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A description is given of a novel method for the formal analysis of multilocus DNA fingerprints, the so-called 'genetic factor model'. Using this model, multilocus DNA fingerprints can be shown to be a robust means for both paternity testing and pedigree reconstruction.
Very little is known about the structure and sequence of the genomic DNA and kDNA of T. rangeli and no highly polymorphic markers are known. In this paper, we show that the Jeffreys' multilocal probe 33.15 produces characteristic DNA fingerprints with these trypanosomes. The multiband patterns can be used to differentiate T. cruzi from T. rangeli and for recognizing relationships between strains of the latter from widely different geographic areas and different hosts. The topology of a UPGMA phenetic tree constructed from band-sharing data suggests the existence of two groups of T. rangeli: one encompassing parasites from Central America and the northern part of South America and another with the parasites from southern Brazil. This splitting was confirmed by the use of both nuclear and kinetoplast unique sequence probes. Among strains of T. rangeli, band sharing was generally negatively correlated with geographical distance. This work confirms the usefulness of DNA fingerprints as a potent technique for the analysis of relationships in trypanosomatid populations.
In an unselected control line of a selection experiment in chicken the following traits were evaluated: number of eggs, average egg weight, egg mass, age at first egg, body weight at 20 weeks, body weight at 40 weeks, feed consumption and feed efficiency (feed consumption/egg mass). Data and blood samples of 143 females of the same age were available for analysis. The animals were ranked for each trait according to the phenotypic performance adjusted for hatch and laying house effect rather than the breeding value. DNA mixes of the top 5 and the bottom 5 hens were compared to each other by DNA fingerprinting. The most striking differences could be observed with the probe pV47 in the mixes for body weight at 20 weeks and the feed efficiency. In the following the study focused on feed efficiency. Five groups of 10 hens each, around quantiles 0%, 25%, 50%, 75% and 100%, were analyzed with respect to the occurrence of 7 particular DNA fingerprint bands. The association of the groups' mean feed efficiency with the groups' band frequencies was assessed by linear regression. For 2 bands significant regression coefficients (P < 0.005) were found.
In the last years, DNA fingerprinting became the most powerful tool for identification and paternity testing in man. The success of this method encouraged the German Federal Ministry of Environment, Natural Protection and Reactor Safety to apply DNA fingerprinting in the field of protection of endangered species of birds, such as birds of prey or parrots. In the last three years, we received more than 400 blood and tissue samples of 23 species of birds of prey or parrots, most of them obtained by confiscation, to establish paternity and legal breeding success. We used digoxigenated oligonucleotide probes, mainly (GGAT)4 and (GACA)4 for hybridization. In most cases of confiscated families of birds, paternity testing showed exclusions of nestlings.
Multilocus DNA fingerprinting with oligonucleotide probes (GTG)5, (GATA)4, and (CA)8 was applied in order to determine paternity in one birth cohort (15 infants) of social group (S) from the free-ranging colony of rhesus macaques (Macaca mulatta) on Cayo Santiago. While sires could be identified in 11 cases, all males tested (N = 19) could be excluded from paternity for the remaining four infants. Data revealed marked discrepancies between actual paternity and paternity as inferred from the observation of copulation behavior. Thus, a dominant social rank does not appear to be strongly associated with reproductive success. Furthermore, alternative reproductive strategies were found to yield comparable net benefits in reproduction. A second group of animals (M) was translocated from Cayo Santiago to the Sabana Seca Field Station in 1984. They have continuously resided together in a large outdoor enclosure since then. Here paternity assessment was seriously impeded by a reduced number of discriminating bands, i.e. offspring bands which were unequivocally derived from the sires. This was initially held to be indicative of a smaller degree of heterozygosity in Group M, and was attributed to inbreeding due to a lack of male immigration or extra-group fertilizations. However, a comparison of the DNA fingerprint patterns obtained in Group S and Group M lends only partial support to this idea.
Individual-specific DNA fingerprints of crocodilians were obtained by the use of Bkm-2(8) probe. Pedigree analyses of Crocodylus palustris, C. porosus and Caiman crocodilus revealed that the multiple bands (22-23 bands with Aludigest) thus obtained were inherited stably in a Mendelian fashion. Unique fingerprints permitted us to identify individuals, assign parentage, and reconstruct the DNA profile of a missing parent. Average band sharing between unrelated crocodiles was found to be 0.37. Band sharing between animals of known pedigrees increased predictably with relatedness and provided a basis for distinguishing relatives from non-relatives. Similar results obtained in other species/genera, using the same probe, suggest that this approach may be applicable to all species of crocodilians, and could facilitate genetic studies of wild and captive populations.
