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PubMed1992

Deletions, duplications and novel restriction fragment length polymorphism in Duchenne and Becker muscular dystrophies.

To determine the mutations of Southern Chinese with Duchenne and Becker muscular dystrophies (DMD, BMD), we analysed 28 DMD and BMD patients in 24 unrelated families for intragenic deletions and duplications by using cDNA probes covering the entire 14 kb of the dystrophin gene. Deletions were detected in nine unrelated patients (seven patients by probe 8 and two by probe 2b-3). Gene duplications were detected by probe 1-2a in two patients with the duplication bands confirmed in both Hind III and Bgl II digests and by densitometry. A third patient was found to have a junction fragment with Bgl II and a duplication band with Hind III by probe 5b-7. Therefore 50% of the 24 unrelated families were found to have either deletions or duplications. A previously undescribed restriction fragment length polymorphism (RFLP) was found in one family with probe 5b-7 in Bgl II digests which was found to segregate with the disease phenotype. This new RFLP was not detected in over 70 unrelated X chromosomes we have examined so far, and appeared to be "private" for this family. The presence of this new restriction site may or may not be the mutation responsible for the disease phenotype.

Asian People↗

PubMed2004

Phylogenetic analysis and PCR-restriction fragment length polymorphism identification of Campylobacter species based on partial groEL gene sequences.

The phylogeny of 12 Campylobacter species and reference strains of Arcobacter butzleri and Helicobacter pylori was studied based on partial 593-bp groEL gene sequences. The topology of the phylogenetic neighbor-joining tree based on the groEL gene was similar to that of the tree based on the 16S rRNA gene. However, groEL was found to provide a better resolution for Campylobacter species, with lower interspecies sequence similarities (range, 65 to 94%) compared with those for the 16S rRNA gene (range, 90 to 99%) and high intraspecies sequence similarities (range, 95 to 100%; average, 99%). A new universal reverse primer that amplifies a 517-bp fragment of the groEL gene was developed and used for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of 68 strains representing 11 Campylobacter species as well as reference strains of A. butzlerii and H. pylori. Digestion with the AluI enzyme discriminated all Campylobacter species included in the study but showed more intraspecies diversity than digestion with the ApoI enzyme. A hippurate-negative variant of Campylobacter jejuni with a high level of groEL sequence similarity to both C. jejuni (96%) and C. coli (94%) gave a unique AluI profile and an ApoI profile identical to those of other C. jejuni strains. In conclusion, groEL gene sequencing and PCR-RFLP analysis are recommended as valuable tools for the identification of Campylobacter species.

Animals↗

PubMed1994

Genomic DNA fingerprinting of clinical Haemophilus influenzae isolates by polymerase chain reaction amplification: comparison with major outer-membrane protein and restriction fragment length polymorphism analysis.

Non-capsulate strains of Haemophilus influenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing: (i) the fingerprints of 16 colonies of a single H. influenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. influenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. influenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. influenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H. influenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.

Bacterial Outer Membrane Proteins↗

PubMed1999

Rapid identification and typing of Staphylococcus aureus by PCR-restriction fragment length polymorphism analysis of the aroA gene.

The Staphylococcus aureus aroA gene, which encodes 5-enolpyruvylshikimate-3-phosphate synthase, was used as a target for the amplification of a 1,153-bp DNA fragment by PCR with a pair of primers of 24 and 19 nucleotides. The PCR products, which were detected by agarose gel electrophoresis, were amplified from all S. aureus strains so far analyzed (reference strains and isolates from cows and sheep with mastitis, as well as 59 isolates from humans involved in four confirmed outbreaks). Hybridization with an internal 536-bp DNA fragment probe was positive for all PCR-positive samples. No PCR products were amplified when other Staphylococcus spp. or genera were analyzed by using the same pair of primers. The detection limit for S. aureus cells was 20 CFU when the cells were suspended in saline; however, the sensitivity of the PCR was lower (5 x 10(2) CFU) when S. aureus cells were suspended in sterilized whole milk. TaqI digestion of the PCR-generated products rendered two different restriction fragment length polymorphism patterns with the cow and sheep strains tested, and these patterns corresponded to the two different patterns obtained by antibiotic susceptibility tests. Analysis of the 59 human isolates by our easy and rapid protocol rendered results similar to those of other assays.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed1996

Linkage of strain-specific nicotinic receptor alpha 7 subunit restriction fragment length polymorphisms with levels of alpha-bungarotoxin binding in brain.

Inbred mouse strains have been shown to differ in their levels of brain alpha-bungarotoxin binding. These differences in alpha-bungarotoxin receptors have been shown to correlate with an animal's sensitivity to nicotine-induced seizures. Recent studies have shown that the alpha 7 nicotinic acetylcholine receptor subunit is the major alpha-bungarotoxin binding site in rodent brain. In this report, we examined whether mouse strains that differ in levels of alpha-bungarotoxin binding and sensitivity to nicotine-induced convulsions also differ for the alpha 7 subunit. A full-length murine alpha 7 cDNA was cloned and sequenced and found to be identical to that of a mouse alpha 7 cDNA recently reported. Subsequently, a comparison of alpha 7 cDNA sequences and RNA species was performed between two strains (C3H/2 and DBA/2) that differ in levels of brain alpha-bungarotoxin binding and sensitivity to nicotine-induced seizures. The only difference observed was a single nucleotide difference in the open reading frame of alpha 7 that does not affect the primary amino acid sequence. Inbred strains were also surveyed for restriction fragment length polymorphisms at the alpha 7 locus. Strain-specific polymorphisms were identified, and F2 and backcross animals from a classic genetic cross between C3H/2 and DBA/2 mice were compared for the inheritance of alpha 7 genotype and alpha-bungarotoxin receptor levels. A significant association between genotype and receptor levels was observed in both, the F2 and backcross generations. These results indicate that alpha 7 genotype is an important determinant of alpha-bungarotoxin receptor levels.

Animals↗

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↗

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↗

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↗