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Results for “Polymorphism, Restriction Fragment Length”

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PubMed1998

Development of a PCR-restriction fragment length polymorphism assay using the nucleotide sequence of the Helicobacter hepaticus urease structural genes ureAB.

Infection with Helicobacter hepaticus causes chronic active hepatitis in certain strains of mice and is associated with hepatocellular carcinoma in A/JCr mice. Like the gastric helicobacters, H. pylori and H. mustelae, H. hepaticus possesses a high level of urease activity. However, the H. hepaticus urease structural gene sequences have not been previously determined, and the role of the urease enzyme in colonization and in pathogenesis is not known. PCR was used to amplify a portion of the urease structural genes from H. hepaticus genomic DNA. Amplified DNA fragments were cloned, and the nucleotide sequence was determined. The deduced amino acid sequence of the partial H. hepaticus ureA gene product was found to exhibit 60% identity and 75% similarity to the predicted H. pylori UreA. The deduced amino acid sequence of a partial H. hepaticus ureB gene product exhibited 75% identity and 87% similarity to the predicted H. pylori UreB. Diversity among H. hepaticus isolates was evaluated by means of a restriction fragment length polymorphism (RFLP) assay. The 1.6-kb fragments within the ureAB open reading frames, amplified from 11 independent isolates, were digested with the restriction endonuclease HhaI. Three distinct RFLP patterns were observed. Identical RFLP profiles were noted in sequential isolates of one strain of H. hepaticus during an 18 month in vivo colonization study, suggesting that the urease genes of H. hepaticus are stable. The urease genes among H. hepaticus strains were also well conserved, showing 98.8 to 99% nucleotide sequence identity among three isolates analyzed. These findings indicate that H. hepaticus has urease structural genes which are homologous to those of the gastric Helicobacter species and that these gene sequences can be used in a PCR and RFLP assay for diagnosis of this important murine pathogen.

Amino Acid Sequence↗

PubMed1986

Human restriction fragment length polymorphisms and cancer risk assessment.

The polymorphic restriction fragments of the human Ha-ras locus, produced by the variable tandem repetition (VTR) of a short consensus sequence, fall into three classes based on allelic frequencies. Alleles of the "rare" class (individual frequencies less than 0.5%) have been detected only in white blood cell and tumor DNA of cancer patients. This phenomenon is independent of ethnic origin. No significant association of rare alleles with cancer patients has been demonstrated at an independent tandem repeat locus, VTR4.1. The results suggest that the Ha-ras restriction fragment length polymorphism is useful in cancer risk assessment.

Alleles↗

PubMed2006

Use of multiplex terminal restriction fragment length polymorphism for rapid and simultaneous analysis of different components of the soil microbial community.

A multiplex terminal restriction fragment length polymorphism (M-TRFLP) fingerprinting method was developed and validated for simultaneous analysis of the diversity and community structure of two or more microbial taxa (up to four taxa). The reproducibility and robustness of the method were examined using soil samples collected from different habitats. DNA was PCR amplified separately from soil samples using individual taxon-specific primers for bacteria, archaea, and fungi. The same samples were also subjected to a multiplex PCR with the primers for all three taxa. The terminal restriction fragment length polymorphism profiles generated for the two sets of PCR products were almost identical not only in terms of the presence of peaks but also in terms of the relative peak intensity. The M-TRFLP method was then used to investigate rhizosphere bacterial, fungal, and rhizobial/agrobacterial communities associated with the dwarf shrub Calluna vulgaris growing in either open moorland, a mature pine forest, or a transition zone between these two habitats containing naturally regenerating pine trees. Rhizosphere microbial communities associated with Vaccinium myrtillus collected from the native pine forest were also investigated. In this study, individual PCR products from the three taxa were also pooled before restriction digestion and fragment size analysis. The terminal restriction fragment length polymorphism profiles obtained with PCR products amplified individually and with multiplexed and pooled PCR products were found to be consistent with each other in terms of the number, position, and relative intensity of peaks. The results presented here confirm that M-TRFLP analysis is a highly reproducible and robust molecular tool for simultaneous investigation of multiple taxa, which allows more complete and higher resolution of microbial communities to be obtained more rapidly and economically.

Archaea↗

PubMed1988

Use of restriction fragment length polymorphisms (RFLPs) to distinguish between nematodes of pathogenic significance.

