequence; D003198:Computer Simulation; D016172:DNA Fingerprinting / Q000379:methods*; D004279:DNA, Viral / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D008745:Methylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011340:Problem Solving; D015203:Reproducibility of Results
D004279:DNA, Viral