The efficiency of multilocus DNA fingerprint probes for individualization and establishment of family relationships, determined from extensive casework.

The properties of human DNA fingerprints detected by multilocus minisatellite probes 33.6 and 33.15 have been investigated in 36 large sibships and in 1,702 Caucasian paternity cases involving the analysis of over 180,000 DNA fingerprint bands. The degree of overlap of minisatellite loci detected by these two probes is shown to be negligible (approximately 1%), and the resulting DNA fingerprints are therefore derived from independent sets of hypervariable loci. The level of allelism and linkage between different hypervariable DNA fragments scored with these probes is also low, implying substantial statistical independence of DNA fragments. Variation between the DNA fingerprints of different individuals indicates that the probability of chance identity is very low (much less than 10(-7) per probe). Empirical observations and theoretical considerations both indicate that genetic heterogeneity between subpopulations is unlikely to affect substantially the statistical evaluation of DNA fingerprints, at least among Caucasians. In paternity analysis, the proportion of nonmaternal DNA fragments in a child which cannot be attributed to the alleged father is shown to be an efficient statistic for distinguishing fathers from nonfathers, even in the presence of minisatellite mutation. Band-sharing estimates between a claimed parent and a child can also distinguish paternity from nonpaternity, though with less efficiency than comparison of a trio of mother, child, and alleged father.     

DNA Fingerprinting and Analysis of Population Structure in the Chestnut Blight Fungus, Cryphonectria Parasitica

We analyzed DNA fingerprints in the chestnut blight fungus, Cryphonectria parasitica, for stability, inheritance, linkage and variability in a natural population. DNA fingerprints resulting from hybridization with a dispersed moderately repetitive DNA sequence of C. parasitica in plasmid pMS5.1 hybridized to 6-17 restriction fragments per individual isolate. In a laboratory cross and from progeny from a single perithecium collected from a field population, the presence/absence of 11 fragments in the laboratory cross and 12 fragments in the field progeny set segregated in 1:1 ratios. Two fragments in each progeny set cosegregated; no other linkage was detected among the segregating fragments. Mutations, identified by missing bands, were detected for only one fragment in which 4 of 43 progeny lacked a band present in both parents; no novel fragments were detected in any progeny. All other fragments appeared to be stably inherited. Hybridization patterns did not change during vegetative growth or sporulation. However, fingerprint patterns of single conidial isolates of strains EP155 and EP67 were found to be heterogenous due to mutations that occurred during culturing in the laboratory since these strains were first isolated in 1976-1977. In a population sample of 39 C. parasitica isolates, we found 33 different fingerprint patterns with pMS5.1. Most isolates differed from all other isolates by the presence or absence of several fragments. Six fingerprint patterns each occurred twice. Isolates with identical fingerprints occurred in cankers on the same chestnut stems three times; isolates within the other three pairs were isolated from cankers more than 5 m apart. The null hypothesis of random mating in this population could not be rejected if the six putative clones were removed from the analysis. Thus, a rough estimate of the clonal fraction of this population is 6 in 39 isolates (15.4%).     

DNA fingerprinting analysis of vegetative compatibility groups in Aspergillus caelatus

Forty-three isolates of Aspergillus caelatus, whose vegetative compatibility groups (VCGs) have been identified, were assessed by DNA fingerprinting using a repetitive sequence DNA probe (pAF28) cloned from A. flavus. Thirteen distinct DNA fingerprint groups or genotypes were identified among the 43 isolates. Twenty-four isolates belonging to VCG 1 produced identical DNA fingerprints and included isolates from the United States and Japan. Four other DNA fingerprint groups had multiple isolates sharing identical fingerprints corresponding to VCGs 2, 3, 12 and 13. Eight of the 13 fingerprint groups corresponding to VCGs 4-11 were represented by a single isolate with a unique fingerprint pattern. These results provide further confirmation that the pAF28 probe can distinguish VCGs of species within Aspergillus section Flavi based on DNA fingerprint patterns and that the probe can be used to estimate the number of VCGs in a sample population. Most of the A. caelatus isolates produced fewer restriction fragments and weakly hybridized with the repetitive DNA probe pAF28 compared to hybridization patterns obtained with A. flavus, suggesting less homology of the probe to A. caelatus genomic DNA.     