The availability of restriction fragment length polymorphisms (RFLPs) would be useful for studying the extent of diversity among morphologically indistinguishable populations of filarial parasites. Such polymorphisms may be useful in correlating various physiological and clinical differences with parasite heterogeneity. In order to identify such RFLPs, we isolated DNA from microfilaria of 6 filarial species (Acanthocheilonema viteae, Brugia malayi, Brugia pahangi, Dirofilaria immitis, Litomosoides carinii and Setaria digitatum), digested the DNA with several restriction endonucleases, prepared Southern blots and probed with 32P-labelled DNA probes. The patterns of fragments generated using two restriction endonucleases, Mbo I and Taq I, in combination with two probes, rDNA from the free-living soil nematode Caenorhabditis elegans, and pBM103, an anonymous DNA probe from B. malayi, unequivocally distinguish between all 6 of the species. To ensure that the differences we observed between the species represent true interspecies variation rather than fortuitous individual variations we analysed DNA from several individual B. malayi and B. pahangi worms. The individual B. malayi worms demonstrated restriction profiles that were invariant, as did the individual B. pahangi worms, demonstrating that the differences we observed were true interspecies variations.

Animals↗

PubMed2001

Intraspecific variation and host specificity of Entomophthora muscae sensu stricto isolates revealed by random amplified polymorphic DNA, universal primed PCR, PCR-restriction fragment length polymorphism, and conidial morphology.

The intraspecific variations of Entomophthora muscae s. str. associated with particular host species, Musca domestica and Delia radicum, sampled from different localities and different years in Denmark and the variation of E. muscae s. str. originating from different host taxa were investigated. The isolates were compared both by primary spore morphology and by three molecular methods: random amplified polymorphic DNA, universal primed PCR, and PCR-restriction fragment length polymorphism. Analyses of the different molecular data showed the same overall picture and separated E. muscae s. str. into two main groups with all the M. domestica isolates in one group and isolates from D. radicum, Coenosia tigrina, and Pegoplata infirma in the second group. E. muscae s. str. isolates from M. domestica also differ significantly from the rest of the E. muscae s. str. isolates with regard to the morphology of the primary conidia, which were bigger and contained significantly more nuclei per conidium. Several different E. muscae s. str. genotypes were documented and each type was restricted to a single host species, indicating a very high degree of host specificity at or below the level of the subfamily.

Animals↗

PubMed1988

Heparin cofactor II: cDNA sequence, chromosome localization, restriction fragment length polymorphism, and expression in Escherichia coli.

Heparin cofactor II (HCII) is an inhibitor of thrombin in plasma that is activated by dermatan sulfate or heparin. An apparently full-length cDNA for HCII was isolated from a human liver lambda gt11 cDNA library. The cDNA consisted of 2215 base pairs (bp), including an open-reading frame of 1525 bp, a stop codon, a 3'-noncoding region of 654 bp, and a poly(A) tail. The deduced amino acid sequence contained a signal peptide of 19 amino acid residues and a mature protein of 480 amino acids. The sequence of HCII demonstrated homology with antithrombin III and other members of the alpha 1-antitrypsin superfamily. Blot hybridization of an HCII probe to DNA isolated from sorted human chromosomes indicated that the HCII gene is located on chromosome 22. Twenty human leukocyte DNA samples were digested with EcoRI, PstI, HindIII, KpnI, or BamHI, and Southern blots of the digests were probed with HCII cDNA fragments. A restriction fragment length polymorphism was identified with BamHI. A slightly truncated form of the cDNA, coding for Met-Ala instead of the N-terminal 18 amino acids of mature HCII, was cloned into the vector pKK233-2 and expressed in Escherichia coli. The resultant protein of apparent molecular weight 54,000 was identified on an immunoblot with 125I-labeled anti-HCII antibodies. The recombinant HCII formed a complex with 125I-thrombin in a reaction that required the presence of heparin or dermatan sulfate.

Amino Acid Sequence↗

PubMed1989

Restriction fragment length polymorphism analysis with a cross-reactive HLA class II DR-beta gene probe for the detection of engraftment of MHC-mismatched marrow in the rhesus monkey.

Our interest in MHC-mismatched allogeneic bone marrow transplantation (BMT) in the rhesus monkey prompted us to investigate restriction fragment length polymorphism analysis as a means for detecting lymphohematopoietic chimerism in the primate. A human MHC (HLA) class II DR beta gene cDNA probe was tested on rhesus peripheral blood mononuclear cell DNA digested with any of three restriction enzymes. We found that (1) the human DR beta probe hybridized to as many as 15 restriction fragments per rhesus DNA sample, suggesting cross-hybridization at more than one locus of rhesus MHC class II beta genes; (2) restriction fragment length polymorphisms were common among outbred monkeys as a result of class II beta gene polymorphisms and would be sufficient for chimerism detection in the majority of random pairs of outbred monkeys utilizing only a single restriction enzyme (Bgl II); and (3) sensitivity (5-10% chimerism) was comparable to that reported for restriction fragment length polymorphism assays utilizing non-MHC probes in clinical HLA-identical BMT. Utility of the assay was demonstrated in a preliminary series of experiments in rhesus monkeys conditioned with mixed T cell-depleted MHC-mismatched allogeneic plus T cell-depleted autologous BMT with or without cardiac allograft implantation.

