Two-dimensional DNA Fingerprinting Research Articles

Changes in methylation patterns identified by two-dimensional DNA fingerprinting.

Two-dimensional DNA fingerprinting (2-D fingerprinting) is a sensitive tool for genomic difference analysis between tumor DNA and constitutive DNA of glioma patients. Numerous differences were found even in low-grade gliomas. They can be interpreted as deletions, amplifications, rearrangements, HaeIII restriction site mutations, tandem repeat instabilities, or methylation differences. The influence of methyl groups on the melting behavior of double-stranded DNA fragments in a denaturing gradient gel was demonstrated by analyzing the migration of lambda-phage DNA fragments in 2-D fingerprint gels. A characteristic intensity shift between two neighboring spots in several glioma samples was identified and verified by rehybridization of 2-D filters with a cloned DNA fragment corresponding to the lower spot in 10 out of 11 pilocytic astrocytomas. We hypothesized that this shift may be related to an alteration in the methylation pattern of the tumor DNA. This was specifically tested by analyzing the underlying 750 bp genomic fragment (including 21 CpG dinucleotides) with bisulfite treatment of agarose-embedded DNA. A methylation grade of 88% in tumor DNA as compared to 96% in blood DNA was found. Although only one CpG is located in the melting domain of the cloned fragment, this particular CpG is methylated in all blood samples, but mostly demethylated in the tumor samples. In conclusion, we demonstrate that 2-D fingerprinting may be a powerful tool for the detection of DNA methylation changes in genomic difference analysis.

Cloning of minisatellite-containing sequences from two-dimensional DNA fingerprinting gels reveals the identity of genomic alterations in low-grade gliomas of different patients.

Two-dimensional (2-D) DNA fingerprinting was used to investigate genomic changes in human low-grade gliomas of different subtypes. DNA variations were identified in the 2-D hybridization patterns as spot losses or gains. Computer-aided matching of spot patterns from different patients revealed a clustering of spot changes at particular areas in the gel. Representative spots of each cluster were cloned using a spot cloning protocol which includes the preparation of a duplicate and a master gel. The DNA fragments of the 2-D gels were transferred to DEAE and nylon membrane, respectively. After hybridization of the master blot with a minisatellite core probe, the position of a particular spot was determined with reference to the lambda DNA fragments used as external markers in both gels. The gel spot DNA was recovered from the DEAE membrane by high salt elution and was polymerase chain reaction (PCR)-amplified after ligation of adaptor oligo cassettes. The PCR products were cloned and used as locus-specific probe for the rehybridization of the 2-D blots. One of these probes detected a spot loss in 7 of 28 low-grade gliomas of different subtypes analyzed. Another probe revealed a characteristic intensity shift in 8 of 9 pilocytic astrocytomas between two neighboring spots. The target sequence of this highly specific effect was assigned to chromosome 11q14 by in situ hybridization of a P1 clone harboring the affected genomic region. Thus, we successfully established a spot cloning procedure for the generation of locus-specific probes that may be instrumental in the discovery of the critical early events of glioma pathogenesis.

Detection of DNA variation in cancer.

Detection of DNA variation in cancer is central to the identification of relevant genes and mutations involved in the tumourigenic process. Diverse methods exist for such detection. One category of methods is for the detection of frequent sites for larger DNA alterations in cancer. Such areas may provide clues to the positioning of relevant genes, such as loss of heterozygosity (LOH) as in the case of tumour suppressor genes. Another category of methods is for the detection of single base mutations within specific genes. Frequently, such mutations may obliterate normal protein function. Among the most well-known are DGGE, SSCP, the HOT-method and direct sequencing. The methods for detection of DNA variation of these different levels are discussed. Two methods are presented in more detail. At the large-scale level, two-dimensional DNA fingerprinting has the potential of revealing the extent and location of altered DNA regions. This method is demonstrated using a panel of breast cancer patients. As an example of methods for the small-scale level, a recent development from DGGE, constant denaturant gel electrophoresis (CDGE) is demonstrated. This method has successfully been applied for the detection of mutations in a number of genes. Results with this method in studies of the RB1 gene are given, and its applicability as a screening tool for base mutations is discussed.

Denaturing gradient gel electrophoretic analysis of minisatellite alleles

By two-dimensional DNA fingerprinting, an electrophoretic method which combines separation according to size with separation in a denaturing gradient, virtually all minisatellite sequences detected with a minisatellite core probe can be resolved (Uitterlinden et al., Proc. Natl. Acad. Sci. USA 1989, 86, 2742-2746). To investigate the electrophoretic behavior in denaturing gradient gels of allelic restriction fragments containing minisatellite sequences, we analyzed alleles of the two highly polymorphic minisatellite loci D7S22 and D2S44. The results obtained indicate that for these loci, depending on the restriction enzyme used to digest genomic DNA, alleles of different sizes migrate to regions of similar denaturant concentration, i.e. to isothermal positions in the denaturing gradient. Denaturing gradient gel electrophoresis also allows for the discrimination of restriction fragments which are the result of the presence of internal recognition sites in the minisatellite and, therefore, to distinguish between VNTR and restriction site polymorphisms.

Two-dimensional DNA fingerprinting of human individuals.

The limiting factor in the presently available techniques for the detection of DNA sequence variation in the human genome is the low resolution of Southern blot analysis. To increase the analytical power of this technique, we applied size fractionation of genomic DNA restriction fragments in conjunction with their sequence-dependent separation in denaturing gradient gels; the two-dimensional separation patterns obtained were subsequently transferred to nylon membranes. Hybridization analysis using minisatellite core sequences as probes resulted in two-dimensional genomic DNA fingerprints with a resolution of up to 625 separated spots per probe per human individual; by conventional Southern blot analysis, only 20-30 bands can be resolved. Using the two-dimensional DNA fingerprinting technique, we demonstrate in a small human pedigree the simultaneous transmission of 37 polymorphic fragments (out of 365 spots) for probe 33.15 and 105 polymorphic fragments (out of 625 spots) for probe 33.6. In addition, a mutation was detected in this pedigree by probe 33.6. We anticipate that this method will be of great use in studies aimed at (i) measuring human mutation frequencies, (ii) associating genetic variation with disease, (iii) analyzing genomic instability in relation to cancer and aging, and (iv) linkage analysis and mapping of disease genes.