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.     

Genetic variability among root voles (Microtus oeconomus) from different geographic regions: populations can be distinguished by DNA fingerprinting

Genetic variability among root voles (Microtus oeconomus [Pallas, 1776]) originating from two distantly separate regions of Norway (Valdres and Finnmark) was studied by DNA fingerprinting using the probes 33.15, 33.6 and M13. All three probes revealed polymorphic, although relatively simple, patterns. DNA fingerprint banding patterns were clearly diagnostic of the animals' region of origin. Notably, Valdres animals display a high molecular-weight cluster of bands not found in Finnmark, reflective of the isolation, and possibly an indication of separate colonization events, of the two groups. On the local level in Finnmark, bands associating with a specific trap site were observed in trappings on consecutive years. Comparisons of Finnmark animals taken at three trap sites at approximately 10 km intervals show a gradient of genetic similarity. Captive bred siblings were also compared, yielding average values significantly higher than those seen from same-site comparisons. We suggest that the sensitivity provided by DNA fingerprinting with multi-locus minisatellite probes is appropriate for population genetic studies in M. oeconomus. Also, because band-sharing correlates to spacing in M. oeconomus, we propose that DNA fingerprinting may be used to study dispersal, recruitment and other population processes in this and possibly other rodent species.     

Use of DNA fingerprinting for human population genetic studies

DNA fingerprinting techniques have been used in population genetic studies on many different kinds of organisms. Here, we present new applications for multilocus DNA fingerprint probes in population studies and demonstrate the applicability of DNA fingerprinting to human population genetics, using M13 phage DNA as a probe. The new approach, which is based on a factor method of numerical coding of non-quantitative data (factor correspondence analysis-FCA), shows good agreement between population position, as indicated by the three principal factors, and ethnogenetic proximity.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 x 10(-4) - 7 x 10(-6). The variability derived with the (TG)n probe in horses was higher than what we obtained with several other commonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

Assessment of DNA fingerprinting for determining genetic diversity in sheep populations

Genomic DNA, prepared from 12 animals from four sheep flocks, was digested with either HaeIII or Hin fI and probed with three DNA fingerprinting probes. Mean DNA fingerprint band sharing and band frequency calculated for each flock were used to estimate genetic diversity. Each of the DNA fingerprinting systems showed the same trend in diversity within the sampled flocks, and greater diversity between the flocks than within the flocks. DNA fingerprinting therefore provides a useful measure of genetic diversity in sheep.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)_n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 times 10-⁴-7 times 10-⁶ The variability derived with the (TG)_n, probe in horses was higher than what we obtained with several other commmonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

大豆灰斑病菌DNA指纹图谱初步分析

大豆灰斑病菌具有明显的生理分化现象,鉴定病菌生理小种对于培育和推广抗病品种具有重要意义。从大豆灰斑病菌一毒力较强菌株的DNA基因组文库中筛选出2个含重复顺序的克隆,并用其对16个菌株的基因组DNA进行了Southern分析,获得了基因组特异的指纹图谱,对指纹图谱和毒力间的关系进行了初步比较。     

Checking of individuality by DNA profiling

A review of methods of DNA analysis used in forensic medicine for identification, paternity testing, etc. is provided. Among other techniques, DNA fingerprinting using different probes and polymerase chain reaction-based techniques such as amplified sequence polymorphisms and minisatellite variant repeat mapping are thoroughly described and both theoretical and practical aspects are discussed.     

中国东部栗疫病菌群体的遗传分化*

本实验采用RFLP技术,对中国东部栗疫病菌(Cryphonectria parasitica)进行了群体遗传结构的研究。313个参试菌株来自10个省(市)的16个群体(子群体),样本分布在北纬24°N—41°N。各菌株的DNA分别用限制性内切酶Pst Ⅰ和EcoR Ⅰ酶切,先后以10个低拷贝DNA探针和1个DNA指纹图谱探针进行了杂交和检测。结果表明,两个探针(pCB29和pMS29.1)的杂交图谱呈单态性;探针pCB19的杂交图谱显示,菌株DNA以PstⅠ酶切的为单态性,以EcoR Ⅰ酶切的则呈多态性;其他7个低拷贝探针的杂交图谱都呈多态性(Pst Ⅰ酶切)、指纹图谱探针的检测结果显示,辽宁凤城群体的菌株与中国东部其他群体的菌株相比,具有更多的限制性杂交片段,菌株间的遗传变异性也更大。     

Repetitive plasmid sequences generate DNA fingerprinting patterns in mammals

Bacterial plasmids with stringently regulated copy numbers have directly repeated DNA sequences, termed iterons, in the vicinity of their replication origins. These sequences bind a specific protein exerting a key role in the initiation of plasmid replication. Plasmids P1, pSC101 and RFS1010 have different iteron sequences and belong to three different incompatibility groups. Used as DNA probes each of these plasmids generates specific patterns in mammals similar to those obtained by the DNA fingerprinting technique. The iteron-containing regions were identified as the part of the plasmids responsible for those patterns by using polymerase chain reaction (PCR) amplified DNA segments that contained the iteron regions as probes.     