First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication.

The authenticity and freedom from cross-contaminants of a cell line are important prerequisites for any research, development or production programs involving cell lines. Mini- and microsatellites in the human genome harboring variable-numbers of tandem repeat (VNTR) DNA markers allow individualization at the DNA level and are of practical value for genetic linkage mapping, forensic legal medicine, paternity testing, monitoring of bone marrow transplants, and individualization of established cell lines. We have validated fingerprint techniques of different single- and multiple-locus VNTRs enabling the establishment of a searchable database of DNA profiles. As a result, multiplexed polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of four prominent and highly polymorphic minisatellite VNTR loci was proven as the best tool for screening the uniqueness of DNA profiles in a fingerprint database. In order to avoid false positivity, identical or similar DNA profiles based on AmpFLP VNTR were tested further using a multi-locus fingerprint system. Our data demonstrate that misidentification remains a chronic problem among human continuous cell lines (detailed information at URL http://www.dsmz.de). The combination of rapidly generated DNA profiles based on single-locus VNTR loci, their authentication by screening the fingerprint database, and confirmation of duplicate banding patterns using multilocus fingerprints constitute a highly reliable and robust method, which enables high fidelity and quality of maintenance independent from the quantity of individual cell lines.     

Genetic distances and taxonomic analysis of human populations of the Volga-Ural region based on data on DNA polymorphism]

DNA polymorphism was studied in the human diallelic loci MET and D7S23 linked to the cystic fibrosis gene, diallelic locus PAH (the phenylketonuria gene), polyallelic locus ApoB, and hypervariable DNA sequences identified by means of DNA fingerprinting with phage M13 DNA as a probe. The obtained data were used to calculate genetic distances and perform taxonomic analysis of populations of the Volga-Ural region (Turkic and Finno-Ugric ethnic groups). The DNA polymorphic systems studied were demonstrated to be highly informative; their advantages and disadvantages were revealed. According to the data obtained, the genetic distances that were calculated from DNA fingerprints more adequately reflected the genetic relationships between the populations studied than the distances calculated from the allelic frequencies of four DNA loci. It was also found that, in population studies, it would suffice to analyze only the 3.5-6 kb fingerprint fragment that is most suitable for reading, rather than the entire fingerprint obtained.     

GENETIC VARIABILITY WITHIN A POPULATION AND BETWEEN DIPLOID/HAPLOID TISSUE OF MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

Multi-locus DNA fingerprints using an M13 probe were obtained for eight individuals of giant kelp Macrocystis pyrifera (L.) C. Ag. collected from Monterey Bay, California. For each individual, DNA was extracted from a diploid blade and from ca. 10⁹ haploid spores that were released from four to Jive sporophylls. Viable or swimming spores from one individual were pooled and referred to as a spore group. A total of 34 bands (4–19 kb) was detected in DNA fingerprints from the eight blades and eight spore groups, with individual blade or spore groups exhibiting 7–18 bands (mean = 12.6). One band (4.5 kb) was present in all 16 samples. Eight bands were detected in 11–14 of the 16 samples. Similarity indices were calculated for all pairwise comparisons of fingerprint bands among all possible combinations of blades and spore groups. Mean similarity indices for the eight blades (0.51, SE = 0.032) and spore groups (0.56, SE = 0.031) were significantly lower than for the eight comparisons of the blade and spore groups from a single individual (0.86, SE = 0.052). The data indicate that DNA fingerprints can be used to measure genetic variation within populations of M. pyrifera because variation of DNA fingerprints associated with meiotic products (spores) of a given individual is small relative to variation observed among individuals within the population. Additionally, fingerprint variation between diploid vegetative tissue and haploid meiotic products may be a measure of genetic change due to recombination or DNA turnover mechanisms.     

