DNA amplification fingerprinting of bacteria

Summary We have amplified short arbitrary stretches of total bacterial DNA to produce highly characteristic and complex DNA fingerprints. This DNA amplification fingerprinting (DAF) strategy involves enzymatic amplification of DNA directed by a single arbitrary oligonucleotide primer. Amplification produces a characteristic spectrum of products that is adequately resolved by polyacrylamide gel electrophoresis and visualized by silver staining. Although DAF is simple in concept, we found that amplification parameters must be within an optimal range for reproducibility. We establish a safe window for these parameters, which include magnesium, primer and enzyme concentration as well as cycle number. The refined procedure was used to distinguish between clinical isolates of Streptococcus uberis, Klebsiella pneumoniae, and Escherichia coli. The use of template DNA concentrations higher than 1 ng·l^–1 and high MgCl₂ levels was especially important for reproductibility when amplifying small bacterial genomes. We tested a truncated Thermus aquaticus DNA polymerase, the Stoffel fragment, and found it more tolerant of reaction conditions, more efficient in the amplification of short products, and able to produce more informative fingerprints when compared to the normal thermostable polymerase from which it was derived. Because DAF produces representative fingerprints quickly and reliably from bacteria regardless of prior genetic or biochemical knowledge, we anticipate the general use of this diagnostic tool for bacterial identification and taxonomy.Correspondence to: G. Caetano-Anollés     

DNA amplification fingerprinting of the Azolla-Anabaena symbiosis

The Azolla-Anabaena symbiosis has been used for centuries as a nitrogen biofertilizer in rice paddies. Genetic improvement of the symbiosis has been limited by the difficulty in identifying Azolla-Anabaena accessions and Anabaena azollae strains. The recently developed technique of DNA amplification fingerprinting (DAF) was applied to this problem. DAF uses single, short, oligonucleotide primers of arbitrary sequence to direct amplification of a characteristic set of DNA products by a thermostable DNA polymerase in a thermocycling reaction. The products are separated in polyacrylamide gels and detected by silver staining. DAF could easily distinguish and positively identify accessions of Azolla-Anabaena with DNA extracted from the intact symbioses. The contribution of prokaryotic Anabaena sequences to the fingerprint of the intact symbioses, however, ranged from 0 to 77%, depending on the primer sequence. Therefore, DNA extracted from the intact symbioses would not be suitable for Azolla taxonomy studies. The fingerprints of Anabaena strains isolated by sucrose gradient centrifugation from different species of Azolla could be easily distinguished, and DAF patterns were used to confirm the maternal pattern of transmission of Anabaena in a sexual hybrid. Template DNA extracted from roots was used to produce fingerprints for Azolla without interference from the microsymbiont. Comparison of the patterns from the parents and a hybrid gave strong evidence confirming sexual hybridization.     

Nucleic acid scanning-by-hybridization of enterohemorrhagic Escherichia coli isolates using oligodeoxynucleotide arrays.

Nucleic acid scanning by hybridization (NASBH) is a non-electrophoretic typing strategy that uses gridded oligonucleotides to reproducibly characterize arbitrarily amplified nucleic acid sequences. Membrane-bound arrays of terminally-degenerate oligonucleotides were hybridized to DNA amplification fingerprinting (DAF) products from enterohemorrhagic Escherichia coli O157:H7 isolates. Numerical and cluster analysis of 64 isolates, selected by DAF to represent a single dominant amplification type identified 14 hybridization types. Results show that NASBH is a powerful alternative for the identification of closely related bacteria, can be used successfully in epidemiological studies, and holds potential in general nucleic acid diagnostics.     

Inheritance of polymorphic markers generated by DNA amplification fingerprinting and their use as genetic markers in soybean

DNA amplification fingerprinting (DAF) using a high primer-to-template ratio and single, very short arbitrary primers, was used to generate amplified fragment length polymorphic markers (AFLP) in soybean (Glycine max (L.) Merr.). The inheritance of AFLPs was studied using a cross between the ancestral Glycine soja PI468.397 and Glycine max (L.) Merr. line nts382, F1 and F2 progeny. The amplification reaction was carried out with soybean genomic DNA and 8 base long oligounucleotide primers. Silver-stained 5% polyacrylamide gels containing 7 M urea detected from 11 to 28 DAF products with primers of varying GC content (ranging from 50 to 100% GC). Depending on their intensity, AFLPs were classified into three classes. DAF profiles were reproducible for different DNA extractions and gels. Forty AFLPs were detected by 26 primers when comparing G. soja and G. max. Most AFLPs were inherited as dominant Mendelian markers in F1 and F2 populations. However, abnormal inheritance occured with about 25% of polymorphisms. One marker was inherited as a maternal marker, presumably originating from organelle DNA while another showed apparent paternal inheritance. To confirm the nuclear origin and utility of dominant Mendelian markers, three DAF polymorphisms were mapped using a F11 mapping population of recombinant inbred lines from soybean cultivars Minsoy × Noir 1. The study showed that DAF-generated polymorphic markers occur frequently and reliably, that they are inherited as Mendelian dominant loci and that they can be used in genome mapping.     

