DNA fingerprints of dogs and cats

Human minisatellite probes consisting of tandem repeats of the 'core' sequence, a putative recombination signal in human DNA, cross-hybridize to multiple polymorphic fragments in dog and cat DNA to produce individual-specific DNA 'fingerprints'. Pedigree analysis shows that most of the DNA fragments detected in an individual are heterozygous, and that these fragments are derived from multiple dispersed autosomal loci. DNA fingerprints of cats and dogs should prove suitable for individual identification and for establishing family relationships. They are also suitable for rapid marker generation in large pedigrees and could be applied to linkage analysis in these animals.     

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.     

Minisatellite DNA fingerprints of salmonid fishes

The human minisatellite probes 33.6 and 33.15 cross-hybridized to DNA digests of Atlantic salmon, brown trout and rainbow trout revealing complex multi-banded patterns. These DNA fingerprints (in excess of 40 resolvable fragments in some cases) were highly polymorphic, individual specific and found to be stable, both somatically and in the germline. Pedigree analysis of an Atlantic salmon family confirmed that the minisatellite fragments showed Mendelian inheritance. With only a single occurrence of linkage and allelism being observed it is likely the minisatellite loci are widely distributed throughout the salmonid genome. The potential applications for both multi- and single locus minisatellite probes in salmonid research are discussed.     

DNA fingerprints of poultry

Human minisatellite probes cross-hybridize to DNA of several species of poultry (chicken, duck, turkey and goose), and detect high levels of polymorphism. The resulting DNA fingerprints are individual specific, and allow the discrimination even between closely related birds. The pattern of poultry DNA fingerprints is different from that of humans and other animals, having a higher average proportion of large DNA fragments. Pedigree analysis revealed a low number of allelic pairs of variable DNA fragments, indicating that most of the alleles are unresolved in the DNA fingerprint or too small to be detected. The total number of detectable loci in broilers, using probe 33.6, was estimated as 62, of which 13 loci are on average scoreable and available for use. Poultry DNA fingerprints can be used for individual identification, linkage studies and as an aid in breeding programmes.     

Mouse DNA ''fingerprints'': analysis of chromosome localization and germ-line stability of hypervariable loci in recombinant inbred strains.

Human minisatellite probes cross-hybridize to mouse DNA and detect multiple variable loci. The resulting DNA "fingerprints" vary substantially between inbred strains but relatively little within an inbred strain. By studying the segregation of variable DNA fragments in BXD recombinant inbred strains of mice, at least 13 hypervariable loci were defined, 8 of which could be regionally assigned to mouse chromosomes. The assigned loci are autosomal, dispersed and not preferentially associated with centromeres or telomeres. One of these minisatellites is complex, with alleles 90 kb or more long and with internal restriction endonuclease cleavage sites which produce a minisatellite "haplotype" of multiple cosegregating fragments. In addition, one locus shows extreme germ-line instability and should provide a useful system for studying more directly the rates and processes of allelic variation of minisatellites.     

DNA fingerprints of poultry

Summary. Human minisatellite probes cross-hybridize to DNA of several species of poultry (chicken, duck, turkey and goose), and detect high levels of polymorphism. The resulting DNA fingerprints are individual specific, and allow the discrimination even between closely related birds. The pattern of poultry DNA fingerprints is different from that of humans and other animals, having a higher average proportion of large DNA fragments. Pedigree analysis revealed a low number of allelic pairs of variable DNA fragments, indicating that most of the alleles are unresolved in the DNA fingerprint or too small to be detected. The total number of detectable loci in broilers, using probe 33.6, was estimated as 62, of which 13 loci are on average scoreable and available for use. Poultry DNA fingerprints can be used for individual identification, linkage studies and as an aid in breeding programmes.     

Genetic analysis of fingerprints in Mérinos d''Arles X Booroola Merino crossbred sheep

The M13.13 minisatellite probe, consisting of a polymer of the M13 VNTR consensus sequence, cross-hybridized to ovine DNA and allowed detection of several polymorphic loci. Individual specific patterns were obtained in sheep using this probe. Pedigree analysis showed that individuals were heterozygous for most of the DNA fragments detected (88%). By studying the segregation of male's variable DNA fragments, a minimum of 10 loci were defined. The ovine DNA 'fingerprint' obtained with M13.13 is polymorphic enough to be used efficiently in animal identification, paternity testing, and possibly as a source of genetic markers for linkage analysis.     