Fingerprinting the Asterid species using subtracted diversity array reveals novel species-specific sequences

Background

Asterids is one of the major plant clades comprising of many commercially important medicinal species. One of the major concerns in medicinal plant industry is adulteration/contamination resulting from misidentification of herbal plants. This study reports the construction and validation of a microarray capable of fingerprinting medicinally important species from the Asterids clade.

Methodology/Principal Findings

Pooled genomic DNA of 104 non-asterid angiosperm and non-angiosperm species was subtracted from pooled genomic DNA of 67 asterid species. Subsequently, 283 subtracted DNA fragments were used to construct an Asterid-specific array. The validation of Asterid-specific array revealed a high (99.5%) subtraction efficiency. Twenty-five Asterid species (mostly medicinal) representing 20 families and 9 orders within the clade were hybridized onto the array to reveal its level of species discrimination. All these species could be successfully differentiated using their hybridization patterns. A number of species-specific probes were identified for commercially important species like tea, coffee, dandelion, yarrow, motherwort, Japanese honeysuckle, valerian, wild celery, and yerba mate. Thirty-seven polymorphic probes were characterized by sequencing. A large number of probes were novel species-specific probes whilst some of them were from chloroplast region including genes like atpB, rpoB, and ndh that have extensively been used for fingerprinting and phylogenetic analysis of plants.

Conclusions/Significance

Subtracted Diversity Array technique is highly efficient in fingerprinting species with little or no genomic information. The Asterid-specific array could fingerprint all 25 species assessed including three species that were not used in constructing the array. This study validates the use of chloroplast genes for bar-coding (fingerprinting) plant species. In addition, this method allowed detection of several new loci that can be explored to solve existing discrepancies in phylogenetics and fingerprinting of plants.     

Multilocus DNA fingerprint analysis of cellbanks: Stability studies and culture identification in human B-lymphoblastoid and mammalian cell lines

The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.     

Clonal structure of Rubus chamaemorus populations: comparison of different molecular methods

The clonal structure of Rubus chamaemorus populations was investigated using DNA fingerprinting. The PCR-based methods included the use of 10-base RAPD primers and 16-base simple sequence repeat primers. In the hybridization method variation was studied using hypervariable multilocus probes, one derived from the M13 bacteriophage and the other a synthetic (AC)/(TG) polynucleotide. Although R. chamaemorus expresses clear variation in morphology, the level of genetic differentiation appears to be fairly low. The observed numbers of clones in the three populations examined in Finland varied from 2 to 4. The total number of genotypes across populations was 5, of which one was unique. The results obtained using the two fingerprinting methods were comparable but lead to a slightly different grouping of clones.     

用分子生物学技术对草菇进行菌株鉴别

利用AP-PCR和RAPD技术对三个草菇菌株进行鉴别,其结果与用草菇菌株V34基因文库中的中等重复序列为探针进行限制性内切酶长度多态性分析(RFLP),及对编码核糖体5.8SrRNA的DNA(rDNA)进行PCR扩增后的产物进行限制性内切酶长度多态性分析(PCR-RFLP)的结果相一致。这一结果显示出用这四种方法对草菇菌株进行鉴别具有相似的效果。同时用这四种方法构建的分子生物学标记显示出这三个菌株中的两个菌株在遗传上有着较大程度的相似性。     

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme Not I in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15×2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by M. Johnston     

Use of DNA fingerprinting to determine parentage in muskrats (Ondatra zibethicus).

The detection of high levels of genetic variability by DNA fingerprinting probes has allowed researchers to accurately assess relatedness. Multiple-mating strategies are characteristic of the mating systems of small mammals. As such, techniques that provide an accurate indication of how individuals are related genetically is of great importance to assess the mating system of a species. In this study, we applied the DNA fingerprinting technique to captive and wild muskrats (Ondatra zibethicus) to determine its usefulness for parentage analysis in wild populations. We found that DNA digested with the restriction enzyme Hae III and probed with Jeffrey's minisatellite 33. 15 identified a large amount of polymorphism in both groups of muskrats. The DNA fingerprinting technique correctly assessed parentage within the captive group. In the wild population, paternity was assigned between two adult males based on diagnostic fragments and similarity of banding patterns. The likelihood that paternity could be misassigned to a full sibling was high in this free-ranging population. However, because natal dispersal in muskrats is male biased, it is unlikely that two brothers would associate with the same female.     