Application of DNA amplification fingerprinting (DAF) to mixed culture bioreactors

Summary The use of DNA amplification fingerprinting (DAF) as a tool for monitoring mixed microbial populations in bioreactors was evaluated. Short (8-mer or 10-mer) oligonucleotides were used to prime DNA extracts from various biological reactors during polymerase chain reaction (PCR) amplification. The reactors examined in this study included two sets of anaerobic stirred tank continuous flow bioreactors. One set of anaerobic reactors was operated under methanogenic conditions and one set was operated under sulfate-reducing conditions. The anaerobic reactor communities in the methanol-fed reactors showed extensive DAF homology. DAF was also applied to a fixed-film azo dye degrading reactor to examine the degree of uniformity of colonization of the substratum in representative regions of the reactor. This method is a quick and relatively inexpensive means of monitoring microbial community structure during biological processes. Since no cultivation of the sample is involved, the genetic profile of the community is not biased by outgrowth conditions. DAF profiles may be useful for comparisons of population changes over time or of bench-scale vs pilot-scale reactors but not adequate for assessing community diversity.     

Primer-template interactions during DNA amplification fingerprinting with single arbitrary oligonucleotides

Summary DNA amplification fingerprinting (DAF) is the enzymatic amplification of arbitrary stretches of DNA which is directed by very short oligonucleotide primers of arbitrary sequence to generate complex but characteristic DNA fingerprints. To determine the contribution of primer sequence and length to the fingerprint pattern and the effect of primer-template mismatches, DNA was amplified from several sources using sequence-related primers. Primers of varying length, constructed by removing nucleotides from the 5 terminus, produced unique patterns only when primers were 8 nucleotides or fewer in length. Larger primers produced either identical or related fingerprints, depending on the sequence. Single base changes within this first 8-nucleotide region of the primer significantly altered the spectrum of amplification products, especially at the 3 terminus. Increasing annealing temperatures from 15° to 70° C during amplification did not shift the boundary of the 8-nucleotide region, but reduced the amplification ability of shorter primers. Our observations define a 3-terminal oligonucleotide domain that is at least 8 bases in length and largely conditions amplification, but that is modulated by sequences beyond it. Our results indicate that only a fraction of template annealing sites are efficiently amplified during DAF. A model is proposed in which a single primer preferentially amplifies certain products due to competition for annealing sites between primer and terminal hairpin loop structures of the template.     

Estimation of genetic distance and coefficient of gene diversity from single-probe multilocus DNA fingerprinting data

DNA fingerprinting exhibits multilocus genotypes of individuals, detected by the use of a single multilocus probe. Consequently, population data on DNA fingerprinting do not provide a complete characterization of the genetic variation in terms of allele-frequency distributions, since neither the number of loci nor the locus affiliation of alleles is directly observable. Yet DNA fingerprinting has been proved to be a cost-effective method of detecting hypervariable polymorphisms in several organisms, where the traditional loci fail to detect enough variation for microevolutionary studies. In the present paper we demonstrate that the above-mentioned features of DNA fingerprinting data do not cause any serious problem when they are used in evolutionary studies. Bias-corrected estimators of Nei's standard and minimum genetic distances are derived, and, by an application of this theory to data on seven short tandem repeat loci in three major human populations, it is shown that these modified measures of genetic distances based on DNA fingerprint patterns are quite close to Nei's distances based on locus-specific allele frequencies. Empirical as well as theoretical support of the adequacy of such genetic distances from DNA fingerprinting data is also discussed, and it indicates that the technical limitations of DNA fingerprinting should not deter the use of the method for short-term evolutionary studies.      