A molecular approach to the identification and individualization of human and animal cells in culture: Isozyme and allozyme genetic signatures

Summary The electrophoretic resolution of a group of geneticallymonomorphic gene-enzyme systems that are developmentally and biologically ubiquitous has been used to provide a species-specific and type-specific biochemical characterization of various cultured cells. The relative mobilities of gene-enzyme systems representing nine distinct gene products from cell cultures of 25 species fromDrosophils to man are presented. These isoenzymes effectively discriminate interspecies cell-to-cell contamination and almost invariably serve to identify the contaminating species. The resolution of eightpolymorphic gene-enzyme systems in human cell cultures provides a virtually unique allozyme genetic signature as a monitor of intraspecies cellular contamination. The genetic signatures of 47 commonly used human cells are presented. Included in the test were seven putative HeLa (human cervical carcinoma) contaminants each of which expressed a signature identical with that of HeLa. The probability that an unrelated human cell line will have a signature identical to a typed cell is computed for each line from the genotypic frequencies at each locus in a population of cultured human cells. The gene frequencies of this cell population are comparable to the same frequencies in natural human populations. The most common human signature has a frequency (and therefore a probability) of 0.02. The majority of the 17,010 possible signatures are far less probable. A calculation of the theoretical incidence of chance matching of signatures within test groups of two or more individuals is presented. The probability of a chance match between any two randomly selected individuals is 0.004 and among five randomly selected individuals is 0.034. The allozyme genetic signature represents a definitive monitor of cell identity and is presented as a standard of cell and tissue identification for a variety of biological studies. This work was supported in part by the Virus Cancer Program of the National Cancer Institute.     

Spatial analysis of nuclear and mitochondrial RFLP genotypes in populations of the chestnut blight fungus, Cryphonectria parasitica

Spatial structure of both nuclear and mitochondrial RFLPs were studied in several populations of the chestnut blight fungus, Cryphonectria parasitica, using a variety of spatial autocorrelation tests designed to detect nonrandom patterns. Fungal individuals were sampled from cankers on infected chestnut trees, and the location of each tree was mapped. Single-locus nuclear RFLPs, nuclear fingerprints, and mitochondrial DNA haplotypes were determined for each individual. Individuals with the same DNA fingerprint genotypes occurred closer together than would be expected at random in four of the five plots, while mitochondrial DNA haplotypes were aggregated in all five plots. Genetic distances between individuals, expressed as one minus the proportion of shared restriction fragment size classes for fingerprints and mitochondrial haplotypes, were significantly correlated with Euclidean distances between individuals in four of the five populations, but these correlations were very weak (r < 0.18). The same DNA fingerprint and single-copy nuclear RFLP alleles occurred on the same trees or immediately neighbouring trees more often than would be expected at random. Most of the aggregation for all three genetic markers occurred among individuals within the same cluster of chestnut stems or on neighbouring trees. Lack of spatial autocorrelation in one population was probably due to sampling on a larger scale that was too coarse to detect any patterns. Significant aggregation of genotypes in C. parasitica is most likely caused by some degree of restricted dispersal within populations. The implications of restricted dispersal are discussed in relation to the breeding system and isolation by distance in populations of. C. parasitica.     

