Restriction Endonuclease Fingerprinting Research Articles

Restriction enzyme fingerprinting of enterobacterial plasmids: a simple strategy with wide application.

Restriction enzyme fingerprints were generated from purified plasmid DNA from 324 clinical isolates that belonged to 7 enterobacterial genera and 88 single plasmids in Escherichia coli K 12 according to the following strategy. Purified plasmid DNA was digested with PstI. The number of fragments detected in a 0.8 agarose gel was used to determine which 2 of 6 restriction enzymes including PstI was most likely to provide a fingerprint comprising sufficient fragments to ensure specificity but sufficiently few to allow easy visual assessment and minimize coincidental matching. When PstI produced greater than 20 fragments, EcoRI and HindIII were used; when PstI generated less than 6 fragments Bsp 1286 and AvaII were used and SmaI was employed when between 6 and 20 fragments were obtained from PstI digests. Using a minimum of 12 fragments from a combination of 2 enzymes as the criterion for characterizing a strain/plasmid, satisfactory 2-enzyme fingerprints were obtained from 87% of the strains and plasmids studied using PstI and no more than two additional enzymes per strain. Of the remaining 54 strains, 51 harboured only small plasmids (less than 10 kb) and 3 produced satisfactory fingerprints when digested with a fourth enzyme.

Genomic fingerprinting in the epidemiology of gonorrhoea

We investigated the usefulness of the restriction enzyme (RE) fingerprinting for epidemiological tracing in gonococcal disease. The RE patterns of three paired gonococcal isolates showed corresponding identical fingerprints. Within each of the three pairs of epidemiologically linked isolates the respective restriction patterns were completely identical. Also, the restriction patterns of 6 strains from a larger contact group were identical. Identical restriction patterns were also obtained in each of the two cases where isolates were recovered from both urethra and cervix. The serological findings were in perfect agreement with the genomic fingerprinting as to the identity between all strains of the same epidemiologic chain. Relapse of the original infection could be excluded in one case by the finding of a different RE pattern and also a different serovar pattern of the strain recovered 4 months later.

Differentiation of B15 strains of Neisseria meningitidis by DNA restriction endonuclease fingerprinting.

The restriction endonuclease fingerprinting technique was applied to meningococcal DNA in an attempt to identify individual strains of Neisseria meningitidis B15 (serogroup B, serotype 15), which causes approximately 90% of cases of meningococcal disease in northern Norway. Thirty representative strains (10 each from asymptomatic pharyngeal carriers, patients with septicemia, and patients with meningitis) were investigated with the restriction endonucleases Hind III and Eco RI. The 10 carrier strains showed a remarkable heterogeneity of fingerprints that rendered each strain easily distinguishable from the others. The 10 strains from the blood and the 10 from the cerebrospinal fluid showed similar but not identical restriction patterns. The results obtained with the two endonucleases were in perfect agreement. Our data suggest that a large number of different B15 clones are present in the population of northern Norway, but that only one single clone causes invasive meningococcal disease.

Analysis of bovine herpes virus-type 1 isolates by restriction endonuclease fingerprinting.

In an effort to determine whether distinct types of bovine herpesvirus 1 are responsible for causing specific syndromes, the polypeptides and DNA of 93 BHV 1 isolates from the province of Alberta as well as vaccine strains and numerous other Canadian, U.S. and European isolates were analyzed by PAGE and restriction endonuclease fingerprinting, respectively. The polypeptide patterns showed only slight variations: only six isolates contained polypeptides that varied from the norm in their molecular weight, or were absent. Although on the basis of endonuclease patterns the isolates could be categorized into three "strains" and nine "sub-strains", we were unable to associate any of the strains with specific clinical signs. This suggests that the type of disease caused may be determined more by the route of infection and animal management practices than by the inherent properties of certain types of BHV 1. Most of the Albertan isolates were of one BHV 1 type and sub-type.

Analysis of DNA from various species and strains of malaria parasites by restriction endonuclease fingerprinting

1. 1. DNA from various rodent Plasmodium species and strains and from P. falciparum, the human parasite, were analysed by agarose gel electrophoresis following digestion with restriction endonucleases EcoRI, Hind III and Bam H1. Complex patterns of ethidium-stained bands were obtained, which showed similarity but reproducible differences among the various parasite species ( P. chabaudi, P. yoelii, P. berghei and P. falciparum). 2. 2. No differences could be discerned among two cloned strains of P. yoelii (33X, and YM) and among pyrimethamine-resistant (pyrimethamine + chloroquine)-resistant and the drug-sensitive P. chabaudi clone from which the resistant clones were derived. 3. 3. From the known complexity of Plasmodium DNA it could be concluded that the visible bands were derived from repetitive DNA fractions.