Development of taxon‐specific sequence characterized amplified region (SCAR) markers based on actin sequences and DNA amplification fingerprinting (DAF): a case study in the Phoma exigua species complex

Phoma exigua is considered to be an assemblage of at least nine varieties that are mainly distinguished on the basis of host specificity and pathogenicity. However, these varieties are also reported to be weak pathogens and secondary invaders on non‐host tissue. In practice, it is difficult to distinguish P. exigua from its close relatives and to correctly identify isolates up to the variety level, because of their low genetic variation and high morphological similarity. Because of quarantine issues and phytosanitary measures, a robust DNA‐based tool is required for accurate and rapid identification of the separate taxa in this species complex. The present study therefore aims to develop such a tool based on unique nucleotide sequence identifiers. More than 60 strains of P. exigua and related species were compared in terms of partial actin gene sequences, or analysed using DNA amplification fingerprinting (DAF) with short, arbitrary, mini‐hairpin primers. Fragments in the fingerprint unique to a single taxon were identified, purified and sequenced. Alignment of the sequence data and subsequent primer trials led to the identification of taxon‐specific sequence characterized amplified regions (SCARs), and to a set of specific oligonucleotide combinations that can be used to identify these organisms in plant quarantine inspections.     

Population genetics and spatial structure of the fairy ring fungus Marasmius oreades in a Norwegian sand dune ecosystem

The population genetics and spatial structure of the fairy ring fungus Marasmius oreades (Bolt.?:?Fr.) Fr. was studied by DNA amplification fingerprinting (DAF). Basidiocarp samples were collected from fairy rings from two separate sand dune systems of about 560 m(2) and 1750 m(2), respectively, on the Lista Peninsula in southwestern Norway in 1996. Samples were collected after a careful mapping of fairy rings and a vegetation survey of the composition and spatial structure of vascular plants, bryophytes and lichens. DAF with standard arbitrary oligonucleotide primers was used to examine the genetic relationship between basidiocarp samples. The study showed that the fungal population contained a high number of genotypes and that about 90% of the fairy rings represented a separate genet. Both cluster and phylogenetic analyses of DAF amplification products established relationships between fairy rings and showed that genetically similar basidiocarps were found close to each other. Overall results showed a weak correspondence between genotype and spatial distribution and no correspondence between genotype and composition of the surrounding vegetation. Furthermore, the occurrence of the four dominant sand dune grass species was randomly distributed among the localities housing the various fungal genotypes, indicating that the fungus did not exhibit genotypic specialization to the various grass species that could host it as a pathogen. Results show that establishment of new individuals generally was mediated by basidiospore dispersal and not by fragmenting dikaryotic, vegetative mycelium, as previously proposed.     

Study on DNA Diversity of Liaodong Oak Population at Dongling Mountain Region,Beijing

Random amplified polymorphic DNA(RAPD) and DNA amplification fingerprinting (DAF) markers were used in detecting genetic structure and DNA diversity of two Liaodong oak ( Quercus liaotungensis Koidz. ) populations at Dongling mountain region, a suburb of Beijing City. Shannon's index of phenotypic diversity was used to partition diversity into components within and between populations. Two hundred and five bands from twelve primers were analyzed. The results showed very high genetic variability within the Liaodong oak populations. The diversity in the central population was higher than that of the marginal one. 95 % of total genetic diversity occurred within populations and the coefficient of gene differentiation was 0.05. Significant difference of gene frequency has been detected in a few loci between populations. The study of different age groups of the oak trees implied that deforestation exerted certain impacts on the genetic structure of the Liaodong oak.     

检测植物DNA扩增多态性方法的比较和改进

以辽东栎(Quercus liaotungensis Koidz.)、锦鸡儿(Cargagana ssp.)和野大豆(Glycine soja(L.)Sieb.etZucc.)为材料,比较了随机扩增多态DNA(RAPD)和DNA扩增指纹(DAF)方法。用RAPD的琼脂糖胶电泳和溴乙锭染色,RAPD和DAF谱一般不足10条带。用DAF的变性聚丙烯酰胺凝胶电泳(PAGE)和银染,极大地提高了RAPD的灵敏度和分辨率,多达20～40个产物。用3＇末端完全相同的引物,RAPD和DAF有同样的扩增谱,说明两种方法有相似的机理。降低胶的浓度可提高RAPD和DAF的分辨率,达40～80条带。琼脂糖电泳分离的溴乙锭显示的单荧光带,用PAGE和银染可分辨出多个片段。分子克隆证实单荧光带的分子量异质性。在用Taq DNA多聚酶的条件下,RAPD和DAF的再现性均良好。     

Diversity among Cynodon accessions and taxa based on DNA amplification fingerprinting.