Animals↗

PubMed2005

Intratypic variations among Thai herpes simplex virus (HSV) isolates determined by restriction fragment length polymorphism (RFLP) analysis.

Whole genomic polymorphisms for 20 HSV-1 and 20 HSV-2 isolates from Thai patients were analyzed by means of Restriction Fragment Length Polymorphism (RFLP) analysis using 4 restriction endonucleases: BamHI, Kpnl, HindIII, and EcoRI. Variations in cleavage sites among the HSV-1 and HSV-2 isolates were compared to the cleavage patterns of standard HSV-1 strain KOS and HSV-2 strain Baylor 186. Although 70% of HSV-1 isolates with BamHI digestion, 50% with Kpnl, 75% with HindIII and 70% with EcoRI digestion were found to be similar to the standard HSV-1 (KOS) pattern, new BamHI restriction sites were detected in some HSV-1 isolates. For HSV-2 isolates, 85% had the same pattern as the standard HSV-2 (Baylor 186) after digestion with BamHI, HindIII, and EcoRI. No difference was observed with Kpnl digestion. When the patterns from the 4 enzymes were combined, HSV-1 isolates showed more divergence than the HSV-2 isolates. HSV-1 isolates found in both non-genital and genital lesions had more variety than the HSV-2 isolates. This suggests that intratypic variations in HSV-2 are fewer than in HSV-1.

DNA, Viral↗

PubMed1999

Identification of Trichinella isolates by polymerase chain reaction--restriction fragment length polymorphism of the mitochondrial cytochrome c-oxidase subunit I gene.

We developed a polymerase chain reaction based approach using restriction fragment length polymorphisms of the mitochondrial cytochrome c-oxidase subunit I to identify nine genotypes (Trichinella spiralis, Trichinella britovi-European strains, Trichinella britovi-Japanese strains, Trichinella nativa, Trichinella nelsoni, Trichinella T5, Trichinella T6, Trichinella T8 and Trichinella pseudospiralis) in the genus Trichinella. Partial mitochondrial cytochrome c-oxidase subunit I genes of nine genotypes were amplified by polymerase chain reaction, sequenced, and digested with three restriction endonucleases (Mse I, Alu I and Bsp1248 I). This polymerase chain reaction based restriction fragment length polymorphism method allowed the identification of Trichinella genotypes. Trichinella spiralis, Trichinella britovi-Japanese strains, Trichinella nelsoni, T5 and Trichinella pseudospiralis were distinguishable by digestion with Mse I. Trichinella britovi-European strains and Trichinella T8 were distinguishable by digestion using Alu I, and Trichinella nativa and Trichinella T6 were distinguishable by double-digestion with Mse I and Bsp1286 I. The results obtained with this polymerase chain reaction based restriction fragment length polymorphism assay confirmed those previously reported by others and support the separation of the Japanese isolates as a new genotype, namely Trichinella T9.

Animals↗

PubMed1995

Evaluation of a restriction fragment length polymorphism typing method for Listeria monocytogenes.

The results of an evaluation of restriction fragment length polymorphism (RFLP) typing for Listeria monocytogenes are presented. The method depends on the use of cloned DNA fragments from an L. monocytogenes (serovar 4b) strain to probe Southern blotted NciI restriction fragments derived from L. monocytogenes strains. Analyses of 862 isolates of serogroups 1/2, 3 and 4 were performed and a total of 32 RFLP patterns were observed. Interstrain RFLPs were common within serogroup 1/2 and the numerical index of discriminatory power for this group was 0.883. Serogroup 4 strains were divided into two major and three minor RFLP types, suggesting a high level of genotypic homogeneity, and the numerical index of discriminatory power was correspondingly low. The technique was found to be highly reproducible, and the stability of patterns was demonstrated by analyses of passaged strains and multiple isolates of the same strain from related specimens. RFLP typing is of value in epidemiological investigations involving strains of serogroup 1/2.

Blotting, Southern↗

PubMed2003

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

Amino Acid Sequence↗

PubMed1998

Genetic polymorphism of Leishmania species using kinetoplast DNA restriction fragment length polymorphism and cDNA probe of Leishmania donovani.