Characterization of a set of X-linked sequences and of a panel of somatic cell hybrids useful for the regional mapping of the human X chromosome

Summary We have characterized 19 DNA fragments originating from the human X chromosome. Most of them have been isolated from an X chromosome genomic library (Davies et al. 1981) using a systematic screening procedure. These DNA probes have been used to search for restriction fragment length polymorphisms (RFLP). The frequency of restriction polymorphisms (1 per 350 bp analysed) was lower than expected from data obtained with autosomal fragments. The various probes have been mapped within 12 subchromosomal regions using a panel of human-rodent hybrid cell lines. The validity of the panel was established by hybridization experiments performed with 27 X-specific DNA probes, which yielded information on the relative position of translocation break-points on the X chromosome. The DNAs from the various hybrid lines are blotted onto a reusable support which allows one to quickly map any new X-specific DNA fragment. The probes already isolated should be of use to map unbalanced X chromosome aberrations or to characterize new somatic cell hybrid lines. The probes which detect RFLPs define new genetic markers which will help to construct a detailed linkage map of the human X chromosome, and might also serve for the diagnosis of carriers or prenatal diagnosis.     

DNA fingerprints from blood mixes in chickens and in Turkeys

Abstract

Although DNA fingerprints are useful in individual identification and genetic linkage studies, expensive and time‐consuming laboratory procedures limit their practical application. By mixing blood from individuals within a population, a DNA fingerprint pattern representing the population can be obtained. The pattern was identical to that in which DNA from individuals was mixed, and was not improved by adjusting blood volumes according to hemoglobin levels.     

DNA fingerprinting in roe deer using the digoxigenated probe (GTG)5

The digoxigenin-labelled oligonucleotide (GTG)₅ was used as a multilocus probe to detect hypervariable microsatellites in roe deer DNA digested with Hae III. The resulting fingerprints of 24 animals belonging to four subpopulations were characterized with regard to within-subpopulation as well as between-subpopulation similarity. The mean number of polymorphic fragments was 20 and the average band-sharing rate for unrelated animals 0.27. A mean probability of 91.5% for a fragment to be present in the heterozygous state was evaluated and the probabilities of identical band patterns in unrelated individuals were estimated to be in the range 1.3 times 10^-16 - 2.5 times 10^-18. Though band-sharing rates of animals belonging to different subpopulations (range 0.18-0.24) were lower than those of within-subpopulations, several measures of population subdivision and the genetic distance do not reveal a striking differentiation of the subpopulations studied.     

GENETIC VARIABILITY AND POPULATION DIFFERENTIATION INFERRED FROM DNA FINGERPRINTING IN SILVEREYES (AVES: ZOSTEROPIDAE)

This study evaluated DNA fingerprinting as a tool for estimating population genetic diversity and differentiation by comparing minisatellite variation in island and mainland populations of silvereyes (Aves: Zosterops lateralis). Three populations with different recent histories were compared: (1) Heron Island and neighboring islands, colonized 3000 to 4000 yr ago; (2) Lady Elliot Island, colonized within the past two decades; and (3) an adjacent mainland population, which presumably has existed for thousands of years. The degree of genetic variability within the three populations reflected both their size and the time since their colonization. Minisatellite diversity was highest in the mainland population, intermediate in the Capricorn Island group (which was shown to represent a single admixture), and lowest in the Lady Elliot Island population, possibly because of a recent population bottleneck during colonization. Mean band sharing between any two populations was less than the mean within either of those populations, and four fingerprint bands common to island birds were rare or absent in the fingerprints of mainland birds. In the absence of significant gene flow between the mainland and the islands, the populations have apparently become distinct at minisatellite loci, as evidenced by differences in both allelic diversity and in the frequencies of specific fragments. Within the Heron Island population, cohort analyses demonstrated the temporal stability of the fingerprint profile over 6 yr. This study demonstrates that length polymorphisms at minisatellite loci may be stable enough over time to retain information about recent historical and demographic effects on the relative genetic variability and differentiation of small, closely related populations.     

