Unique DNA Fingerprints Research Articles

Identification of Pistachio (Pistacia vera L.) Nuts with Microsatellite Markers

A genomic DNA library enriched for dinucleotide (CT)n and (CA)n and trinucleotide (CTT)n microsatellite motifs has been developed from `Kerman' pistachio (Pistacia vera L.). The enrichment method based on magnetic or biotin capture of repetitive sequences from restricted genomic DNA revealed an abundance of simple sequence repeats (SSRs) in the pistachio genome which were used for marker development. After an enrichment protocol, about 64% of the clones contained (CT)n repeats while 59% contained (CA)n for CT and CA enriched libraries, respectively. In the (CT)n enriched library, compound sequences were 45% while for (CA)n it was 13.5%. In both dinucleotide enriched libraries, about 80% of the clones having microsatellites have a repeat length in the range of 10 to 30 units. A library enriched for trinucleotide (CTT)n contained <19% of the clones with (CTT)n repeats. Of the clones that contained microsatellites, 62% had sufficient flanking sequence for primer design. An initial set of 25 pairs of primers was designed, out of which 14 pairs amplified cleanly and produced an easily interpretable PCR product in the commercially important American, Iranian, Turkish, and Syrian pistachio cultivars. The efficient DNA extraction method developed for pistachio kernels and shells (roasted and nonroasted) yielded DNA of sufficient quality to use PCR to create DNA fingerprints. In total, 46 alleles were identified by 14 primer pairs and a dendrogram was constructed on the basis of that information. The SSR markers distinguished most of the tested cultivars from their unique DNA fingerprint. An UPGMA cluster analysis placed most of the Iranian samples in one group while the Syrian samples were the most diverse and did not constitute a single distinct group. The maximum number of cultivar specific markers were found in `Kerman'(4), the current industry standard in the United States, and the Syrian cultivar Jalab (5). The technique of using extracted DNA from pistachio kernal or shell coupled with the appropriate marker system developed here, can be used for analyses and measurement of trueness to type.

Influence of Adjunct Use and Cheese Microenvironment on Nonstarter Bacteria in Reduced-Fat Cheddar-Type Cheese

This study investigated population dynamics of starter, adjunct, and nonstarter lactic acid bacteria (NSLAB) in reduced-fat Cheddar and Colby cheese made with or without a Lactobacillus casei adjunct. Duplicate vats of cheese were manufactured and ripened at 7°C. Bacterial populations were monitored periodically by plate counts and by DNA fingerprinting of cheese isolates with the random amplified polymorphic DNA technique. Isolates that displayed a unique DNA fingerprint were identified to the species level by partial nucleotide sequence analysis of the 16S rRNA gene. Nonstarter biota in both cheese types changed over time, but populations in the Colby cheese showed a greater degree of species heterogeneity. The addition of the L. casei adjunct to cheese milk at 104 cfu/ml did not completely suppress “wild” NSLAB populations, but it did appear to reduce nonstarter species and strain diversity in Colby and young Cheddar cheese. Nonetheless, nonstarter populations in all 6-mo-old cheeses were dominated by wild L. casei. Interestingly, the dominant strains of L. casei in each 6-mo-old cheese appeared to be affected more by adjunct treatment and not cheese variety.

An Assessment of Variability of Pulp Mill Wastewater Treatment System Bacterial Communities using Molecular Methods

Abstract The DNA fingerprinting techniques, 16S-restriction fragment length polymorphism (16S-RFLP), ribosomal intergenic spacer analysis (RISA) and repetitive extragenic palindrome PCR (Rep-PCR), were used for analyzing the bacterial communities of seven pulp and paper wastewater treatment systems. All three methods generate DNA fingerprints that can be compared using the computer-assisted program, Gelcompar©. Community similarity coefficients were based on quantitative determinations of both the positions of the DNA bands and the band intensities in order to compare the relative differences in the populations. Unique 16S-RFLP DNA fingerprints were observed for each mill suggesting that individual mills contained phylogenetically different communities. However this method was not sensitive enough to detect differences within a mill treatment system from different locations or from different sampling times. The RISA method, which generated more complex fingerprints than 16S-RFLP, could, for some mills, discern differences between samples. The Rep-PCR technique, however, showed the highest degree of resolution and produced not only distinct patterns for each mill but also distinct fingerprints for the temporal and spatial samples from some of the treatment systems. The sensitivity of this method might potentially be used to monitor the stability of the bacterial community within a secondary treatment system.