Leishmaniasis represents a group of diseases that range from simple cutaneous lesions through metastasizing diffused cutaneous to severe systemic infection depending upon the taxon to which the causative parasite belongs. Therefore, it is important to identify the infecting Leishmania. Methods presently being used, including immunology, biochemistry and molecular biology have one or the other limitations, leaving scope for the search for newer probes. This study reports the characterization of leishmania isolates both by restriction fragment length polymorphism of kinetoplast DNA (kDNA) and genomic DNA. The genomic DNA was probed with a cDNA probe B2a1. Using a kDNA restriction pattern technique, different isolates of Leishmania donovani could be differentiated from the UR6 strain of L. tropica, but it was not possible to differentiate between newer local isolates of L. donovani with most of the restriction enzymes except AluI. However, the B2a1 cDNA probe was able to differentiate these isolates effectively. Both of these techniques could differentiate newer local isolates of L. donovani from the older isolates of L. donovani from India, i.e., DD8, RMRI and SS. The Indian isolates of L. donovani could also be differentiated from isolates of L. donovani from Jeddah and Germany using both techniques. The present study indicates that the cDNA probe B2a1 can be used as an important adjunct to kDNA restriction analysis for the characterization of Leishmania species.

Animals↗

PubMed1986

An overview of the restriction fragment length polymorphism of the HLA-D region: its application to individual D-, DR- typing by computerized analyses.

HLA-D/DR alleles as defined by cellular and serological typing can also be identified by biochemical methods. The Southern blot technique provides an additional typing facility which can be applied to DNA obtained from any source of nucleated cells. The polymorphism revealed by Southern blot analyses, the so-called restriction fragment length polymorphism (RFLP), depends upon the restriction enzyme and cDNA probes used. To identify HLA-DR specificities a protocol was developed based on the use of the results of southern blot analyses with several restriction enzymes and cDNA probes within a panel of HLA-D/DR homozygous cells representing the DR1 to DRw8 alleles. First, hybridizations with the 3' untranslated sequence of the DR beta cDNA probe, after digestion of the DNA with PvuII (PvuII-DR beta 3') allows the selective identification of DR1, DR2 and DRw8; DR3, DR5 and DRw6 are found as one group as well as DR4 and DR7 as another. Second, TaqI-DQ alpha hybridization allows the splitting of DRw6-Dw18, DRw6-Dw19 and DRw6-Dw9 from the DR3, DR5 and DRw6 group. The other alleles DR3, DR4, DR5, DRw6-Dw16 and DR7 are revealed by dehybridization and rehybridization of the blot with a DR beta cDNA probe. This protocol was used to test whether in a panel of 30 randomly chosen individuals the HLA-DR typing could be performed. The results were highly concordant to the serotyping. Furthermore by adding the Pst-DR beta and TaqI-DQ alpha RFLPs, most of the MLC defined Dw specificities could also be identified. An overview of the specific fragments described here has been summarized in matrices which can be used as references for DNA-typing in computerized analyses.

Collodion↗

PubMed2004

PrP polymorphisms in Basque sheep breeds determined by PCR-restriction fragment length polymorphism and real-time PCR.

Two new PCR-based methods were developed to decode prion protein (PrP) gene polymorphisms at codons 136, 154 and 171: a PCR-restriction fragment length polymorphism (RFLP) analysis consisting of two PCR reactions followed by three enzymatic digestions, and a real-time PCR consisting of four reactions with seven fluorogenic probes. Both methods were used to study the distribution of PrP gene polymorphisms in a representative sample (1297 animals) of the populations of the two native breeds of sheep of the Spanish Basque Country, Latxa and Carranzana. Fourteen genotypes were found in the Latxa breed, in which ARQ/ARQ was the genotype most frequently observed (49.3 per cent), followed by ARR/ARQ (32.6 per cent) and ARQ/ARH (5.8 per cent). The genotype associated with the highest resistance to scrapie (ARR/ARR) was present in 5 per cent of the animals analysed. Similar results were observed in the Carranzana sheep.

Animals↗

PubMed1998

Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations.

Ascomycota↗

PubMed1999

Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment.

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.

Angiomatosis, Bacillary↗

PubMed2006

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates.

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.

Animals↗

PubMed2000

DNA fingerprints of Trichinella as revealed by restriction fragment length polymorphism and single-strand conformational polymorphism (RFLP-SSCP).

A method was developed for gene fingerprinting, combining the principles of restriction fragment length polymorphism and single-strand conformational polymorphism (PCR-RFLP-SSCP). Taking advantage of this method, we analysed the genotypes of 20 isolates from five species of Trichinella (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella murrelli and Trichinella pseudospiralis) and two uncertain genotypes (Trichinella T6 and Trichinella T8). Target genes for the analysis included three kinds of random amplified polymorphic DNA (RAPD), the gene encoding 43 kDa excretory-secretory protein, the gene encoding mitochondrial cytochrome c -oxidase subunit I and the gene of 18 S rRNA. The genotype revealed by this method was in good concordance with the taxonomy of Trichinella (six species and two uncertain genotypes which is currently accepted based on morphology, isozyme pattern and reproductive isolation). More important, this method revealed intraspecies polymorphism among geographical isolates of T. spiralis and T. britovi.

Animals↗