As part of an integrated mapping and sequencing analysis of genomes, we have developed an approach allowing the characterization of large numbers of cDNA library clones with a minimal number of experiments. Three basic elements used in the analysis of cDNA libraries are responsible for the high efficiency of this new approach: (1) high-density library arrays allowing thousands of clones to be screened simultaneously; (2) hybridization fingerprinting techniques to identify clones abundantly expressed in specific tissues (by hybridizations with labeled tissue cDNA pools) and to avoid the repeated selection of identical clones and of clones containing noncoding inserts; and (3) a computerized system for the evaluation of hybridization data. To demonstrate the feasibility of this approach, we hybridized high-density cDNA library arrays of human fetal brain and embryonal Drosophila with radiolabeled cDNA pools derived from whole mouse tissues. Fingerprints of the library arrays were generated, localizing clones containing cDNA sequences from mRNAs expressed at middle to high abundance (> 0.1-0.15%) in the respective tissue. Partial sequencing data from a number of clones abundantly expressed in several tissues were generated to demonstrate the value of the approach, especially for the selection of cDNA clones for the analyses of genomes based on expressed sequence tagged sites. Data obtained by the technique described will ultimately be correlated with additional transcriptional and sequence information for the same library clones and with genomic mapping information in a relational database.
Minisatellites, microsatellites, and short random oligonucleotides all uncover highly polymorphic DNA fingerprint patterns in Southern analysis of genomic DNA that has been digested with a restriction enzyme having a 4-bp specificity. The polymorphic nature of the fragments is attributed to tandem repeat number variation of embedded minisatellite sequences. This explains why DNA fingerprint fragments are uncovered by minisatellite probes, but does not explain how it is that they are also uncovered by microsatellite and random oligonucleotide probes. To clarify this phenomenon, we sequenced a large bovine genomic BamHI restriction fragment hybridizing to the Jeffreys 33.6 minisatellite probe and consisting of small and large Sau3A-resistant subfragments. The large Sau3A subfragment was found to have a complex architecture, consisting of two different minisatellites, flanked and separated by stretches of unique DNA. The three unique sequences were characterized by sequence simplicity, that is, a higher than chance occurrence of tandem or dispersed repetition of simple sequence motifs. This complex repetitive structure explains the absence of Sau3A restriction sites in the large Sau3A subfragment, yet provides this subfragment with the ability to hybridize to a variety of probe sequences. It is proposed that a large class of interspersed tracts sharing this complex yet simplified sequence structure is found in the genome. Each such tract would have a broad ability to hybridize to a variety of probes, yet would exhibit a dearth of restriction sites. For each restriction enzyme having 4-bp specificity, a subclass of such tracts, completely lacking the corresponding restriction sites, will be present. On digestion with the given restriction enzyme, each such tract would form a large fragment.(ABSTRACT TRUNCATED AT 250 WORDS)
In the present report the potential of (CAC)5 is demonstrated for DNA fingerprinting in rice. Based on the fingerprint data, (CAC)5 was estimated to differentiate up to 6.3 x 10(8) genotypes of rice. In a search for homologous sequences in rice transcripts, four oligonucleotide probes, namely, (CAC)5, (GATA)4, (GACA)4, and (TG)10, were used. Among these probes, (CAC)5 showed hybridization to total rice RNA in a tissue-specific manner; while a band at 1.2 kb was common in both seed and leaf RNA, there were additional prominent bands at 1.9 and 2.3 kb in seed and leaf RNA, respectively. When (CAC)5 was hybridized to poly(A)+ RNA, in addition to a band at 1.2 kb in seed and leaf, hybridization was observed only in seed to heterogeneously sized RNAs.
Electrophoretic banding patterns of total soluble cell proteins, DNA restriction fragments and chromosomal DNA were used to characterise ten strains of Saccharomyces cerevisiae used for commercial production of wine. These fingerprinting procedures provided unique profiles for all the different yeast strains and can therefore be used to identify and control industrial strains. Furthermore, the protein profiles, restriction fragments banding patterns and electrophoretic karyotyping by contour clamped homogeneous electric field electrophoresis (CHEF), were valuable to differentiate hybrid and parental strains in yeast breeding programmes. Hybrid strains, with desirable oenological properties, were obtained by mass spore-cell mating between a heterothallic killer yeast and two homothallic sensitive strains and all were shown to have unique DNA fingerprints and electrophoretic karyotypes.
In analogy to the polygene determined morphological features, the DNA-fingerprint is also not suitable for statistical processing. Statements about the individuality are merely speculative. Frequencies of genes cannot be found, since it is impossible to determine which combinations of bands belong to one gene locus. Hence the DNA fingerprint enables the recognition of exclusions from paternity; it does not, however, allow a statistical analysis, no matter which method be employed.