The genus Cynodon (Gramineae), comprised of 9 species, is geographically widely distributed and genetically diverse. Information on the amounts of molecular genetic variation among and within Cynodon taxa is needed to enhance understanding of phylogenetic relations and facilitate germplasm management and breeding improvement efforts. Genetic relatedness among 62 Cynodon accessions, representing eight species, was assessed using DNA amplification fingerprinting (DAF). Ten 8-mer oligonucleotides were used to amplify specific Cynodon genomic sequences. The DNA amplification products of individual accessions were scored for presence (1) or absence (0) of bands. Similarity matrices were developed and the accessions were grouped by cluster (UPGMA) and principal coordinate analysis. Analyses were conducted within ploidy level (2x = 18 and 4x = 36) and over ploidy levels. Each primer revealed polymorphic loci among accessions within species. Of 539 loci (bands) scored, 496 (92%) were polymorphic. Cynodon arcuatus was clearly separated from other species by numerous monomorphic bands. The strongest species similarities were between C. aethiopicus and C. arcuatus, C. transvaalensis and C. plectostachyus, and C. incompletus and C. nlemfuensis. Intraspecific variation was least for C. aethiopicus, C. arcuatus, and C. transvaalensis, and greatest for C. dactylon. Accessions of like taxonomic classification were generally clustered, except the cosmopolitan C. dactylon var. dactylon and C. dactylon var. afganicus. Within taxa, accessions differing in chromosome number clustered in all instances indicating the 2x and 4x forms to be closely related. Little, if any, relationship was found between relatedness as indicated by the DAF profiles and previous estimates of hybridization potential between the different taxa.     

Genetic variation detected by DNA fingerprinting with a rice minisatellite probe in Oryza sativa L.

A rice minisatellite probe detecting DNA fingerprints was used to assess genetic variation in cultivated rice (Oryza sativa L.). Fifty-seven cultivars of rice, including 40 closely related cultivars released in the US, were studied. Rice DNA fingerprinting revealed high levels of polymorphism among distantly related cultivars. The variability of fingerprinting pattern was reduced in the closely related cultivars. A genetic similarity index (S) was computed based on shared fragments between each pair of cultivars, and genetic distance (D) was used to construct the dendrograms depicting genetic relationships among rice cultivars. Cluster analysis of genetic distance tended to group rice cultivars into different units corresponding with their varietal types and breeding pedigrees. However, by comparison with the coefficients of parentage, the criterion of relatedness based on DNA fingerprints appeared to overestimate the genetic relationships between some of the closely related US cultivars. Although this may reduce the power of fingerprints for genetic analysis, we were able to demonstrate that DNA fingerprinting with minisatellite sequences is simpler and more sensitive than most other types of marker systems in detecting genetic variation in rice.This paper reports the results of research only. Mention of a proprietary product does not consititute an endorsement or a recommendation for its use by the USDA or the University of Missouri. Contribution from the US Department of Agriculture, Agricultural Research Service, Plant Genetics Research Unit, and the University of Missouri Agricultural Experiment Station Journal Series No. 12178.     

A Robust DNA Amplification Fingerprinting System Applied to Analysis of Genetic Variation Within Fusarium oxysporum f.sp. cubense

Genetic variation among isolates of F. oxysporum f.sp. cubense (Foc) was analysed using a DNA amplification fingerprinting (DAF) system modified to improve reproducibility and transportability. This analysis was done after determining the widest tolerance range (or 'window of reproducibility') for each component in amplification reaction. Reproducible polymerase chain reactions (PCRs) were achieved with between 25 and 250 ng of template DNA, 9–15 μM primer, 4–6 mM MgCl² and 2–4 units of Stoffel Fragment enzyme. For experimental work we used the middle value of these ranges which allowed at least 20% error tolerance for each component. Similarly, thermocycling and electrophoresis conditions were also improved. Manual scoring of the DNA fingerprints was compared to analysis of scanned gel images using the Gel Compar program (Applied Maths, Kortrijk, Belgium). The data were clustered by unweighted pair group method analysis (UPGMA) based on the Jaccard similarity coefficient. Isolates of Foc representing all known vegetative compatibility groups (VCGs) were examined and the genetic relationships between the VCGs were determined. Isolates of Foc were divided into two major groups with 30% genetic similarity. These optimized DNA amplification, thermocycling, and electrophoresis conditions were suitable for analysis of other organisms and should be applicable to other techniques that use arbitrary primers such as random amplified polymorphic DNA (RAPD) and arbitrarily primed-PCR (AP-PCR).     

Genetic diversity and phylogenetic relationships in Vigna Savi germplasm revealed by DNA amplification fingerprinting.