DNA fingerprinting of Lactobacillus crispatus strain CTV-05 by repetitive element sequence-based PCR analysis in a pilot study of vaginal colonization.

Lactobacillus crispatus is one of the predominant hydrogen peroxide (H(2)O(2))-producing species found in the vagina and is under development as a probiotic for the treatment of bacterial vaginosis. In this study, we assessed whether DNA fingerprinting by repetitive element sequence-based PCR (rep-PCR) can be used to distinguish the capsule strain of L. crispatus (CTV-05) from other endogenous strains as well as other species of vaginal lactobacilli. Vaginal and rectal lactobacilli were identified to the species level by using whole-chromosome probe DNA hybridization. The DNAs from L. crispatus, L. jensenii, L. gasseri, and an as-yet-unnamed H(2)O(2)-negative Lactobacillus species designated 1086V were subjected to rep-PCR. The results of gel electrophoresis and ethidium bromide staining of the DNA fingerprints obtained were compared. L. crispatus CTV-05 had a unique DNA fingerprint compared to all other lactobacilli. DNA fingerprints for 27 production lots of L. crispatus sampled from 1994 through 2001 were identical to that of the original strain isolated in 1993, suggesting strain stability. In a pilot study of nine women, this DNA fingerprinting method distinguished CTV-05 from other endogenous vaginal lactobacilli prior to and after vaginal capsule use. rep-PCR DNA fingerprinting is useful for strain typing and for evaluating longitudinal loss or acquisition of vaginal lactobacilli used as probiotics.

DNA fingerprinting of sister blastomeres from human IVF embryos.

Previously published single cell DNA fingerprinting systems have been plagued by high rates of allele drop-out (ADO) and preferential amplification (PA) preventing clinical application in preimplantation genetic diagnosis. Tetranucleotide microsatellite markers with high heterozygosity, known allelic size ranges and minimal PCR stutter artefacts were selected for chromosomes X, 13, 18 and 21 and optimized in a multiplex fluorescent (FL)-PCR format. FL-PCR products were analysed using the ABI Prism 377 DNA sequenator and Genescan software. Validation of the DNA fingerprinting system was performed on single diploid (n = 50) and aneuploid (n = 25) buccal cells and embryonic blastomeres (n = 21). The optimized pentaplex PCR DNA fingerprinting system displayed a high proportion of successful amplifications (>91%) and low ADO and PA (<6%) when assessed on 50 human buccal cells. DNA fingerprints of single cells from a subject with Down's syndrome detected the expected tri-allelic pattern for the chromosome 21 marker, confirming trisomy 21. In a blind study on 21 single blastomeres, all embryos were identifiable by their unique DNA fingerprints and shared parental alleles. A highly specific multiplex FL-PCR based on the amplification of five highly polymorphic microsatellite markers was developed for single cells. This finding paves the way for the development of a more complex PCR DNA fingerprinting system to assess aneuploidy and single gene mutations in IVF embryos from couples at genetic risk.

DNA Fingerprinting Analysis of Vegetative Compatibility Groups in Aspergillus flavus from a Peanut Field in Georgia.