A rapid direct-extraction method was used to obtain DNA from environmental soil samples. Heat, enzymes, and guanidine isothiocyanate were utilized to lyse cells. The DNA was purified by agarose gel electrophoresis, amplified with 16S rRNA-based primers by use of the polymerase chain reaction, and then digested with the restriction endonuclease PalI. The extraction method was used to obtain DNA from a variety of plants, bacteria, and fungi including Gossypium hirsucum (cotton), Pseudomonas, Bacillus, Streptomyces, and Colletotrichum. Up to 100 micrograms DNA/g (wet weight) of soil and 400 micrograms DNA/g of plant material were recovered. Restriction endonuclease analysis patterns of amplified rDNA from pure microbial cultures and plant species contained three to five different DNA fragments. Amplified rDNA of mixed population DNA extracts from soil samples, digested with the restriction endonuclease PalI, contained 12-20 DNA fragments, appearing as sample "fingerprints." Results from eight environmental soil samples that were analyzed suggest that the amplified rDNA fingerprints can be used to help characterize the genetic and biological diversity of the microbial populations in these samples.
During an outbreak of Serratia marcescens from May to November 1993 43 strains obtained from 27 ICU patients infected or colonized with multiresistant S. marcescens were genotypically characterized with random amplified polymerase chain reaction (RAPD-PCR)-fingerprinting. In addition, 43 epidemiologically unrelated control isolates were selected. PCR-fingerprinting identified ten different genotypes of S. marcescens among the outbreak related strains. One predominant genotype was demonstrated in 21/43 isolates of 11/27 patients. A cluster of this genotype was found in seven/eight patients on the cardiosurgical ICU. The epidemiologically unrelated strains all showed different genotypes as compared to the predominant type. This survey proved RAPD-PCR to be a highly discriminatory and reproducible method for epidemiological studies of S. marcescens strains in nosocomial outbreaks.
Ninety-five Neisseria gonorrhoeae organisms isolated in Valencia, Spain, were characterized by antibiotic sensitivity testing, auxotyping, serotyping, plasmid analysis and restriction endonuclease fingerprinting (HindIII digestion). Cluster analysis of the restriction patterns revealed that 31 isolates (32.6%) formed 12 clearly defined clusters. Penicillinase-producing Neisseria gonorrhoeae strains formed four of these groups. Eight groups of gonococcal strains were identified by auxotyping, although 83% of isolates belonged to two auxotypes (Proto, Pro-). Twenty-three different serovars were identified by serotyping. The serovar pattern IB/rop was found in 38% of isolates. A 60% coincidence was found between gonococcal groupings obtained by combination of auxotyping, serotyping and plasmid analysis and those obtained with the restriction enzyme fingerprinting technique. The specificity of enzyme restriction patterns of Neisseria gonorrhoeae is confirmed to be of practical importance in the epidemiologic study of gonorrhoea.
Variation amongst strains of Helicobacter pylori was examined by 35S-methionine-labelled protein SDS-PAGE (Radio-PAGE), immunoblot and DNA fingerprinting techniques. These methods allowed discrimination amongst isolates and showed total correlation. Pig and baboon gastric Helicobacter pylori-like organisms were found to be very similar to Helicobacter pylori by both Radio-PAGE and immunoblotting, whereas a Helicobacter mustelae isolate was markedly different. The HindIII restriction endonuclease digest patterns of Helicobacter pylori DNA illustrated the considerable genomic variation of this organism. The Radio-PAGE and immunoblot typing methods both gave precise identification of Helicobacter pylori strains, but Radio-PAGE was found to give higher resolution and represents a standardised universally applicable fingerprinting method for Helicobacter pylori.
A statistical framework is proposed for analysing DNA fingerprint data from experiments aimed at constructing ordered clone physical maps of chromosomes. The fingerprint data consists of the lengths and hybridization states of restriction digest fragments and the paper develops a solution to the fundamental problem of deciding whether or not two randomly selected clones overlap. Overlap probabilities are calculated using Bayes' rule together with appropriate statistical descriptions of the chromosome and experimental procedure. The analysis is flexible, allowing a variety of assumptions to account for experimental errors and difficulties, such as unobserved fragments. The approach described here provides a basis for predicting the rate of progress of an experimental protocol and hence for comparing alternate protocols. It is readily generalized to related problems with a wide range of possible data. Results are presented for the clone mapping protocol currently being employed at Los Alamos National Laboratory on human chromosome 16 (Stallings et al., 1990, Proc. natl. Acad. Sci. U.S.A., 87, 6218-6222).
The detection of polymorphic regions of the human DNA provides individual specific markers, DNA fingerprints (DNA-F), for the identification of individuals. DNA-F can also be used for the detection of cell clones and serve as molecular markers of malignant disease. In leukemia DNA-F analysis can be used for the follow-up studies during remission or after bone marrow transplantation. Recent studies on DNA fingerprinting in leukemia are reviewed.
Oligonucleotide fingerprinting was applied to investigate the relatedness of several cell lines that were established between 1973 and 1977 from a teratocarcinoma. We were able to distinguish cell lines derived at different times. In addition, sublines from one cell line (PYS-2) could be discriminated by using a combination of different probes. Therefore multilocus fingerprinting with oligonucleotides is a useful method for monitoring changes in cell lines kept in culture for many generations.