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PubMed2003

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB).

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.

DNA, Bacterial↗

PubMed1999

Gel electrophoretic restriction fragment length polymorphism analysis of DNA derived from individual nematodes, using the PhastSystem.

The DNA sequences constituting the internal transcribed spacer region, located between 18S and 26S rDNA genes within the rRNA operon, derived from single nematodes of two genera (Steinernema and Heterorhabditis) were amplified by polymerase chain reaction (PCR) and subjected to digestion by four restriction enzymes. The digests were analyzed by restriction fragment length polymorphism (RFLP) gel electrophoresis on the PhastSystem, using 7.5%T, 5%C(Bis) polyacrylamide. The downscaling from conventional agarose to PhastSystem gels permitted the analysis to be done on individual nematodes, rather than on mixed samples with average properties. The analysis time was reduced so as to allow for the electrophoretic separation on 200 samples/workday. The resulting patterns of DNA fragments differed from those obtained by agarose gel electrophoresis under conventional conditions by an increased number of detected fragments. The PhastSystem gel analysis provides the basis for taxonomical revisions.

Animals↗

PubMed1999

Identification of Mycobacterium species by PCR-restriction fragment length polymorphism analyses using fluorescence capillary electrophoresis.

We developed a scheme for the rapid identification of Mycobacterium species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) using HaeIII and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5' end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.

Bacterial Proteins↗

PubMed1990

Analysis of restriction fragment length polymorphisms of the major histocompatibility complex of 15I5-B-congenic chicken lines.

Eight 15I5 B-congenic White Leghorn chicken lines, containing haplotypes B2, B5, B12, B13, B15, B19, and B21, were subjected to molecular genotyping with chicken B-F (Class I) and B-L (Class II) major histocompatibility complex (MHC) probes. Genomic DNA was digested with restriction enzymes, hybridized with a Class I or Class II probe, and analyzed for restriction fragment length polymorphisms. Digestion with HindIII or EcoRI yielded no B-L polymorphisms. Digestion with PvuII or BglII and hybridization with a B-L or B-F probe produced polymorphisms that were shared between several haplotypes, although the haplotypes with similar patterns were clustered differently between Class I and Class II probes. The genetic variation seen for B-L and B-F probe hybridization of PvuII digests was much less than that previously demonstrated for B-G probing of PvuII digests of the same lines. Description of MHC Class I and II restriction patterns of the well-characterized 15I5 B-congenic lines will aid in identification of genes important in disease resistance.

Animals↗

PubMed1996

Genetic characterization of Epimedium species using random amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (RFLP) diagnosis.

Total DNA was extracted from the leaves of seven Epimedium species grown in different places in Japan. Their genetic characterization was performed by DNA analyses of random amplified polymorphic DNA (RAPD) using 32 random primers having 10 base sequences, and by restriction fragment length polymorphism (RFLP). E. sagittatum and E. koreanum were easily distinguished by a representative amplified band pattern. It became evident that E. sagittatum had extremely different genetic composition compared to the other species. A dendrogram obtained from the similarity matrix by cluster analysis indicates that E. sagittatum can be completely isolated from the other species. Moreover, it became evident that E. grandiflorum var. higoense, E. trifoliatobinatum and E. koreanum are independent species, contrary to the previous assumption that they are subspecies or a variety. The geographical variation of E. sempervirens was confirmed by cluster analysis. E. diphyllum showed wide genetic variations, in spite of sampling from the same area.

Base Sequence↗

PubMed1994

A restriction fragment length polymorphism of a p53-related sequence in rat (Rattus norvegicus).

Polymorphisms of the p53 tumor-suppressor gene and its related sequences were investigated among inbred rat strains. In a series of Southern blot hybridizations using the human p53 cDNA probe with various restriction endonucleases, HindIII digests and PstI digests showed interstrain variation in the length of hybridizing fragments. Distribution of the HindIII variant fragments among 17 inbred rat strains was identical with that of PstI variant fragments. The variant fragments were segregated as codominant alleles in backcross progeny, and the locus for this restriction fragment length polymorphism of the p53-related sequence was found to correspond to a p53-related sequence located on rat chromosome 9.