DNA fingerprints of farm animals generated by microsatellite and minisatellite DNA probes

A multi-locus DNA probe, R18.1, derived from a bovine genomic library, detected DNA fingerprints of highly polymorphic loci in hybridization to genomic DNA from poultry and sheep, and of moderate polymorphic loci in cattle and human DNA. The average numbers of detected bands in chickens and sheep were 27.8 and 21.4, and the average band sharing levels were 0.25 and 0.33, respectively. In hybridization to cattle and human DNA, the results were less polymorphic; nevertheless, individual identification is feasible using probe R18.1. The results obtained by R18.1 were compared to results obtained by Jeffreys minisatellite probe 33.6 and two microsatellite oligonucleotides, (GT)12 and (GTG)5. The total number of detected loci using probes R18.1 and 33.6 were estimated in chickens through family analysis of broilers and the maximal number of detectable loci was calculated.     

The use of synthetic tandem repeats to isolate new VNTR loci: cloning of a human hypermutable sequence.

Synthetic tandem repeats (STRs) of oligonucleotides have previously been shown to detect polymorphic loci in the human genome. Here, we report results from the use of three such probes to screen a human cosmid library. Nine of the 45 positive clones that were analyzed appear to contain highly polymorphic minisatellite or VNTR loci. The degree of enrichment for minisatellite sequences varied with the choice of STR: one provided a 15- to 20-fold enrichment (4 polymorphic loci among 10 clones), whereas 2 others gave a 3- to 5-fold enrichment (5 polymorphic probes in a total of 35 clones) compared to random screening. The 9 VNTR markers have been localized by linkage analysis in the CEPH panel and/or by in situ hybridization. Eight probes identify new loci, one of which maps to an interstitial region. One of the VNTR loci (identified by probe CEB1) was found to be hypermutable, with 52 mutation events identified among 310 children characterized in 40 CEPH families. The parental origin of the mutation could be identified in all instances, and only one mutation was found to be of maternal origin. The mutation rate in males was estimated to be approximately 15%. Segregation analysis of flanking markers suggests that mutations are not associated with crossing over. As the only previously described hypermutable minisatellite loci in humans have equal rates of male and female mutations, these observations establish that a second type of hypermutable minisatellite exists in the human genome. In neither case does the generation of new alleles appear to be associated with unequal crossing over.     

Systematic cloning of human minisatellites from ordered array charomid libraries

We present a rapid and efficient method for the isolation of minisatellite loci from human DNA. The method combines cloning a size-selected fraction of human MboI DNA fragments in a charomid vector with hybridization screening of the library in ordered array. Size-selection of large MboI fragments enriches for the longer, more variable minisatellites and reduces the size of the library required. The library was screened with a series of multi-locus probes known to detect a large number of hypervariable loci in human DNA. The gridded library allowed both the rapid processing of positive clones and the comparative evaluation of the different multi-locus probes used, in terms of both the relative success in detecting hypervariable loci and the degree of overlap between the sets of loci detected. We report 23 new human minisatellite loci isolated by this method, which map to 14 autosomes and the sex chromosomes.     

Cloning a selected fragment from a human DNA ''fingerprint'': isolation of an extremely polymorphic minisatellite.

A large hypervariable DNA fragment from a human DNA fingerprint was purified by preparative gel electrophoresis and molecular cloning. The cloned fragment contained a 6.3 kb long minisatellite consisting of multiple copies of a 37 bp repeat unit. Each repeat contained an 11 bp copy of the "core" sequences, a putative recombination signal in human DNA. The cloned minisatellite hybridized to a single locus in the human genome. This locus is extremely polymorphic, with at least 77 different alleles containing 14 to 525 repeat units per allele being resolved in a sample of 79 individuals. All alleles except the shortest are rare and the resulting heterozygosity is very high (approximately 97%). Cloned minisatellites should therefore provide a panel of extremely informative locus-specific probes ideal for linkage analysis in man.     

