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Results for “Polymorphism, Restriction Fragment Length”

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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↗

PubMed1997

Restriction fragment length polymorphism analysis of Akabane virus nucleoprotein gene.

The nucleoprotein genes of Akabane virus S RNA segment from 21 Japanese and two Australian isolates were amplified by the reverse transcriptase-polymerase chain reaction (RT-PCR) using a primer set containing the initiation and termination codon of the gene. The RT-PCR products were sufficiently produced from the purified virion RNAs of all the isolates, and then analyzed by enzymatic digestion with 11 restriction endonucleases. Digestion with the eight restriction enzymes revealed sequence variation of the isolates. Restriction fragment length polymorphism (RFLP) profiles obtained by digestion revealed the existence of four major groups (genogroups) among the isolates. The two Australian isolates had extremely different RFLP profiles than the Japanese isolates. The data demonstrate the usefulness of analyzing the RFLP patterns to understand the genetic variability of AKA virus isolated in Japan and Australia.

Animals↗

PubMed1995

Identification of the antithrombin III Kyoto mutation by restriction fragment length polymorphism analysis.

Clarification of the molecular biologic abnormality underlying antithrombin III (AT III) deficiency is important to a complete understanding of the coagulation system. We have reported previously the deficiency of AT III Kyoto in which there has been a single substitution of thymine for guanine at codon 406 of exon VI. This results in the amino acid substitution of methionine for arginine. The nucleotide substitution at codon 406 in the mutant allele eliminates a Hae III restriction site. Accordingly, Hae III restriction analysis of the PCR fragments in this family could segregate the deficient members from the normal members, enabling the detection of the existence of Hae III restriction fragment length polymorphism of the PCR product (PCR-RFLP). Moreover, we confirmed by direct DNA sequencing with the polymerase chain reaction that the tested deficient members in an AT III Kyoto family inherit the same type of mutation in their genes. In conclusion, Hae III PCR-RFLP can be a useful method for screening of this mutant gene in the family.

Adult↗

PubMed2001

Agilent 2100 bioanalyzer for restriction fragment length polymorphism analysis of the Campylobacter jejuni flagellin gene.

The Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, Calif.) utilizes capillary electrophoresis on a microchip device (LabChip 7500; Caliper Technologies, Mountain View, Calif.) that is capable of rapidly sizing small DNA fragments. To determine whether the system could replace conventional restriction fragment length polymorphism (RFLP) typing by agarose gel electrophoresis, we compared the analyzer with conventional flagellin RFLP for typing Campylobacter jejuni. Ninety-seven isolates representing 46 Fla types were initially analyzed. Correct Fla types were detected in 59% of the isolates. The major problem with the system was in resolving samples containing multiple DNA fragments differing from 8 to 20 bp. Overall, the bioanalyzer has the potential to replace conventional RFLP analysis by gel electrophoresis, but improvements in the chip separation are needed.

Autoanalysis↗

PubMed2003

PCR-restriction fragment length polymorphism analysis for detection of point mutations associated with macrolide resistance in Campylobacter spp.

A 23S rRNA gene fragment in domain V was sequenced from 30 clinical isolates of Campylobacter spp., including 22 resistant to macrolides. Two point mutations associated with erythromycin resistance were identified at positions 2074 and 2075 on the 23S rRNA gene (homologous to A2142C and A2143G mutations in Helicobacter pylori) in which an adenine residue is also replaced with a cytosine and a guanine residue, respectively. A combined PCR-restriction fragment length polymorphism technique was developed to detect these mutations by using the BsaI and BceAI enzymes.

Anti-Bacterial Agents↗

PubMed2001

Evaluation of PCR-restriction profile analysis and IS2404 restriction fragment length polymorphism and amplified fragment length polymorphism fingerprinting for identification and typing of Mycobacterium ulcerans and M. marinum.

Mycobacterium ulcerans and M. marinum are emerging necrotizing mycobacterial pathogens that reside in common reservoirs of infection and exhibit striking pathophysiological similarities. Furthermore, the interspecific taxonomic relationship between the two species is not clear as a result of the very high phylogenetic relatedness (i.e., >99.8% 16S rRNA sequence similarity), in contrast to only 25 to 47% DNA relatedness. To help understand the genotypic affiliation between these two closely related species, we performed a comparative analysis including PCR restriction profile analysis (PRPA), IS2404 restriction fragment length polymorphism (RFLP), and amplified fragment length polymorphism (AFLP) on a set of M. ulcerans (n = 29) and M. marinum (n = 28) strains recovered from different geographic origins. PRPA was based on a triple restriction of the 3' end region of 16S rRNA, which differentiated M. ulcerans into three types; however, the technique could not distinguish M. marinum from M. ulcerans isolates originating from South America and Southeast Asia. RFLP based on IS2404 produced six M. ulcerans types related to six geographic regions and did not produce any band with M. marinum, confirming the previous findings of Chemlal et al. (K. Chemlal, K. DeRidder, P. A. Fonteyne, W. M. Meyers, J. Swings, and F. Portaels, Am. J. Trop. Med. Hyg. 64:270-273, 2001). AFLP analysis resulted in profiles which grouped M. ulcerans and M. marinum into two separate clusters. The numerical analysis also revealed subgroups among the M. marinum and M. ulcerans isolates. In conclusion, PRPA appears to provide a rapid method for differentiating the African M. ulcerans type from other geographical types but is unsuitable for interspecific differentiation of M. marinum and M. ulcerans. In comparison, whole- genome techniques such as IS 2404-RFLP and AFLP appear to be far more useful in discriminating between M. marinum and M. ulcerans, and may thus be promising molecular tools for the differential diagnosis of infections caused by these two species.