The ability of species-specific DNA probe pAF28 to correctly match 75 strains of Aspergillus flavus isolated from a peanut field in Georgia with 1 of 44 distinct vegetative compatibility groupings (VCGs) was assessed. Multiple strains belonging to the same VCG typically produced identical DNA fingerprints, with the exception of VCG 17 and VCG 24, which contained strains that showed 83 and 87% similarity, respectively. A. flavus isolates sharing more than 80% of the fragments are recognized as belonging to the same DNA fingerprint group. Each VCG represented by a single isolate produced unique DNA fingerprints. The results provide further evidence that the pAF28 probe is able to distinguish A. flavus VCGs based on DNA fingerprints and can be used to predict the approximate number of VCGs in a sample population. The DNA probe also hybridized strongly and displayed multiple and distinct bands with other species in Aspergillus section Flavi: A. bombycis, A. caelatus, A. nomius, A. pseudotamarii, and A. tamarii. Although individual strains representing Aspergillus spp. in section Flavi produced DNA fingerprints with multiple bands, the banding patterns could not be used to classify these strains according to species.

Characterisation of Australian isolates of Fusarium oxysporum f. sp. cubense by DNA fingerprinting analysis

Genetic variation among Australian isolates of the fungus Fusarium oxysporum f. sp. cubense (Foc), which causes Fusarium wilt in banana, was examined using DNA amplification fingerprinting (DAF). Ninety-four isolates which represented Races 1, 2, 3, and 4, and vegetative compatibility groups (VCGs) 0120, 0124, 0125, 0128, 0129, 01211, 01213/16, and 01220 were analysed. The genetic relatedness among isolates within each VCG, and between the 8 different VCGs of Foc present in Australia was determined. The DNA fingerprint patterns were VCG-specific, with each VCG representing a unique genotype. The genetic similarity among isolates within each VCG ranged from 97% to 100%. Among the different VCGs of Foc, 3 major clusters were distinguished which corresponded with race. All Race 1 and 2 isolates (VCGs 0124, 0125, 0128, and 01220) were closely related and clustered together, the Race 3 isolates from Heliconia clustered separately, and all Race 4 isolates (VCGs 0120, 0129, 01211, and 01213/16) clustered together. Fifteen isolates from Alstonville, NSW, were characterised because although they were classified as Race 2 based on their recovery from cooking banana cultivars, they belonged in VCG 0124, which had previously contained only Race 1 isolates. The occurrence of more than one race within a VCG means that vegetative compatibility grouping cannot be used to assign pathotype to pathogenic race as previously thought. It was possible to distinguish the Race 1 and Race 2 isolates within VCG 0124 using DNA fingerprinting, as each race produced a unique DNA fingerprint pattern. Among the Australian isolates, DNA fingerprinting analysis identified 9 different VCGs and genotypes of Foc.

Phenotypic and genetic characterization of a novel Borrelia burgdorferi sensu lato isolate from a patient with lyme borreliosis.

Borrelia burgdorferi sensu lato A14S was cultured from a skin biopsy specimen of a patient with erythema migrans in The Netherlands. This isolate had a unique DNA fingerprint pattern compared to 135 other B. burgdorferi sensu lato isolates. In this study, the isolate A14S was further characterized by protein analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and reactivity with various monoclonal antibodies. In addition, the 16S rRNA, ospA, and ospC genes, as well as the 5S-23S rRNA intergenic spacer DNA, were amplified by PCR, cloned, and sequenced. SDS-PAGE protein profiles and phylogenetic analysis based on all of the analyzed genes confirmed that B. burgdorferi sensu lato A14S was phenotypically and genetically different from the three human pathogenic species B. burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii, as well as from other B. burgdorferi sensu lato species. Our findings indicate that Borrelia genomic groups or isolates other than the three well-known human pathogenic species may also cause human Lyme borreliosis.

The application of microsatellites in molecular pathology.

