Hubert Pöche

Oligonucleotide Fingerprinting Using Simple Repeat Motifs: A Convenient, Ubiquitously Applicable Method to Detect Hypervariability for Multiple Purposes

A panel of simple repetitive oligonucleotide probes has been designed and tested for multilocus DNA fingerprinting in some 200 fungal, plant and animal species as well as man. To date at least one of the probes has been found to be informative in each species. The human genome, however, has been the major target of many fingerprinting studies. Using the probe (CAC)5 or (GTG)5, individualization of all humans is possible except for monozygotic twins. Paternity analyses are now performed on a routine basis by the use of multilocus fingerprints, including also cases of deficiency, i.e. where one of the parents is not available for analysis. In forensic science stain analysis is feasible in all tissue remains containing nucleated cells. Depending on the degree of DNA degradation a variety of oligonucleotides are informative, and they have been proven useful in actual case work. Advantages in comparison to other methods including enzymatic DNA amplification techniques (PCR) are evident. Fingerprint patterns of tumors may be changed due to the gain or loss of chromosomes and/or intrachromosomal deletion and amplification events. Locus-specific probes were isolated from the human (CAC)5/(GTG)5 fingerprint with a varying degree of informativeness (monomorphic versus truly hypervariable markers). The feasibility of three different approaches for the isolation of hypervariable mono-locus probes was evaluated. Finally, one particular mixed simple (gt)n(ga)m repeat locus in the second intron of the HLA-DRB genes has been scrutinized to allow comparison of the extent of exon-encoded (protein-) polymorphisms versus intronic hypervariability of simple repeats: adjacent to a single gene sequence (e.g. HLA-DRB1*0401) many different length alleles were found. Group-specific structures of basic repeats were identified within the evolutionarily related DRB alleles. As a further application it is suggested here that due to the ubiquitous interspersion of their targets, short probes for simple repeat sequences are especially useful tools for ordering genomic cosmid, yeast artificial chromosome and phage banks.

DNA Fingerprinting with the oligonucleotide probe (CAC)5/(GTG)5: somatic stability and germline mutations

SummaryDNA fingerprints were generated from various human somatic tissues and from peripheral blood of 179 children and their 80 parents using (CAC)5/(GTG)5 oligonucleotide probes. Whereas somatic stability of the fingerprint patterns was demonstrated, the average rate for germline mutations was estimated to be approximately 0.001 per DNA locus and gamete, with the three different restriction enzymes used. Seven out of eight mutations observed appeared to be of paternal origin.

Paternity testing with oligonucleotide multilocus probe (CAC)5/(GTG)5 : a multicenter study

The statistical analysis is reported of 256 paternity cases referred to seven different German laboratories for multilocus DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5 and restriction enzyme HinfI. All parameters characteristic of multilocus DNA fingerprints were found to differ significantly between the contributing centres: the number of analyzed gel positions, the number of bands scored per individual, the probability of occurrence of a band at a particular position, and the band-sharing probabilities between the mother and both child and alleged father. Despite these differences, paternity cases could be divided clearly into two distinct subgroups on the basis of (i) offspring bands that could not be assigned to either the mother or the alleged father and (ii) the extent of band-sharing between child and alleged father. This partitioning, which is likely to correspond to true and false paternity, confirms previous findings for other multilocus probes. A goodness-of-fit test on the normalized number of bands scored per individual revealed no systematic deviations from commonly adopted analytical models regarding electrophoretic bands as independent entities. Log10-likelihood ratios of paternity vs. non-paternity were calculated utilizing one of these models, and a clear-cut partitioning was again obtained which coincides with that mentioned before. Only one case could not be decided unambiguously, and was either due to two independent mutations or to a close relative of the alleged father being the true father.

Ribosomal protein synthesis in cultured skin fibroblast cells obtained from patients with Duchenne muscular dystrophy

Ribosomes and ribosomal subunits were extracted from cultured skin fibroblasts from patients with Duchenne muscular dystrophy (DMD). A poly(U)-directed polyphenylalanine synthesis system was used to test 80S ribosomes from DMD cells and normal controls as well as hybrid 80S couples of subunits from DMD cells and control cells. The activity of ribosomes extracted from the patients was 38-62% lower than that of normal controls. Of the 80S hybrid ribosomes, only those consisting of 40S subunits from DMD cells and 60S subunits from the control cells, showed a similar decrease in activity. That means that the defect is exclusively based on an alteration in the small ribosomal subunit.

Oligonucleotide DNA fingerprinting: Results of a multi-center study on reliability and validity

We report the results of an empirical study of 256 paternity cases referred to 7 different German laboratories for DNA fingerprinting with oligonucleotide probe (CAC)5/(GTG)5. All parameters characteristic of such multilocus DNA fingerprints were found to differ significantly between the contributing centres. Despite these differences, clear-cut decisions between paternity and non-paternity could be made in all but one case. Furthermore, we found no systematic deviation of the gel-phenotype distribution among trios from random expectation as derived from commonly adopted analytical models. Thus, we conclude that oligonucleotide DNA fingerprinting is a robust and reliable means for the resolution of paternity cases.

The Identification of a Charred Body by Oligonucleotide Fingerprinting with the (GTG)5 Probe

DNA-fingerprinting is a powerful tool for testing consanguinity in complete trios, as well as in deficiency cases. Restriction fragment length polymorphism associated with interspersed simple repetitive DNA sequences arise from different DNA fragment length that contain variable number of the repeated motifes [3]. These hypervariable simple repeat fragments are stably inherited in Mendelian fashion with an exactly defined mutation rate for (GTG)5/(CAC)5 [4]. The simple repetitive probe (GTG)5/(CAC)5 produces a highly informative band pattern in the range from 3.0 to 23.1 kilobases and if discriminates theoretically between all persons on earth, except for monozygotic twins [5, 6].

DNA fingerprinting with simple repetitive oligonucleotide probes in forensic medicine

DNA technology in forensic medicine (determination of the origin of biological materials and of consanguinity) has advanced dramatically over the last five years. Individual-specific fingerprints in man can be obtained using oligonucleotide probes specific for simple repetitive DNA sequences (Ali et al. 1986, Schafer et al. 1988, Nurnberg et al. 1989). Simple repetitive DNA sequences consist of short, tandemly repeated sequence motives. They are spread all over the human chromosomes, i.e. in intergenic spacers and introns; they show extensive polymorphism (Epplen 1988, Epplen et al. 1989). The simple repetitive DNA elements do not exert any sequence dependent function. They do neither contribute to the phenotype nor to the behaviour in man. Therefore the only information obtained concerns the individual-specific hybridization pattern of an individual.

Genetic Typing of Biological Evidence Using PCR

In three separate crime cases identification was achieved using three different PCR systems D1S80 (Budowle et al., 1991), TC11 (Edwards et al., 1992) and SE33 (Polymeropoulos et al., 1992). Although more than 27 alleles are known in the D1S80 system the value of the findings is limited, because of the high frequency of the alleles 18 and 24.