Animals↗

PubMed1991

Restriction fragment length polymorphism of the L-myc gene and susceptibility to metastasis in genitourinary cancers.

We examined Southern blot analysis of genomic DNAs from 70 patients with sporadic renal cell carcinoma, using the human L-myc oncogene fragment as a hybridization probe. Our purpose was to study the relationship between the restriction fragment length polymorphism (RFLP) of the L-myc and the frequencies of metastasis. The patients were classified into 3 genotypes according to the polymorphic patterns defined by two alleles (L-L:17, L-S:31, S-S:22). The relative ratios of the 3 genotypes in the renal cancer patients were similar to those seen in healthy Japanese. However, of 20 patients who exhibited distant metastases at diagnosis, only 2 belonged to the L-L type. The incidence of distant metastasis in L-L type patients was significantly lower than that in L-S and S-S patients (p = 0.068, by Fisher's exact probability test). These results basically correspond to the previous findings in the lung cancer patients [Kawashima et al.: Proc. natn. Acad. Sci. USA 85: 2353-2356, 1988]. On the other hand, L-myc RFLP analysis in 50 prostatic cancer patients revealed that the incidence of metastasis at diagnosis did not correlate with L-myc genotypes. L-myc RFLP seems to be less promising in prostatic cancer than in lung or kidney cancer.

Blotting, Southern↗

PubMed1999

Genetic variation of Cryphonectria hypoviruses (CHV1) in Europe, assessed using restriction fragment length polymorphism (RFLP) markers.

A total of 72 hypovirus-infected isolates of the chestnut blight fungus Cryphonectria parasitica were sampled from nine European countries between 1975 and 1997. The double-stranded RNA of the Cryphonectria hypoviruses (CHV1) was isolated and reverse transcription (RT)-PCR products were obtained for two different regions of the viral genome (ORF A and ORF B) using primer sequences of the type species CHV1-EP713. Both PCR products of each viral isolate were digested with four restriction endonucleases recognizing sequences of four nucleotides. The restriction fragment length polymorphism (RFLP) analysis revealed 41 genetically distinct RFLP types of CHV1 with 10 types occurring more than once. Identical RFLP types were detected nine times among viruses collected in the same location. Cluster analysis based on the RFLP banding patterns separated the viral isolates into five CHV1 clusters or subtypes. Most viral isolates (64 out of 72) grouped into one large cluster which comprised all viruses from Italy (including CHV1-EP747), Switzerland, Crotia, Bosnia, Hungary, Greece, and the French island Corsica, as well as five out of 11 isolates from continental France. Two additional subtypes of CHV1 were found in France (one related to CHV1-EP713) and one each in Spain and Germany. The Swiss samples collected over a period of 20 years showed that very little RFLP variation has evolved during this time. The results of this study are consistent with the hypothesis of multiple introductions of CHV1 into Europe.

Ascomycota↗

PubMed2004

New method of differentiating wild-type varicella-zoster virus (VZV) strains from Oka varicella vaccine strain by VZV ORF 6-based PCR and restriction fragment length polymorphism analysis.

A new method was developed to distinguish accurately wild-type varicella-zoster virus (VZV) strains from the Oka vaccine strain. Several DNA fragments covering open reading frame (ORF) 1-37 were amplified from wild-type VZV strains including the Oka parent strain and from the Oka vaccine strain. Restriction fragment length polymorphisms of these regions were compared, and nucleotide differences between the vaccine virus and other wild-type VZV strains were noted in ORFs 6, 10, and 35. In addition, variations of the R2 and R4 reiterated structures of the vaccine and its parent strains were examined. The Oka vaccine strain used in Japan was shown to be a mixture of viruses with different nucleotide sequences that had variations in at least three nucleotide positions in ORF 1-37 and had variable polymorphisms at R2 and R4 repeat regions (two and three patterns, respectively). The Oka parent strain on the other hand showed a single sequence and had only one reiterated structure at these regions. When VZV ORF 6 was amplified and its product was digested with AluI, the Oka vaccine strain could be precisely differentiated from its parent and from 56 other Japanese clinical isolates.

Chickenpox Vaccine↗

PubMed1987

Restriction fragment length polymorphism of the C1 inhibitor gene in hereditary angioneurotic edema.