Present within the genome are large numbers of seemingly unimportant DNA segments arranged in repetitive units. Furthermore, these stretches of DNA contain variations or polymorphisms that are characteristic for an individual and results in a unique DNA fingerprint. Approximately 30% of the DNA repeat sequences are arranged as short tandem repeat sequences, which are called microsatellites. Microsatellites may consist of 1, 2 or 3 nucleotides; dinucleotides being the commonest. Microsatellites are characterised by being: stably inherited and hence highly conserved from one generation to the next, and unique to an individual and the same in different cells from the same individual. As a result of the above features, microsatellites can be used for personal identification, population genetic analysis and construction of evolutionary trees. In addition, they are located in several important gene loci and this allows microsatellites to be used as markers of disease and to provide information about individual gene status, especially in tumors. This can be accomplished by assessing allelic imbalance or loss of heterozygosity of a particular gene by analysing microsatellites located at specific loci in the gene. Recently, mutations within microsatellites have been described as a result of defective DNA repair mechanisms, resulting in the phenomenon of microsatellite instability. This has been implicated in the aetiopathogenesis of several hereditary and non-hereditary conditions. There are several ways of analysing microsatellites, the popular using radioactively-labelled primers and autoradiography. This method has several drawbacks, especially the use of radioactivity and interpretative/technical problems. The use of fluorescently-labelled primers, automated DNA sequencing coupled with a computer software package obviates these problems. This technique has the added advantage of analysing several microsatellites in large numbers of cases, simultaneously. Thus, microsatellite analysis has become an important investigative tool for the molecular biologist and has provided new information in many diseases.

Optimization of the arbitrarily-primed polymerase chain reaction (AP-PCR) for intra-species differentiation of Vibrio vulnificus

Optimal parameters were established for obtaining unique and reproducible DNA fingerprints of selected clinical and environmental isolates of Vibrio vulnificus by the arbitrarily primed polymerase chain reaction (AP-PCR). Genomic DNA from selected strains was subjected to AP-PCR amplification using a single, arbitrarily selected oligonucleotide primer, R-PSE420. Amplified DNA was analyzed by agarose gel electrophoresis and ProRFLP® computer software. Reproducibility of the fingerprint was dependent upon the concentrations of the purified genomic DNA, MgCl 2 and oligonucleotide primer, and on PCR cycling parameters. Using 1 μg of purified genomic DNA, 2.5 mM MgCl 2, 1.04 μM of R-PSE420 oligonucleotide primer and thermal cycling protocols with stepwise increases in the annealing temperatures, DNA fingerprints which were reproducible and free of primer artifacts were generated. By following the optimized AP-PCR amplification protocol, unique DNA fingerprint profiles for each V. vulnificus strain tested were produced. These AP-PCR generated unique DNA fingerprint profiles can be used in the identification, and investigation of the distribution and diversity of various strains of V. vulnificus in bivalve shellfish and their surrounding waters.

Spread of the multiresistant Iberian clone of methicillin-resistant Staphylococcus aureus (MRSA) to Italy and Scotland.

The multidrug-resistant "Iberian" clone of methicillin-resistant Staphylococcus aureus (MRSA) was first identified on the basis of its unique DNA fingerprints as the strain responsible for the massive 1989 outbreak of MRSA disease in the hospital Princeps d'Espanya, Barcelona, Spain. Most Iberian MRSA carry a constitutive beta-lactamase. They are resistant to most beta-lactam antibiotics, macrolides, aminoglycosides, tetracycline, rifampin and ciprofloxacin and are susceptible to fosfomycin, fusidic acid, mupirocin, sulfamethoxazole/trimethoprim and vancomycin. The characteristic DNA fingerprints of the clone include the mecA polymorph I, Tn554 pattern E (or its variants), a chromosomal macrorestriction pattern (pulsed-field gel electrophoretic type) A (or its subtype variants), the lack of the mecI regulatory gene and a homogeneous, high level of expression of methicillin resistance. Molecular surveillance studies have documented the extensive spread of this clone to many Portuguese hospitals during the 1990s. In this article, we describe the spread of the Iberian MRSA to hospitals in Rome, Italy, and Scotland.

Molecular epidemiology of Staphylococcus aureus and Enterococcus faecalis in endophthalmitis.