Alleles↗

PubMed1988

Segregation of HLA-DR, -DQ and -DP phenotypes and restriction fragment length polymorphic fragments in two recombinant families.

Members of two families were typed for HLA-DR, -DQ and -DP specificities by means of sera and local PLT bulk reagents. One B:C and one DR:DP cross-over were identified in both families. The restriction fragment length polymorphism (RFLP) was analyzed by southern blotting and the use of DR beta, DR alpha, DQ beta, DQ alpha, DP beta and DP alpha probes. Previous observations concerning typical RFLP patterns for serological DR/DQ- and cellular DP-specificities were confirmed. With respect to recombinant haplotypes, RFLP was informative in both families. All polymorphic fragments-(DRB, DQA and B as well as DPA and B)-segregated together in the HLA-B:DR cross-overs. In the DR:DP recombinant children, the DR and DQ fragments were separated from DP fragments, demonstrating that DP-types as defined by PLT and RFLP map close together. No cross-hybridization between the segregating fragments was detected with the various probes.

Female↗

PubMed1995

The PML gene is linked to a megabase-scale insertion/deletion restriction fragment length polymorphism.

The PML gene located on chromosome band 15q22 is involved with the RAR alpha locus (17q21) in a balanced reciprocal translocation uniquely observed in acute promyelocytic leukemia. Physical mapping studies by pulsed-field gel electrophoresis revealed that the PML gene is flanked by two CpG islands that are separated by a variable distance in normal individuals. Several lines of evidence demonstrate that this is the consequence of a large insertion/deletion polymorphism linked to the PML locus: (1) overlapping fragments obtained with a variety of rare-cutting restriction enzymes demonstrated the same variability in distance between the flanking CpG islands; (2) mapping with restriction enzymes insensitive to CpG methylation confirmed that the findings were not a consequence of variable methylation of CpG dinucleotides; (3) the polymorphism followed a Mendelian inheritance pattern. This polymorphism is localized 3' to the PML locus. There are five common alleles, described on the basis of BssHII fragments, ranging from 220 to 350 kb with increments of approximately 30 kb between alleles. Both heterozygous (61%) and homozygous (39%) patterns were observed in normal individuals. Megabase-scale insertion/deletion restriction fragment length polymorphisms are very rare and have been described initially in the context of multigene families. Such structures have been also reported as likely regions of genetic instability. High-resolution restriction mapping of this particular structure linked to the PML locus is underway.

Alleles↗

PubMed1990

A restriction fragment length polymorphism for human topoisomerase II: possible relationship to drug-resistance.

In previous studies we used Southern blotting to examine the topoisomerase II locus (on chromosome 17) in human leukemia cell lines and noted a difference in the XmnI restriction endonuclease digestion pattern between an m-AMSA-resistant line and its m-AMSA-sensitive parent line (Zwelling, L. A.; Hinds, M,; Chan, D.; Mayes, J.; Sie, K. L.; Parker, E.; Silberman, L.; Radcliffe, A.; Beran, M.; Blick, M. Characterization of an amsacrine-resistant line of human leukemia cells. Evidence for a drug-resistant form of topoisomerase II. Journal of Biological Chemistry 264:16411-16420; 1989). We now demonstrate that the variable XmnI digestion pattern represents a normal restriction fragment length polymorphism (RFLP) which is observed in subjects without malignant disease and exhibits an autosomal pattern of inheritance. These data suggest that the previously described deviation in the genomic structure of topoisomerase II in the m-AMSA-resistant cell line did not reflect a new mutation, but rather a reduction to homozygosity at the topoisomerase II locus. This reduction to homozygosity is not due to chromosomal loss, as chromosome 17-specific gene probes clearly identify two chromosome 17's in the sensitive line and four in the resistant line, using chromosome painting with a chromosome 17-specific library. Some other genetic change must be the cause of the resistance of HL-60/AMSA and its topoisomerase II to the inhibiting actions of m-AMSA.

Alleles↗

PubMed2004

[Detection and identification of Mycobacteria with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) from patients with Mycobacterial skin infections].