A comparative genetic analysis of the Irish greyhound population using multilocus DNA fingerprinting, canine single locus minisatellites and canine microsatellites

Pairwise analysis of Hin fI/33·6 DNA fingerprints from a total of one hundred and fifty-three Irish greyhounds of known pedigree were used to determine band-share estimates of unrelated, first-degree and second-degree relationships. Forty-eight unrelated Irish greyhounds were used to determine allele frequencies for three single-locus minisatellites, and following a preliminary screen, eight of the most polymorphic tetra-nucleotide microsatellites from a panel of 15. The results indicated that both band-share estimates by DNA fingerprinting and microsatellite allele frequencies are highly effective in resolving parentage in this greyhound population, while single-locus minisatellites showed limited polymorphism and could not be used alone for routine parentage testing in this breed. The present study also demonstrated that, to obtain optimal resolution of parentage, sample sets of known pedigree status are required to determine the band-share distribution and/or microsatellite allele frequencies.     

Characterization of Zebu cattle breeds in Tanzania using random amplified polymorphic DNA markers

A total of 141 short primers, of arbitrary nucleotide sequence, were used singly in poly-merase chain reactions to amplify DNA fingerprints in pools of DNA representing three Zebu cattle breeds. Two primers, which discriminated between the breed-specific DNA pools were used further to amplify individual pool components in order to establish band frequencies of the amplified fingerprints. One of the primers (ILO 1127) amplified a RAPD fingerprint in 61%of TSZ animals but less than 6% in the other breeds, while another primer (ILO 1065) revealed a DNA sequence common to 89% of the Boran animals and less than 30% in the other two breeds. Bandsharing and mean average percentage difference calculated within and between the three breeds using RAPD fingerprint data showed a higher degree of homogeneity within than across the breeds and indicated measurable divergence between the three breeds. It is concluded that RAPD polymorphisms are useful as genetic markers for cattle breed differentiation.     

Genetic markers linked to quantitative traits in poultry

This study utilized DNA fingerprints and crosses of two genetically distinct lines of layer-type chickens to identify genetic markers linked to quantitative trait loci (QTL). In phase I, back-cross (BC¹) hens were separately ranked for each of eight traits and then blood pools were produced in groups along each phenotypic distribution. The DNA was isolated from the blood pools and used in a gradient analysis to screen for DNA fingerprint bands that exhibited intensity gradients associated with the phenotypic traits. To identify linkage of bands with QTL and to estimate band effects, F₂ progeny were produced in phase II from the phase I BC, population. A single-trait animal model was used for analysis of associations of all individual DNA fingerprint bands of sires and their progeny phenotypic performance. Twenty fingerprint bands, only two of which had shown trait-associated gradients in phase I, were identified by the animal model analysis of the progeny test as QTL linked (P≤005) to specific traits of growth, reproduction and egg quality. These 20 bands warrant further study as potentially valuable molecular markers for QTL.     

DNA "fingerprints" and segregation analysis of multiple markers in human pedigrees

Tandem-repetitive DNA hybridization probes based on a putative human recombination signal detect multiple polymorphic minisatellite fragments in human DNA. The genetic complexity of the resulting individual-specific DNA "fingerprints" was investigated by studying a large sibship affected by neurofibromatosis and a more extensive pedigree segregating for two different hemoglobinopathies. The segregation of up to 41 different heterozygous DNA fragments from each parent could be analyzed in a single sibship, using two different repeat probes. Most of these variable DNA fragments could not be paired as alleles, to an extent which suggests that the DNA fingerprints are together derived from approximately 60 heterozygous loci (approximately 120 variable fragments), only a proportion of which can be scored in a given individual. Two or three of the DNA fragments detected by one probe showed tight linkage and may be derived from long minisatellite(s) that are cleaved to produce more than one polymorphic DNA fragment. Excluding allelic and linked DNA fragments, almost all remaining scorable fragments segregated independently, allowing up to 34 unlinked loci to be examined simultaneously. These loci are scattered over most or all of the human autosomes. Minisatellite probes are therefore suitable for rapid marker generation and can be applied to linkage analysis in human pedigrees.     