Genomic DNA fingerprint analysis was performed on 39 Staphylococcus aureus and 28 Enterococcus faecalis endophthalmitis isolates collected from multiple clinical centers. Among 21 S. aureus genomic DNA fingerprint patterns identified, five clonotypes were recovered from multiple unrelated patients and accounted for 58.9% (23 of 39) of the isolates analyzed. Compared with strains having unique genomic DNA fingerprint patterns, the S. aureus clonotypes occurring more than once were more likely to result in visual acuities of 20/200 or worse (P = 0.036 [chi2 test]). In contrast to the S. aureus isolates, the E. faecalis endophthalmitis isolates were a clonally diverse population, enriched for the expression of a known toxin, cytolysin, which is plasmid encoded.

Unique DNA fingerprint patterns of Paenibacillus larvae subsp. larvae strains

SUMMARYPaenibacillus larvae subsp. larvae, the causative agent of American foulbrood, is an economically important pathogen in Argentina. Using the polymerase chain reaction (PCR) with primers that anneal to dispersed repetitive bacterial sequences, we generated genomic fingerprints of a collection of 100 P. I. larvae isolates. By using BOX-primers, the strains were grouped into three clusters named A, B and C, respectively. Genotypes A and C were closely related, and all three patterns were highly similar. P. I. larvae banding patterns were distinctive from those of all other Paenibacillus, Bacillus and Brevibacillus species from apiarian sources (n = 41) examined in this work. Paenibacillus larvae subsp. pulvifaciens, the causative agent of powdery scale disease, was closely related to P. I. larvae genotype B, where the only difference found between these subspecies was a band of about 2000 bp. When using ERIC-primers, band differences between both subspecies were not detectable whereas clear differences were noticed with REP-primers. The fingerprints obtained with BOX- and REP-primers appeared unique to P. I. larvae in relation to those generated by other Paenibacillus, Bacillus and Brevibacillus species that share the same habitat.

Clonality in Sclerotinia sclerotiorum on Infected Cabbage in Eastern North Carolina

ABSTRACT Eighty-four isolates of Sclerotinia sclerotiorum from four cabbage production fields in North Carolina and 16 isolates from an experimental cabbage field plot in Louisiana were DNA-fingerprinted and tested for mycelial compatibility. In a comparison with 594 unique DNA fingerprints of S. sclerotiorum from Canadian canola, no fingerprints were shared among Canadian, North Carolina, and Louisiana populations. DNA fingerprints from the North Carolina sample were distinctive from those of the Canadian and Louisiana samples, with significantly more hybridizing fragments in the 7.7- to 18-kilobase range. Forty-one mycelial compatibility groups (MCGs) and 50 unique DNA fingerprints were identified from the North Carolina sample. Three MCGs and three fingerprints were identified from the Louisiana sample. From the North Carolina sample, 32 MCGs were each associated with a unique fingerprint; of these, there were 11 clones (i.e., cases in which two or more isolates belonged to the same MCG and shared the same DNA fingerprint). Six clones sampled from two or more fields represented approximately 29% of the total sample (24 of 84 isolates), with six clones recovered from fields 75 km apart. There were 10 cases in which one MCG was associated with more than one DNA fingerprint and two cases in which one DNA fingerprint was associated with more than one MCG. The small sample from Louisiana was strictly clonal. The North Carolina sample had a clonal component, but deviated from one-to-one association of MCG with DNA fingerprint to an extent consistent with more recombination or transposition than the other two populations sampled.

Comparison of three varieties of Leptographium wageneri using Random Amplified Polymorphic DNA

Leptographium wageneri (Kendrick) Wingfield is the cause of black stain root disease in western North America. The fungus has been separated into three host-specialised morphological variants, namely L . wageneri var. wageneri (Kendrick) Wingfield, var. pseudotsugae Harrington & Cobb and var. ponderosum (Harrington & Cobb) Harrington & Cobb. These varieties have been well-characterised on the basis of both morphological and molecular data. The aim of this study was to determine whether HAPD analysis might be used in distinguishing between the three varieties of L . wageneri . Screening was performed for thirty primers, and four primers that produced unique DNA fingerprints for each variety were selected. RAPD analysis separated the three varieties of L. wageneri into distinct groups. The RAPD analysis was reliable, reproducible and might be useful in separating the three varieties of L . wageneri , which are morphologically very similar.

Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains.

PCR DNA fingerprinting using repetitive intergenic DNA sequences (rep-PCR) was investigated as a means of differentiating between closely related strains of Streptomyces which were, in some cases, indistinguishable by other classification methods. Our results demonstrated that the majority of strains had unique rep-PCR DNA fingerprints and established that the technique could be a very useful tool in rapidly determining strain identity.

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay

Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shiitake mushroom, Lentinula edodes. Seven primers produced polymorphisms in all 15 strains tested, producing 12–19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applications in mushroom breeding and strain improvement programs.

Strain typing of Lentinula edodes by random amplified polymorphic DNA assay.

Single 10-base primers were used to generate randomly amplified polymorphic DNA (RAPD) markers in the shittake mushroom, Lentinula edodes. Seven primers produced polymorphisms in all 15 strains tested, producing 12-19 bands ranging from 0.34 to 2.52 kb. Thirteen of the 15 strains had unique DNA fingerprints, whereas L. edodes ATCC 28759 and ATCC 28760 exhibited identical RAPD profiles for all the primers. Molecular-genetic markers obtained with the RAPD assay can be used to differentiate strains of L. edodes and have potential applicatiosn in mushroom breeding and strain improvement programs.

Molecular epidemiology of tuberculosis in Denmark in 1992.

The incidence of tuberculosis (TB) is increasing all over the world, including in countries with a high standard of living and good social security. Denmark represents such a region. Furthermore, it is a small country (5 million inhabitants) with a long tradition in TB control, including a centralization of the bacteriological diagnostic facility. The present study was intended to analyze the transmission of Mycobacterium tuberculosis in a country in which TB has low endemicity by a combination of conventional epidemiological approaches and DNA fingerprinting techniques, whereby individual bacterial strains can be traced. M. tuberculosis isolates from 92% of all new cases of bacteriologically verified TB in Denmark during 1992 were subjected to IS6110 DNA fingerprinting to visualize the DNA restriction fragment length polymorphism (RFLP) patterns of the isolated strains. The data obtained from the RFLP analyses were interpreted by using demographic data, such as age, sex, ethnicity, and residence, for the patients. The risk factors among the patients for being part of an active chain of transmission, as opposed to demonstrating reactivation of a previously acquired latent infection, were estimated by statistical analyses. The magnitude of TB transmission in 1992 in Denmark was determined, and transmitted infections were shown to comprise at least one quarter of the total number of cases. Almost half of the TB cases involved patients of foreign origin. However, most of these isolates showed unique DNA fingerprint patterns and were rarely part of an active chain of transmission. The major chains of recent transmission were localized to distinct geographical regions in the country. TB is frequent among immigrants, especially from Asia and Africa, but it is apparently readily suspected, diagnosed, and treated by the health care system. Danish patients with pulmonary symptoms are not primarily suspected to have TB and, therefore, play an important role in recent TB transmission in Denmark.

Detection of DNA polymorphisms within the genusCattleya (Orchidaceae)

We have adapted methodology necessary for the detection of molecular polymorphisms in the orchid genusCattleya, namely, randomly amplified polymorphic DNA (RAPD). We report a high level of molecular variability among species; each of eight species examined exhibited a unique DNA fingerprint with nine out of ten arbitrary primers used in single-primer RAPD reactions. Among progeny of an intraspecificCattleya cross, 55 percent of major amplification products were found to segregate. Segregation of these markers facilitated the preliminary identification of several linkage intervals. The identification and mapping of DNA polymorphisms by the RAPD technique will facilitate the use of these taxa for the identification of species-specific and genus-specific traits, allow for the measurement of recombination and introgression in hybrid populations, and enable geneticists to address concordance (or lack thereof) in the processes of speciation, morphological evolution, and molecular change in a large and highly advanced plant family.