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  • 1
    ISSN: 0173-0835
    Keywords: Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Germline and somatic instability of the human genome was studied, using synthetic oligonucletides specific for simple repeat motifs. The following probes were used: (GTG)5, (GACA)4, (GATA)4, (CT)8, (TTAGGG)3, (GT)8, (GAA)6 and (GGAT)4. Each of them is unique with respect to the target regions recognized in the genome. Thus compilation of the various fingerprint data provides a complex map of the genome (and its deviations). While the fingerprints of differentiated somatic tissues never showed any alterations, in tumor tissues (namely gliomas) many changes could be detected. Most of the latter reflect secondary karyological aberrations. In nearly one third of the gliomas, drastically amplified and apparently monomorphic DNA fragments were identified. This marker should make it possible to deal with causal pathogenetic mechanisms as well as novel diagnostic strategies.
    Additional Material: 4 Ill.
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  • 2
    ISSN: 0173-0835
    Keywords: DNA fingerprinting ; Two-dimensional DNA typing ; Genome scanning ; Tumor analysis ; Gliomas ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The detection of DNA variation in cancers is an important step in elucidating the mechanism of tumorigenesis. Using the strategy of multipoint genome analysis we detected many differences between glioma-derived and constitutional DNA by customary DNA fingerprinting with simple repetitive oligonucleotide probes. Amplification of the epidermal growth factor receptor (EGFR) gene has been found to be easily detectable as new or highly intensified bands in one-dimensional (1-D) DNA fingerprints of glioblastoma DNA generated with probes (GTG)5 or (GT)8. However, in most low-grade astrocytomas, 1-D DNA fingerprinting has failed to reveal any genomic abnormalities. In these cases a two-dimensional (2-D) technique was successfully employed that is based on size separation in neutral gels followed by sequence-dependent separation in denaturing gradient gels and hybridization with several mini- and microsatellite core probes. The hundreds of spots visualized with this technique were used to detect subtle changes probably occurring as the initial steps of tumorigenesis in human gliomas. On average, five of the approximately 580 sports generated by probes CAC and 33.6 were found to be altered in tumor DNA; 80% of the alterations were spot losses, the rest being spot gains or amplifications. Computer-based image analysis using an external lambda marker provided a stringent way to compare spot patterns generated by 2-D DNA finger-printing. In comparisons performed between typing patterns generated on the same gel, 99% of truly identical spots were confirmed by the sofware. In intergel comparisons 84% of identical spots were matched on the basis of the marker information alone.
    Additional Material: 7 Ill.
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  • 3
    ISSN: 0173-0835
    Keywords: Temperature gradient gel electrophoresis ; Denaturing gradient gel electrophoresis ; Neurofibromatosis gene ; Mutation analysis ; Exon skipping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: We screened a total of 100 unrelated patients with neurofibromatosis type 1 (NF1) for mutations in exons 5 and 8 of the NF1 gene using temperature gradient gel electrophoresis (TGGE). Careful interpretation of exon 5 TGGE patterns was necessary due to interference by an exonic polymorphism. Three novel mutations were identified: a stop mutation in exon 5 (Q239X) caused by a C→T transition at cDNA nucleotide position 715, a transition at the invariant G of the splice accceptor site in intron 4c (G655-1A), and a transversion at the invariant G of the splice donor site in intron 8 (G1185+1T). Analysis of mRNA revealed the predicted abnormal splice products. While skipping of exon 5 causes a shift in the reading frame with a premature stop codon downstream in the middle of exon 6, skipping of exon 8 leads to an in-frame deletion with the predicted protein product being shortened by 41 amino acids.
    Additional Material: 3 Ill.
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  • 4
    ISSN: 0173-0835
    Keywords: DNA fingerprint ; Short tandem marker typing ; Paternity ; Differential reproduction ; Macaca mulatta ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: The fundamental framework for uncovering factors affecting the evolution of social behavior rests upon analyses of variation in reproductive success. In species where females mate with multiple males, paternity is invisible in the absence of genetic data. We determined paternity in two populations of rhesus macaques, Macaca mulatta, using both single locus and multilocus techniques. One troop, Group R, is one of four troops living on a 15 ha island (Cayo Santiago) off the coast of Puerto Rico, while the other troop, Group M, was translocated from Cayo Santiago to the Sabana Seca Field Station (Puerto Rico) in 1984. About a dozen human-derived short tandem repeat (STR) markers have been found to be polymorphic in the study of populations and provide the initial paternity determination. Final evaluation of paternity is then confirmed by multilocus DNA fingerprinting using synthetic oligonucleotide probes. Body condition, age, and dominance rank have an impact on male progeny production, while canine size does not. We suggest that nonagonistic competition in the form of sperm competition and endurance rivalry will modulate male reproductive success. A large body size among males provides them with an advantage in both sperm competition and endurance rivalry. Comparison of the two populations indicated that demographic, social, ecological, and morphological factors interact to regulate variation in reproductive success among male nonhuman primates.