The pantropical genus Vigna (Leguminosae) comprises 7 cultivated species that are adapted to a wide range of extreme agroclimatic conditions. Few data are available on the relationships among these cultivated species or on their importance as sources of resistance against biotic and abiotic stresses. Therefore, we optimized DNA amplification fingerprinting (DAF) to estimate the genetic diversity within, and genetic relationships among, a representative core collection of cowpea, as compared with 16 accessions representing cultivars from 6 Vigna species. A set of 26 primers was selected from 262 tested random primers and used for the characterization of 85 Vigna accessions (6 V. angularis, 4 each of V. mungo and V. radiata, 2 V. umbellata, 1 V. aconitifolia, and 68 V. unguiculata), with Phaseolus vulgaris subsp. vulgaris as outgroup. A total of 212 polymorphic bands were used for maximum parsimony analysis. Our results clearly distinguished Brazilian from African V. unguiculata genotypes. At the species level, V. angularis was the most related and V. radiata the most divergent species relative to V. unguiculata. DAF markers were also informative at the intraspecific level, detecting a large diversity between cowpea cultivars. The implications of the presented results for cowpea breeding programs are discussed.     

Characterization and genetic mapping of a short, highly repeated, interspersed DNA sequence from rice (Oryza sativa L.).

Summary A short, highly repeated, interspersed DNA sequence from rice was characterized using a combination of techniques and genetically mapped to rice chromosomes by restriction fragment length polymorphism (RFLP) analysis. A consensus sequence (GGC)_n, where n varies from 13–16, for the repeated sequence family was deduced from sequence analysis. Southern blot analysis, restriction mapping of repeat element-containing genomic clones, and DNA sequence analysis indicated that the repeated sequence is interspersed in the rice genome, and is heterogeneous and divergent. About 200000 copies are present in the rice genome. Single copy sequences flanking the repeat element were used as RFLP markers to map individual repeat elements. Eleven such repeat elements were mapped to seven different chromosomes. The strategy for characterization of highly dispersed repeated DNA and its uses in genetic mapping, DNA fingerprinting, and evolutionary studies are discussed.     

The evolving technology of DNA fingerprinting and its application to fisheries and aquaculture

In 1985, Alec Jeffreys reported the development of multilocus DNA fingerprinting by Southern blot-detection of hypervariable minisatellites or variable number of tandem repeat (VNTR) loci. This technology found immediate application to various forensic and scientific problems, including fisheries and aquaculture. By 1989, however, it was recognized by many researchers that inherent problems exist in the application of multilocus fingerprinting to large sample sizes as might occur in fisheries and aquaculture genetic studies. As such, individual VNTRs were cloned for single-locus DNA fingerprinting. Although single-locus fingerprinting ameliorates many of the problems associated with multilocus DNA fingerprinting, it suffers from the problem that electrophorectic anomalies of band migration within and between gels necessitates binning of alleles, thus underestimating genetic variability in a given population. Amplification of microsatellite loci by the polymerase chain reaction, however, solved many of the problems of Southern blot-based DNA fingerprinting. Moreover, microsatellites exhibit attributes that make them particularly suitable as genetic markers for numerous applications in aquaculture and fisheries research: (1) they are abundant in the genome; (2) they display varying levels of polymorphism; (3) alleles exhibit codominant Mendelian inheritance; (4) minute amounts of tissue are required for assay (e.g., dried scales or otoliths); (5) loci are conserved in related species; (6) potential for automated assay. Recent innovations in DNA fingerprinting technology developed over the past 5 years are discussed with special emphasis on microsatellites and their application to fisheries and aquaculture, e.g., behavioural and population genetics of wild species, and selection and breeding programmes for aquaculture broodstock.     

DNA fingerprinting of eukaryote genomes by synthetic oligodeoxyribonucleotide probes

An extreme level of DNA sequence polymorphism, the basis of DNA fingerprinting, was first demonstrated using genome derived cloned probes. Subsequently, it was shown that DNA fingerprinting can also be carried out using short synthetic oligodeoxyribonucleotide probes specific for simple repetitive sequences. Further, in addition to radioactively labeled probes, non-radioactive oligonucleotides generate equally informative hybridization patterns. We discuss the development in the area of DNA fingerprinting and its future scope with respect to plant, animal and the human DNA.     

莲藕品种DNA指纹图谱的绘制

采用RAPD技术对14个莲藕品种进行遗传多态性分析,用5个Operon引物和80个SBS的RAPD引物进行筛选,从中选出来自SBS的RAPD-C13和RAPD-D15扩增出的8条多态性条带,绘制了14个品种的DNA指纹图谱,在该图谱中每个品种均有各自特异的DNA指纹。     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998