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.     

On the use of DNA fingerprints for linkage studies in cattle

To find a marker for the bovine "muscular hypertrophy" gene and for the "roan" locus, we have typed six cattle pedigrees totaling 540 animals for nine blood group systems, for 12 biochemical markers, for RFLPs at four loci, and with five probes revealing multilocus DNA fingerprints. Segregation analysis of the fingerprint bands showed that, in cattle, a fingerprint probe will reveal a mean of 7.6 clearly resolvable bands, behaving as simple, highly informative Mendelian entities characterized by a mean mutation rate of +/- 1/4500 gametes. For one of the bands, we observed a "mutation burst" generating germline mosaicism. Because some of the fingerprint bands were allelic or corresponded to clustered minisatellites, a mean of only 5.7 independent loci is explored per probe. Fingerprint bands revealed by different probes also show a clear propensity for close linkage, pointing toward nonrandom distribution of minisatellite sequences or the existence of minisatellite clusters. Although this reduces the power of fingerprints for linkage analysis substantially, we were able to demonstrate genetic linkage between fingerprint bands and at least three of the classical markers, to exclude the roan locus from 4.5 Morgans of the bovine genome with the DNA fingerprints and for an additional 2.5 Morgans with the classical markers, and to identify a solid candidate marker for the bovine muscular hypertrophy gene, yielding a lod score greater than or equal to 2.84 without any obliged recombinant.     

Linkage analysis by two-dimensional DNA typing.

In two-dimensional (2-D) DNA typing, genomic DNA fragments are separated, first according to size by electrophoresis in a neutral polyacrylamide gel and second according to sequence by denaturing gradient gel electrophoresis, followed by hybridization analysis using micro- and minisatellite core probes. The 2-D DNA typing method generates a large amount of information on polymorphic loci per gel. Here we demonstrate the potential usefulness of 2-D DNA typing in an empirical linkage study on the red factor in cattle, and we show an example of the 2-D DNA typing analysis of a human pedigree. The power efficiency of 2-D DNA typing in general is compared with that of single-locus typing by simulation. The results indicate that, although 2-D DNA typing is very efficient in generating data on polymorphic loci, its power to detect linkage is lower than single-locus typing, because it is not obvious whether a spot represents the presence of one or two alleles. It is possible to compensate for this lower informativeness by increasing the sample size. Genome scanning by 2-D DNA typing has the potential to be more efficient than current genotyping methods in scoring polymorphic loci. Hence, it could become a method of choice in mapping genetic traits in humans and animals.     

Preparation of synthetic tandem-repetitive probes for DNA fingerprinting

DNA fingerprints are generated using probes that hybridize to hypervariable minisatellites, also known as variable number tandem repeat loci. Cloned minisatellites have served as the predominant source of DNA fingerprinting probes. A short segment within the repeat units of minisatellites, called the "core" sequence, is highly conserved within a family of related minisatellites, thereby allowing a single-cloned minisatellite to cross-hybridize to 20 to 40 other minisatellites. In this article, we describe a method for the synthetic preparation of polymeric core sequence probes for DNA fingerprinting. Unlike "monomeric" oligonucleotide probes, the polymeric probes mimic the tandem-repetitive structure of minisatellites, and thus each probe molecule can potentially form many sites of hybridization with a target minisatellite. The synthetic probes are cloned into plasmid DNA to provide a perpetual source of probe material.     

Hypervariable minisatellite DNA sequences in the Indian peafowl Pavo cristatus.

We report here for the first time the large-scale isolation of hypervariable minisatellite DNA sequences from a non-human species, the Indian peafowl (Pavo cristatus). A size-selected genomic DNA fraction, rich in hypervariable minisatellites, was cloned into Charomid 9-36. This library was screened using two multilocus hypervariable probes, 33.6 and 33.15 and also, in a "probe-walking" approach, with five of the peafowl minisatellites initially isolated. Forty-eight positively hybridizing clones were characterized and found to originate from 30 different loci, 18 of which were polymorphic. Five of these variable minisatellite loci were studied further. They all showed Mendelian inheritance. The heterozygosities of these loci were relatively low (range 22-78%) in comparison with those of previously cloned human loci, as expected in view of inbreeding in our semicaptive study population. No new length allele mutations were observed in families and the mean mutation rate per locus is low (less than 0.004, 95% confidence maximum). These loci were also investigated by cross-species hybridization in related taxa. The ability of the probes to detect hypervariable sequences in other species within the same avian family was found to vary, from those probes that are species-specific to those that are apparently general to the family. We also illustrate the potential usefulness of these probes for paternity analysis in a study of sexual selection, and discuss the general application of specific hypervariable probes in behavioral and evolutionary studies.     