    Additional Material: 2 Ill.
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  • 5
    ISSN: 0173-0835
    Keywords: Two-dimensional DNA fingerprinting ; Gliomas ; Genomic changes ; Spot cloning ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: 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 ciritical early events of glioma pathogenesis.
    Additional Material: 5 Ill.
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  • 6
    ISSN: 0173-0835
    Keywords: Microsatellites ; DNA-sequence analysis ; Genetic variability ; Macaca mulatta ; Primate evolution ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Human (GATA)n microsatellites D12S66 and D12S67 could be successfully amplified by polymerase chain reaction (PCR) in various species of apes and monkeys. In 86 unrelated animals of the most intensively studied species Macaca mulatta we demonstrated five alleles at “D12S66” differing in size in increments of 4 bp (159-175 bp), whereas 17 alleles were observed at locus “D12S67”. The alleles of the latter locus are distributed in two separate groups with no alleles of intermediate size. Six alleles were found between 108-128 bp and 11 alleles between 181-249 bp. Mendelian inheritance of the codom-inant alleles was proven by family studies. Sequencing of the “D12S67” locus revealed that the shorter alleles are characterized by a single perfect (GATA)n stretch whereas the longer alleles consist of two blocks of (GATA)n repeats separated by an intervening sequence of 9 bp. The composite structure of the longer alleles closely resembles that of the 12 human D12S67 alleles (229-273 bp). The enormous species variation in the fragment size range, with the smallest allele found in Macaca mulatta (108 bp) and the largest (364 bp) in Gorilla gorilla gorilla strongly indicates that D12S67 has been subjected to recurrent mutations over the course of primate evolution including a large deletion and/ or insertion event.
    Additional Material: 4 Ill.
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  • 7
    ISSN: 0173-0835
    Keywords: Two-dimensional DNA fingerprinting ; Denaturing gradient gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: 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 intergel 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 paterns, 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.
    Additional Material: 6 Ill.
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  • 8
    ISSN: 0173-0835
    Keywords: DNA fingerprinting ; Two-dimensional DNA electrophoresis ; Numerical iteration ; Relaxation method ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Two-dimensional (2-D) DNA fingerprinting is a technique that allows for parallel genome analysis through the simultaneous detection of up to 500 minior 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.
    Additional Material: 6 Ill.
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  • 9
    ISSN: 0173-0835
    Keywords: Temperature gradient gel electrophoresis ; Psoralen ; Bipolar clamping ; Heteroduplex ; Melting ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Temperature gradient gel electrophoresis (TGGE) is a rapid and sensitive screening method for point mutations and other small DNA alterations. Usually a polymerase chain reaction (PCR)-product of 150 to 500 bp that has been clamped at one end by a psoralen molecule or a “GC-clamp” is tested for abnormal melting characteristics by electrophoresis in a temperature gradient. Under optimal conditions, a heterozygous mutation within the fragment is detected through the presence of three additional bands in the TGGE gel, the mutant homoduplex and two heteroduplex bands. However, the ideal pattern of four sharp bands is not always found due to inconsistencies in melting behavior along the sequence of the DNA fragment under study. Some of these fragments show fuzzy bands that may impede or even prevent the detection of a mutation. Here, we describe a method to overcome this problem by utilizing one psoralen clamp at each end of the PCR product. Using TGGE assays established for exons 16, 17, and 18 of the NF1 gene and for exon 14 of the FBN1 gene as examples, we show that bipolar clamping may transform blurred bands into sharp ones and may visualize mutations that could not be detected by conventional single-sided clamping.
    Additional Material: 4 Ill.
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  • 10
    ISSN: 1432-1203
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary A search for crossover events was performed in seven families with two phenylketonuria-affected children applying seven enzymes for the detection of eight restriction fragment length polymorphisms (RFLPs) by hybridisation with a human phenylalanine hydroxylase (PAH) cDNA probe. A discordance was detected within the PvuIIb-RFLP pattern of two affected sibs. This observation is most likely the result of a cross-over event in the PAH locus. Alternative explanations are discussed. Non-paternity could be excluded by DNA fingerprinting using the oligonucleotide probe (GTG)5.
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