OBJECTIVE: To establish a rapid approach to the detection and identification of Mycobacteria from lesions of patients with suspected Mycobacterial infections. METHODS: Specimens were obtained from five patients suspected to have Mycobacterial infections. DNA extracted from clinical samples was amplified by nested PCR. The PCR products were digested with HhaI, MboI, and BstUI restriction enzymes and applied to PAGE. The species of Mycobacteria were determined by restriction fragment length polymorphism (RFLP) analysis. Identification of Mycobacteria culture was also performed in 3 patients. RESULTS: M. marinum was found in two patients diagnosed as swimming pool granuloma. M. tuberculosis was found in one patient diagnosed as infectious skin granuloma. All these 3 Mycobacteria were confirmed by Mycobacteria culture. A strain of M. tuberculosis and a strain of M. fortuitum were detected in remain two patients. CONCLUSION: The results above indicate that PCR-RFLP analysis is rapid and reliable in detection and identification of different Mycobacteria species from skin tissues. Application of this method will be helpful for early diagnosis and treatment of Mycobacteria skin infections.

Bacterial Proteins↗

PubMed1993

Restriction fragment length polymorphism of DQB and DRB class II genes of the ovine major histocompatibility complex.

The ovine major histocompatibility complex (MhcOvar) class II region was investigated by Southern blot hybridizations using ovine probes specific for the second exons of Ovar-DRB and Ovar-DQB genes. Multiple bands were revealed when genomic DNA was digested with each of five restriction enzymes (BamHI, EcoRI, HindIII, PvuII and TaqI), and successively hybridized with the two radiolabelled ovine probes. Restriction fragment length polymorphisms (RFLPs) were analysed in 89 sheep originating from six inbred families and the inheritance of the fragment patterns was determined. Forty-one fragments were recorded with the DQB probe; 32 were detected with the DRB probe. They constituted 9 DQB and 10 DRB allelic patterns. Twelve DQB-DRB haplotypes were resolved in this study.

Alleles↗

PubMed1992

Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.

Recently, the insertion element IS1081 from Mycobacterium bovis was identified. In this study, the usefulness of IS1081 in the epidemiology of tuberculosis was investigated. The host range of this insertion sequence was found to be restricted exclusively to the group of Mycobacterium tuberculosis complex bacteria, whereas none of the 10 mycobacterial species which do not belong to the M. tuberculosis complex contained IS1081-homologous DNA. All 99 M. tuberculosis complex strains investigated carried five or six copies of IS1081, and very limited IS1081-associated restriction fragment length polymorphisms were observed among the strains. Seven different IS1081-containing bands were distinguished in each strain, and the patterns differed only in one or two insertion sequence-containing bands. The banding pattern of M. bovis BCG differed in the presence of a 8.0-kb IS1081-containing PvuII fragment which was absent from all other M. tuberculosis complex strains.

Base Sequence↗

PubMed1993

PCR-based restriction fragment length polymorphism analysis of DNA sequence diversity of flagellin genes of Campylobacter jejuni and allied species.

Genotypes were analysed within 29 geographically diverse strains of Campylobacter jejuni mainly from human enteritis cases and four strains representing C. coli, C. lari and C. helveticus. A 1723 bp DNA fragment, amplified by the polymerase chain reaction (PCR) from the flagellin (fla) genes, was used to probe genomic Southern blots for restriction site variation at homologous loci. Internal Hin fl restriction fragment length polymorphisms (RFLP) of the fla gene PCR amplification product were also used to characterize isolates. Copy number and polymorphisms in and around the fla genes were detected, and strains were grouped into 10 genotypes on the basis of fla restriction site similarities. Campylobacter jejuni had a fla gene copy number of two and the majority (64%) of strains in the species had common internal fla gene sequences as indicated by Hin fl restriction analysis. A greater degree of diversity was detected around the fla loci in Hae III and Bgl II genomic Southern blots. The results showed that analysis of fla genes provided a novel and fundamental approach to grouping strains and offered a basis for defining a predominant clonal evolutionary line within C. jejuni.

Base Sequence↗

PubMed1996

[Restriction fragment length polymorphism assays of clinical isolated human cytomegalovirus strain genomic DNA].