Hereditary angioneurotic edema (HANE) results from the deficiency of the inhibitor of the first component of human complement (C1-INH). It is inherited as an autosomal dominant trait. Heterogeneity of this defect has been shown at the protein and mRNA level. Southern blot analysis of genomic DNA was performed after digestion with six different restriction endonucleases in 24 families affected with type 1 HANE (low antigenic and functional C1-INH levels) and five with type 2 (low functional C1-INH levels and normal or elevated levels of dysmorphic C1-INH). Blots were hybridized with a C1-INH cDNA probe of 1,227 bp. With one enzyme (Pst I), two different patterns of restriction fragment length polymorphism (RFLP) were detected. One was present in one kindred with type 1 HANE and the other appeared the same in one type 1 and in one type 2 family, thus indicating that each RFLP resulted from a different mutation. Analysis of a total of 34 members of these three families suggested that the polymorphisms are tightly linked to the mutation responsible for the disease. Using a 170-bp probe we showed that the three different mutations leading to these polymorphisms are located in the same region of the C1-INH gene. These data suggest that different mutations in the same region of the C1-INH gene are responsible for C1-INH deficiency in these families. Most of these mutations are probably point mutations or other "minor" defects and do not appear to be due to major deletions or rearrangements.

Angioedema↗

PubMed1993

Genetic evidence for the existence of cryptic species in the Anopheles albitarsis complex in Brazil: allozymes and mitochondrial DNA restriction fragment length polymorphisms.

Allozyme and mitochondrial DNA (mtDNA) restriction studies were undertaken to determine the extent of genetic divergence among field populations of Anopheles albitarsis in Brazil. Two sympatric species, An. deaneorum and An. marajoara, were identified in collections from Costa Marques (CM), Rondonia. Genetic evidence includes (1) the presence of two types of individuals, each with diagnostic allelic clusters (for Had-1, Pgi-1, Pep-1, Mpi-1, and Idh-1), (2) a deficiency of heterozygotes, and (3) characteristic mtDNA haplotypes. In addition, two allopatric cryptic species of An. marajoara were identified, one from Iguape (An. marajoara form IG), Sao Paulo state, and the other from the Island of Marajo (An. marajoara form MA). Though form IG and form-MA resemble form CM in wing spot morphology, they differ from it in diagnostic allozymes and mtDNA haplotypes. An. marajoara form CM had a higher variability (mean heterozygosity, H = 0.22, and percentage of polymorphic loci, P = 66.7) than did form IG and form MA (H = 0.08 in both, and P = 25.0 and 33.3, respectively). Form MA and form IG are genetically more similar to each other than both are to form CM. Based on wing morphology, estimates of F statistics, and genetic similarities, we propose that An. albitarsis in Brazil is a species complex. It comprises at least two morphologically distinguishable species: (1) An. deaneorum (currently one taxon) and (2) the An. marajoara species complex, which further consists of at least three cryptic forms, marajoara form MA, marajoara form IG, and marajoara form CM.

Alleles↗

PubMed1986

Immunoglobulin heavy chain switch region restriction fragment length polymorphisms are associated with renal disease.

We describe here, to our knowledge for the first time, associations between polymorphisms at the genomic DNA level in the immunoglobulin gene region and renal diseases which lead to chronic renal failure. Recent studies have shown that protein polymorphisms, present in immunoglobulin (Ig) heavy chains (Gm allotypes) are associated with certain forms of renal disease and with end stage renal failure per se. To investigate this association at the DNA level we have used probes which recognize Ig heavy chain genes and this report describes results obtained with one of these, the S mu switch region probe. With the restriction endonuclease Sst 1 (or the isoschizomer; Sac I) a number of restriction fragment length polymorphisms (RFLP) can be obtained which are recognized by this probe and there is a highly significant association between certain of these and renal disease. This is the first report of Ig switch region polymorphisms being associated with disease, yet our results suggest that S mu RFLP are more closely linked to renal disease than Ig protein polymorphisms.

Humans↗

PubMed1989

Restriction fragment length polymorphism of the L-myc gene and susceptibility to metastasis in renal cancer patients.

We examined Southern blot analyses of normal and tumor DNAs from 50 patients with sporadic renal cancer, using the human L-myc oncogene fragment as a hybridization probe. Our purpose was to study the relationship between the restriction fragment length polymorphism (RFLP) of the L-myc and the frequencies of metastases. There was no individual difference in patterns of L-myc RFLP between normal and tumor-tissue DNAs digested with EcoRI. The patients were classified into 3 genetic types according to the polymorphic patterns defined by the 2 alleles [10-kilobase (kb) and 6.6-kb fragments]. The relative ratios of the 3 genotypes in the renal cancer patients were similar to those seen in healthy Japanese. However, of 16 patients who exhibited distant organ metastases at the time of surgery, only one was a 10-kb fragment homozygote. The incidence of distant metastases in 10-kb homozygotes was significantly lower than that in 6.6-kb homozygotes plus heterozygotes (p = 0.06). These results basically correspond to the previous findings in the lung cancer patients, and suggest that L-myc RFLP is a widely applicable genetic marker to predict prognosis in cancer patients.

Adult↗