4396713 Restriction endonuclease fingerprinting of kinetoplast DNA minicircles: Larry P Simpson, Carlos M Morel assigned to The Regents of the University of Calif

4396713 Restriction endonuclease fingerprinting of kinetoplast DNA minicircles: Larry P Simpson, Carlos M Morel assigned to The Regents of the University of Calif

4396713 Restriction endonuclease fingerprinting of kinetoplast DNA minicircles: Larry P Simpson, Carlos M Morel assigned to The Regents of the University of Calif

4396713 Restriction endonuclease fingerprinting of kinetoplast DNA minicircles: Larry P Simpson, Carlos M Morel assigned to The Regents of the University of Calif

Fingerprinting and sequence homology of plasmids from different virulent strains of Agrobacterium rhizogenes

Several wild-type virulent strains of Agrobacterium rhizogenes, belonging to both biotypes 1 and 2, were analyzed for plasmid content. All the strains were found to harbor at least one large plasmid, of a size comparable to that of A. tumefaciens Ti plasmids as determined by electron microscopy. The plasmids were characterized by restriction endonuclease finger-printing and compared for sequence homology by the Southern blot-hybridization technique. Despite the diversity of the restriction cleavage patterns, the plasmids of the various strains share rather extensive sequence homology. All of the biotype 1 organisms analyzed were shown to harbor one common plasmid.

Characterization of pathogenic trypanosomatidae by restriction endonuclease fingerprinting of kinetoplast DNA minicircles.

A simple protocol was developed for the routine preparation of a kinetoplast DNA fraction from trypanosomatids. The digestion of this DNA with selected restriction endonucleases, followed by the electrophoretic analysis of the fragments on polyacrylamide gradient gels, yielded characteristic patterns that could be used for the intrinsic characterization of stocks (populations derived by serial passage in vivo and/or in vitro from a primary isolation, without any implication of homogeneity or characterization), strains (sets of populations originating from a group of trypanosomes of a given species or subspecies present at a given time in a given host or culture, and defined by the possession of one or more designated characters), and clones (trypanosomes derived from a single individual by binary fission) of certain pathogenic hemoflagellates.

Sequential outbreaks of infection due to Klebsiella pneumoniae in a neonatal intensive care unit: implication of a conjugative R plasmid.

Sequential outbreaks of infection in a neonatal intensive care unit were due to multiple antibiotic-resistant strains of Klebsiella pneumoniae of different serotypes. In investigations of these outbreaks, the transfer of resistance to gentamicin, ampicillin, cephalothin, carbenicillin, and kanamycin from gentamicin-resistant organisms to standard laboratory recipients and between recipients was observed. Purified plasmid DNA, isolated from all multiple antibiotic-resistant strains, was analyzed by agarose gel electrophoresis, which revealed a common, large plasmid component with a molecular size of 71 megadaltons. Analysis of drug-resistant progeny suggested this plasmid encoded resistance to antibiotics and the information needed for its transmission. The identity of the plasmid from three different sources was established by the use of restriction-enzyme fingerprinting. The dissemination and persistence of this plasmid in environmental and fecal organisms, despite the disappearance of multiple antibiotic-resistant K. pneumoniae, provided a potential source for spread to other bacteria.

Genetic transformation of Streptococcus sanguis (Challis) with cryptic plasmids from Streptococcus ferus.

By using the basic methodology initially published by Kretschmer et al. (J. Bacteriol. 124:225-231, 1975), we have been able to introduce phenotypically cryptic plasmids from Streptococcus ferus (formerly Streptococcus mutans subsp. ferus) into Streptococcus sanguis by genetic transformation. In this system, the entry of the cryptic plasmids is selected indirectly. This is effected with transforming deoxyribonucleic acid mixtures in which the cryptic plasmid deoxyribonucleic acid is present in an approximate 10-fold molar excess with respect to a plasmid (pVA1) known to confer erythromycin resistance. Under such conditions, 5 to 10% of the pVA1-containing erythromycin-resistant transformants were cotransformed with cryptic plasmid deoxyribonucleic acid. pVA1 may be selectively eliminated by growth of its S. sanguis host strain at 42 degrees C, enabling the construction of isogenic strains with and without S. ferus cryptic plasmids. Comparative physiological studies of such strains have failed to reveal any plasmid-conferred phenotypes in S. sanguis. With this procedure, we have been able to physically separate two small cryptic plasmids (2.4 x 10(6) and 2.8 x 10(6) daltons) of S. ferus. Although these plasmids were found naturally to exist in a single S. ferus host, they were able to replicate independently of one another in S. sanguis. Restriction enzyme fingerprinting indicated that these plasmids did not share a common ancestry.

Demonstration of Exogenous Genital Reinfection with Herpes Simplex Virus Type 2 by Restriction Endonuclease Fingerprinting of Viral DNA

Analyses of 17 coded isolates of herpes simplex virus type 2 (HSV-2) from genital and perigenital lesions of eight patients showed that recrudescent lesions could be the consequence of exogenous infection. The experimental design was based on earlier observations that no two epidemiologically unrelated isolates of herpes simplex virus type 1 or HSV-2 were identical as determined by the fingerprinting of viral DNAs with restriction endonucleases-enzymes that cleave DNA at specific sites. After 32P-labeled DNAs were extracted from infected cells, cleaved with restriction endonucleases (KpnI, HsuI, EcoRI, or BglII), and subjected to electrophoresis in agarose gels, analyses of DNA fragment patterns established that each member of seven pairs of isolates was identical to the other member but different from the isolates in other pairs. After the code was broken, it was found that of the remaining three isolates, two were successive isolates from one patient and one was a third, nonidentical isolate from a different patient. Thus, one patient yielded three isolates, of which the first two were identical and the third was different. A second patient yielded one isolate that contained one strain of HSV-2 and a second isolate that contained the original strain of HSV-2 and another, genetically distinct strain of virus. Thus, infection with HSV-2 in the same or nearby site can occur in the face of a prior infection with a genetically different strain of the same serotype.