Ten clinical isolated strains of Human cytomegalovirus (HCMV) were obtained from 73 urine specimens of different people. Isolations from urine were carried out in human embryolung fibroblasts. Viral isolates were passaged four times. HCMV DNAs of laboratory strain AD169 and 10 clinical isolated strains were extracted with Hirt method, digested with each of the restriction enzymes EcoRI, Hind II. Comparison of restriction fragment length polymorphism (RFLP) of AD169 and isolated strains were made by hybridizing digested DNA with 32P labeled with HCMV Hind II cloned subgenomic fragments as the probe (pCM1035, pCM1015). pCM1035 is located in the joining region between the long(L) and short (S) unique sequences of the virus (L-S junction) pCM1015 is located in the terminal sequences of the virus. The results showed the genomic high degree of homology existed among all strains and the variable restriction site was in the L-S junction and terminal portion. The RFLP patterns of the clinical isolates which did not have relation in epidemiology were different, but the patterns of clinical isolates related in epidemiology were quite similar. Polymorphism frequently occurred in this case of EcoRI digested fragment hybridized with the probe of pCM1035. Southern hybridization of HCMV isolations is useful to researches into the molecular epidemiology and pathogenesis of HCMV infection.

Adult↗

PubMed1990

Similar frequencies of renin gene restriction fragment length polymorphisms in hypertensive and normotensive subjects.

A prospective study was conducted to compare the frequency of renin gene polymorphisms in normotensive and hypertensive subjects. Hypertensive (n = 102, blood pressure 168 +/- 17/103 +/- 9 mm Hg) and normotensive (n = 120, blood pressure 122 +/- 10/75 +/- 9 mm Hg) subjects were white, had similar age and sex distributions (hypertensive group, 45 +/- 10 years old and 52% female; normotensive group, 44 +/- 9 years old and 55% female) and similar body mass index (hypertensive group, 23.2 +/- 2.6; normotensive group, 22.5 +/- 2.4 kg/m2, p = 0.048). The familial susceptibility to hypertension was defined as at least one parent and one sibling who were hypertensive before age 65; subjects in the normotensive group had no familial history of hypertension. Renin gene polymorphisms located throughout the renin gene were identified by using three restriction enzymes (Taq I, HinfI, HindIII). For each polymorphic restriction site, allele frequencies were similar in the hypertensive and the normotensive groups. In the absence of parental genotypes, the haplotype frequencies combining the three restriction fragment length polymorphisms were estimated by using maximum likelihood techniques and were similar in both groups (hypertensive group, 0.429, 0.277, and 0.177; normotensive group, 0.453, 0.245, and 0.195 for the three most common haplotypes). A rare haplotype detected by Taq I/Hind III was apparently more frequent in the hypertensive than in the normotensive group (hypertensive group, tH 0.086, th 0.022; normotensive group, tH 0.038, th 0.050), but the difference was not statistically significant. In conclusion, no association between renin gene polymorphisms and essential hypertension was demonstrated in the present study.

Adult↗

PubMed2004

PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli.

A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.

Animals↗

PubMed2006

Molecular characterization of Streptococcus suis strains by 16S-23S intergenic spacer polymerase chain reaction and restriction fragment length polymorphism analysis.

We developed a new molecular method of typing Streptococcus suis based on polymerase chain reaction (PCR) amplification of a large fragment of rRNA genes, including a part of the 16S and 23S genes and the 16S-23S intergenic spacer region (ISR), followed by restriction fragment length polymorphism (RFLP) analysis with RsaI or MboII endonuclease. The 16S-23S ISRs of 5 S. suis isolates were sequenced and compared. Size and sequence polymorphisms were observed between the S735 reference strain and the 4 wild-type strains. The genetic relationships between 138 independent S. suis strains belonging to various serotypes, isolated from swine or human cases, were determined. The discriminatory power of the method was > 0.95, the threshold value for interpreting typing results with confidence (0.954 with RsaI and 0.984 with RsaI plus MboII). The in vitro reproducibility was 100%. The strains isolated from humans were less genetically diverse than the strains isolated from pigs. For the first time, 2 molecular patterns (R6, M9) were significantly associated with S. suis serotype 2 strains. This genetic tool could be valuable in distinguishing individual isolates of S. suis during epidemiologic investigations.

Animals↗

PubMed1999

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A-F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full-genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full-genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.

Base Sequence↗