DNA fingerprinting in cattle using oligonucleotide probes

Oligonucleotide probes specific for simple tandem repeat sequences produce individual specific DNA fingerprints in man and all animal species tested so far. Here 11 different synthetic probes were hybridized to bovine genomic DNAs which had been digested with the restriction endonucleases HinfI, AluI and HaeIII. Two of these probes gave DNA fingerprint patterns which were analysed for three German breeds. Different parameters were calculated, such as the average number of bands per individual or the probability of finding identical fingerprints in two unrelated individuals. The number of polymorphic bands varies from 11 to 23 in the different breeds and the probability of finding the same banding pattern in two unrelated individuals ranges from 1.5 x 10(-7) to 2.4 x 10(-7). Hence this DNA fingerprinting procedure allows precise identification of individuals. It is also a useful additional method for paternity testing in cattle.     

Genetic structure of Lake Magadi tilapia populations

The control region of the mitochondrial DNA haplotype frequencies were significantly different among the two separate lagoon populations of Oreochromis alcalicus grahami in Lake Magadi and of O. a. alcalicus from lake Natron, and DNA fingerprint similarity indices were significantly higher for intra-population comparisons of the two Magadi lagoon populations and the Lake Natron population than the inter-population similarity indices among these populations. A modified F _st measure indicated population sub-division and the phylogeographic partitioning of the VNTR fragments observed were unique to specific populations further indicating substantial genetic differentiation. The lagoon populations within Lake Magadi demonstrated the same degree of genetic differentiation as either of these populations did to the outgroup (the Lake Natron population). There appears to be limited gene flow between Lake Magadi tilapia populations and this population structure has important implications for protecting locally adapted populations within this unique ecosystem.     

Minisatellite DNA markers in the chicken genome

This paper reports the detailed characterization of multilocus minisatellite DNA fingerprints in the chicken. Results are presented of DNA fingerprint segregation analyses carried out in three chicken pedigrees, calculating the number of detected loci, testing for Mendelian inheritance, and cosegregation among fingerprint bands. Two pedigrees (families 1 and 2) were analysed using the Jeffreys probes 33.6 and 33.15 only, and one pedigree (family 3) was analysed using 33.6, 33.15. 3′α-globin HVR and M13 protein III gene repeat. Mean band transmission frequencies in families 1 and 2 were near to the Mendelian expectation of 0.5 and no mutations were observed. Family 3 showed transmission frequencies slightly exceeding 0.5. Linkage among bands was higher than observed in some other avian species, with each allele represented by a mean of 1.48 HaeIII fragments. Cosegregation of heterozygous parental fragments representing distinguishable loci followed the expected binomial distribution. The number of minisatellites detectable by the four probes was estimated to be 217. The pattern of cosegregation among those minisatellite loci was tested against that expected for different levels of recombination through the use of a simulation model. We conclude that most minisatellites are unlinked and probably widely dispersed in the chicken genome.     

Fingerprinting human chromosomes by polymerase chain reaction-mediated DNA amplification.

We describe here a method for DNA fingerprinting of human chromosomes by Alu-polymerase chain reaction (PCR) amplification of DNA from monochromosomal hybrids, following digestion with restriction endonucleases. DNA digestion with restriction enzymes prior to PCR amplification reduces the total number of amplified fragments. The number and pattern of bands of PCR products observed in an electrophoretic medium are chromosome specific and provide a "fingerprint signature" for individual human chromosomes. Using this approach, we have produced fingerprints for human chromosomes 2, 5, 7, 9, and 12. The applicability of this approach to chromosome identification was assessed by comparing the fingerprints obtained for two different hybrids containing chromosome 7. DNA fragments specific for the long and the short arms of human chromosome 12 have also been identified. In addition, Alu-PCR-generated DNA fragments, specific for different chromosomes, were used to probe Southern blots of a hybrid cell panel to identify human chromosomes present in hybrid cell lines. The chromosomal specificity of these probes permits the identification of intact as well as rearranged chromosomes composed of segments arising from more than one chromosome.