Fingerprinting closely related xanthomonas pathovars with random nonamer oligonucleotide microarrays

Current bacterial DNA-typing methods are typically based on gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes are required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments. Furthermore, statistical comparison of gel-based fingerprints is complex and nonstandardized. To overcome these limitations of gel-based microbial DNA fingerprinting, we developed a prototype, 47-probe microarray consisting of randomly selected nonamer oligonucleotides. Custom image analysis algorithms and statistical tools were developed to automatically extract fingerprint profiles from microarray images. The prototype array and new image analysis algorithms were used to analyze 14 closely related Xanthomonas pathovars. Of the 47 probes on the prototype array, 10 had diagnostic value (based on a chi-squared test) and were used to construct statistically robust microarray fingerprints. Analysis of the microarray fingerprints showed clear differences between the 14 test organisms, including the separation of X. oryzae strains 43836 and 49072, which could not be resolved by traditional gel electrophoresis of REP-PCR amplification products. The proof-of-application study described here represents an important first step to high-resolution bacterial DNA fingerprinting with microarrays. The universal nature of the nonamer fingerprinting microarray and data analysis methods developed here also forms a basis for method standardization and application to the forensic identification of other closely related bacteria.     

PCR primed with minisatellite core sequences yields DNA fingerprinting probes in wheat

 Four minisatellite core sequences were used as primers in a polymerase chain reaction (PCR) technique, known as the directed amplification of minisatellite-region DNA (DAMD), to detect polymorphisms in three pairs of hexaploid/tetraploid wheat cultivars. In each pair, the tetraploid cultivar (genomic formula AABB) was extracted from its corresponding hexaploid (genomic formula AABBDD) parent. Reproducible profiles of the amplified products revealed characteristic bands that were present only in the hexaploid wheats but not in their extracted tetraploids. Some polymorphisms were observed among the hexaploid cultivars. Twenty-three DAMD-PCR amplified fragments were isolated and screened as molecular probes on the genomic DNA of wild wheat species, hexaploid wheat and triticale cultivars. Subsequently, 8 of the fragments were cloned and sequenced. The DAMD-PCR clones revealed various degrees of polymorphism among different wild and cultivated wheats. Two clones yielded individual-specific DNA fingerprinting patterns which could be used for species differentiation and cultivar identification. The results demonstrated the use of DAMD-PCR as a tool for the isolation of informative molecular probes for DNA fingerprinting in wheat cultivars and species. Received: 13 May 1996/Accepted: 11 October 1996     

Repetitive, genome-specific probes in wheat (Triticum aestivum L. em Thell) amplified with minisatellite core sequences

The detection and analysis of DNA polymorphisms in crops is an essential component of marker-assisted selection and cultivar identification in plant breeding. We have explored the direct amplification of minisatellite DNA by PCR (DAMD-PCR) as a means for generating DNA probes that are useful for detecting DNA polymorphisms and DNA fingerprinting in wheat. This technique was facilitated by high-stringency PCR with known plant and animal minisatellite core sequences as primers on wheat genomic DNA. The products of DAMD-PCR from Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii showed a high degree of polymorphism and the various genomes could be identified. Cloning of the DAMD-PCR products and subsequent Southern hybridization frequently revealed polymorphic probes showing a good degree of genome specificity. In addition, polymorphic, single locus, and moderately dispersed PCR products were cloned that may have a potential for DNA fingerprinting. Our experiments were limited primarily to diploid wheats and the results indicated that DAMD-PCR may isolate genome-specific probes from wild diploid wheat species that could be used to monitor genome introgression into hexaploid wheat.This paper reports the results of research only. Mention of a proprietary product does not constitute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the University of Missouri, the Agricultural Experimental Station and U.S. Department of Agriculture-Agricultural Research Service, Plant Genetics Research Unit, journal series No. 12523     

DNA “fingerprints” and paternity ascertainment in chimpanzees (Pan troglodytes)

Highly variable regions of DNA are found in a wide diversity of organisms and are typically composed of alleles consisting of a variable number of tandem repeats (VNTRs) of a short core sequence. DNA fingerprinting probes are VNTR probes that simultaneously detect a large number of similar VNTRs in the target DNA. The highly polymorphic pattern observed in a DNA fingerprint allows resolution of questions concerning individual identification. M13 phage was used to fingerprint captive chimpanzees for paternity ascertainment. Although the probability of band sharing among captive chimps appears to be higher than among some other reported captive and feral animal populations, the probe is highly useful and can be expected to become more widely used in the genetic management of captive populations.