A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene.

The human genome contains approximately 50,000 copies of an interspersed repeat with the sequence (dT-dG)n, where n = approximately 10-60. In humans, (TG)n repeats have been found in several sequenced regions. Since minisatellite regions with larger repeat elements often display extensive length polymorphisms, we suspected that (TG)n repeats ("microsatellites") might also be polymorphic. Using the polymerase chain reaction to amplify a (TG)n microsatellite in the human cardiac actin gene, we detected 12 different allelic fragments in 37 unrelated individuals, 32 of whom were heterozygous. Codominant Mendelian inheritance of fragments was observed in three families with a total of 24 children. Because of the widespread distribution of (TG)n microsatellites, polymorphisms of this type may be generally abundant and present in regions where minisatellites are rare, making such microsatellite loci very useful for linkage studies in humans.     

Minisatellite DNA fingerprints of Atlantic cod (Gadus morhua)

The human minisatellite probes 33.6 and 33.15 cross–hybridized to Hae III and Hinf I digested cod DNA, revealing complex fragment patterns in both Arctic and coastal cod. The DNA fingerprints were highly polymorphic, individual specific and stable in the germline. The potential applications of multi locus probes in cod research are discussed.     

"Major minisatellite loci" detected by minisatellite clones 33.6 and 33.15 correspond to the cognate loci D1S111 and D7S437

G. Chimini et al. (1989, Genomics 5: 316-324) have recently reported that the two multilocus DNA fingerprinting probes 33.6 and 33.15 each detect a single major site in the human genome, at 1q23 and 7q35-q36, respectively, and speculate that these sites represent particularly large loci homologous to these probes. However, the human minisatellite loci cloned in 33.6 and 33.15 can themselves be assigned by somatic cell hybrid analysis to 1cen-q24 and 7q31.3-qter, respectively, corresponding to the "major loci" of Chimini et al. Furthermore, under their hybridization conditions, both 33.6 and 33.15 act largely as locus-specific minisatellite probes. The "major minisatellite loci" postulated by Chimini et al. do not therefore appear to represent major localized clusters of minisatellites in the human genome, but rather the loci cloned in 33.6 and 33.15.     

Application of DNA fingerprints for cell-line individualization.

DNA fingerprints of 46 human cell lines were derived using minisatellite probes for hypervariable genetic loci. The incidence of 121 HaeIII DNA fragments among 33 cell lines derived from unrelated individuals was used to estimate allelic and genotypic frequencies for each fragment and for composite individual DNA fingerprints. We present a quantitative estimate of the extent of genetic difference between individuals, an estimate based on the percentage of restriction fragments at which they differ. The average percent difference (APD) among pairwise combinations from the population of 33 unrelated cell lines was 76.9%, compared with the APD in band sharing among cell lines derived from the same individual (less than or equal to 1.2%). Included in this survey were nine additional cell lines previously implicated as HeLa cell derivatives, and these lines were clearly confirmed as such by DNA fingerprints (APD less than or equal to 0.6%). On the basis of fragment frequencies in the tested cell line population, a simple genetic model was developed to estimate the frequencies of each DNA fingerprint in the population. The median incidence was 2.9 X 10(-17), and the range was 2.4 X 10(-21) to 6.6 X 10(-15). This value approximates the probability that a second cell line selected at random from unrelated individuals will match a given DNA fingerprint. Related calculations address the chance that any two DNA fingerprints would be identical among a large group of cell lines. This estimate is still very slight; for example, the chance of two or more common DNA fingerprints among 1 million distinct individuals is less than .001. The procedure provides a straightforward, easily interpreted, and statistically robust method for identification and individualization of human cells.