By two-dimensional (2-D) DNA typing a restriction enzyme digest of genomic DNA can be resolved on the basis of both size and base-pair sequence and subsequently analysed by repeat probe hybridization to reveal sequence variants at multiple genomic sites in parallel. The system has been partly automated and allows for large-scale comparative analysis of complex genomes in a cost-effective manner.

The existence of repetitive DNA sequences offers the possibility to assess the mammalian genome for individual variation in its entirety rather than at one or only a few sites. In order to fully explore the various sets of mammalian repeat sequences for this purpose, analytical tools are required which allow many if not all individual members of sets of repetitive elements to be resolved and identified in terms of location and allelic variation. We have applied and further developed an electrophoretic system, two-dimensional DNA typing, which may fulfill these requirements. The two-dimensional system combines separation of DNA fragments by size in a neutral gel, with separation by sequence composition in a denaturing gradient gel. By hybridization with minisatellite- and simple-sequence core probes and by inter-repeat polymerase chain reaction techniques, it is possible to obtain individual--and even chromosome-specific separation patterns that consist of hundreds of spots. Computerized image analysis and matching of such spot patterns allows the rapid assessment of multiple polymorphisms, spread over the genome, to monitor genetic variability in populations. When coupled to databases of polymorphic DNA markers with a known genomic location, two-dimensional DNA typing can greatly accelerate the mapping of genetic traits in humans, animals, and plants.

Summary We have recently used two-dimensional DNA typing to detect genetic alterations in breast tumours. This method, which is based on size separation in neutral gels and sequence separation in denaturing gradient gels followed by hybridisation analysis with mini-and microsatellite core probes, allows the simultaneous analysis of hundreds of allelic fragments in a very short time. Here we demonstrate the potency of this method for total genome scanning of the tumour genome by analysing a small series of breast cancers.Comparison of tumour and normal DNA from ten breast cancer patients, using two-dimensional DNA typing with four core probes, revealed a considerable number of genomic alterations. In contrast, with Southern blot analysis only a few alterations were observed using the same probes. Most of the changes observed (74%) were deletions (absence of spots in the tumour) while 20% corresponded to amplifications (spots of higher intensity in the tumour) and 5% were new spots (gains). About 10% of the genomic changes detected appeared to occur in the tumours of more than one patient.Somatic DNA changes play a critical role in the induction and progression of cancer. The results of molecular and epidemiological studies indicate that the induction of cancer in mammals requires the accumulation of several independent mutations (Peto et al., 1975). Mutations affecting a wide range of cellular functions, including growth control, invasion, metastasis and the rate of mutation accumulation itself, can affect the progression of cancer. Such key genetic lesions in the tumour could be of clinical relevance for diagnosis or as a prognostic indicator. Early detection of the relevant changes, i.e. in the primary malignancy, could provide guidelines for better treatment, which is especially relevant in heterogeneous cancers such as breast cancer, nonsmall cell lung cancer and others. In such tumours combinations of mutations are seen. Although recently much information has been obtained on some of the most frequently occurring genomic changes, there is as yet no complete insight into the individual mutation spectra determining the biological properties of the tumour. Indeed, such basic insight would allow the development of combination therapies targeted to various steps in cancer progression (Russell, 1992).Thus far the lack of genome-scanning methods has effectively constrained the large-scale analysis of individual tumours for specific DNA changes that could be correlated with behavioural characteristics of the malignancy. We have recently demonstrated the usefulness of a two-dimensional (2-D) DNA electrophoretic approach for the rapid analysis of cancers for genomic change (Hovig et al., 1993). In this system, which is based on separation by size followed by sequence-specific separation in denaturing gradient gels, large numbers of polymorphic micro-and minisatellite loci can be screened for deletions and amplifications through sequential hybridisation to core probes (Uitterlinden et al., 1989

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