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PubMed1998

Physical mapping of the liguleless linkage group in Sorghum bicolor using rice RFLP-selected sorghum BACs.

Physical mapping of BACs by fluorescent in situ hybridization (FISH) was used to analyze the liguleless (lg-1) linkage group in sorghum and compare it to the conserved region in rice and maize. Six liguleless-associated rice restriction fragment length polymorphism (RFLP) markers were used to select 16 homeologous sorghum BACs, which were in turn used to physically map the liguleless linkage group in sorghum. Results show a basic conservation of the liguleless region in sorghum relative to the linkage map of rice. One marker which is distal in rice is more medial in sorghum, and another marker which is found within the linkage group in rice is on a different chromosome in sorghum. BACs associated with linkage group I hybridize to chromosome It, which was identified by using FISH in a sorghum cytogenetic stock trisomic for chromosome I (denoted It), and a BAC associated with linkage group E hybridized to an unidentified chromosome. Selected BACs, representing RFLP loci, were end-cloned for RFLP mapping, and the relative linkage order of these clones was in full agreement with the physical data. Similarities in locus order and the association of RFLP-selected BAC markers with two different chromosomes were found to exist between the linkage map of the liguleless region in maize and the physical map of the liguleless region in sorghum.

Basic-Leucine Zipper Transcription Factors↗

PubMedDate not supplied

Comparative linkage maps for the mosquitoes (Culex pipiens and Aedes aegypti) based on common RFLP loci.

We report construction of a comparative linkage map for the mosquito (Culex pipiens) based on restriction fragment length polymorphisms (RFLPs) using cDNA clones from Aedes aegypti as probes to Southern blots of Cx. pipiens genomic DNA. Seventy-one cDNA clones were screened for hybridization and genetic diversity among three Cx. pipiens strains. Fifty-two of 71 cDNA clones, isolated from and previously mapped in Ae. aegypti (73.2%), were hybridized under high-stringency conditions with Cx. pipiens genomic DNA. Thirty-four clones (47.9%) reflected strain-specific polymorphisms. The map consists of 21 cDNA markers that identify 22 loci covering 165.8 cM. The loci mapped in Cx. pipiens cover 7.1 cM on chromosome 1, 80.4 cM on chromosome 2, and 78.3 cM on chromosome 3. Linkage relationships of the RFLP markers for chromosome 1 in Cx. pipiens are the same as chromosome 1 in Ae. aegypti, indicating that chromosome 1 is highly conserved between the two species. The comparative RFLP linkage maps for chromosomes 2 and 3 in Cx. pipiens and Ae. aegypti reflect apparent whole-arm translocations. RFLP markers for chromosome 2 in Ae. aegypti identified homologous loci on one arm of chromosomes 2 and 3 in Cx. pipiens, and RFLP markers for chromosome 3 in Ae. aegypti identified homologous loci on the opposite arms of chromosomes 2 and 3 in Cx. pipiens.

Aedes↗

PubMed1998

Positional cloning without a genome map: using 'Targeted RFLP Subtraction' to isolate dense markers tightly linked to the regA locus of Volvox carteri.

The ability to isolate genes defined by mutant phenotypes has fueled the rapid progress in understanding basic biological mechanisms and the causes of inherited diseases. Positional cloning, a commonly used method for isolating genes corresponding to mutations, is most efficiently applied to the small number of model organisms for which high resolution genetic maps exist. We demonstrate a new and generally applicable positional cloning method that obviates the need for a genetic map. The technique is based on Restriction Fragment Length Polymorphism (RFLP) Subtraction, a method that isolates RFLP markers spanning an entire genome. The new method, Targeted RFLP Subtraction (TRS), isolates markers from a specific region by combining RFLP Subtraction with a phenotypic pooling strategy. We used TRS to directly isolate dense markers tightly linked to the regA gene of the eukaryotic green alga Volvox. As a generally applicable method for saturating a small targeted region with DNA markers, TRS should facilitate gene isolation from diverse organisms and accelerate the process of physically mapping specific regions in preparation for sequence analysis.

Algal Proteins↗

PubMed2005

SNP Cutter: a comprehensive tool for SNP PCR-RFLP assay design.

The Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is a relatively simple and inexpensive method for genotyping single nucleotide polymorphisms (SNPs). It requires minimal investment in instrumentation. Here, we describe a web application, 'SNP Cutter,' which designs PCR-RFLP assays on a batch of SNPs from the human genome. NCBI dbSNP rs IDs or formatted SNPs are submitted into the SNP Cutter which then uses restriction enzymes from a pre-selected list to perform enzyme selection. The program is capable of designing primers for either natural PCR-RFLP or mismatch PCR-RFLP, depending on the SNP sequence data. SNP Cutter generates the information needed to evaluate and perform genotyping experiments, including a PCR primers list, sizes of original amplicons and different allelic fragment after enzyme digestion. Some output data is tab-delimited, therefore suitable for database archiving. The SNP Cut-ter is available at http://bioinfo.bsd.uchicago.edu/SNP_cutter.htm.

DNA Primers↗

PubMedDate not supplied

Location of the self-incompatibility locus in an RFLP and RAPD map of Brassica oleracea.

A single locus controlling self-incompatibility (SI) was located on an RFLP and RAPD linkage map of Brassica oleracea by assaying for SI phenotype and segregation of an RFLP locus detected by the cloned St gene SLG6. The map was developed using an F2 population derived from a cabbage x broccoli cross and included 112 RFLP and 47 RAPD loci arranged into nine main linkage groups covering 921 cM. A portion of the population was assayed for St reaction by pollinating with the broccoli parent. The SI reactions cosegregated precisely with the RFLP locus detected by SLG6, and this locus mapped to linkage group 2.

Brassica↗

PubMed1992

Non-association between 9.2 KB PvuII RFLP and seronegative spondyloarthropathies in Spain.

Several studies of DNA restriction fragment length polymorphism (RFLP) in ankylosing spondylitis (AS) have been carried out. The association between a recently identified class I HLA 9.2 kb PvuII RFLP and AS remain controversial. In order to evaluate this possible association in an Euro-Caucasian population, the genomic DNA of 42 AS patients and 18 patients with Reiter's syndrome (RS) and 42 healthy controls was analysed. Non-association between 9.2 kb PvuII RFLP and AS or RS was observed. As described previously, a strong association between this fragment and HLA-A3 and/or HLA-A9 antigens was demonstrated. A study of two families showed that this RFLP segregates with HLA-A3 and/or HLA-A9 and independently of the HLA-B27. Our findings support the view that the 9.2 kb PvuII fragment is not universally associated with AS.

Arthritis, Reactive↗

PubMed2000

Hot-spot variations of Kaposi's sarcoma-associated herpesvirus latent nuclear antigen and application in genotyping by PCR-RFLP.

Kaposi's sarcoma-associated herpesvirus (KSHV, human herpesvirus-8) is aetiologically associated with Kaposi's sarcoma and several other lymphoproliferative disorders. The latent nuclear antigen (LNA) encoded by KSHV ORF73 has important functions in virus latent infection and shows molecular polymorphism. Sequence variations were identified in the internal repeat domain (IRD) of ORF73. DNA sequencing of ORF73 from one KSHV-infected cell line, PK-1, revealed that there were 558 bp (30.2%) deletions and 66 (3.6%) point mutations located mainly in repeat region 2, the glutamine-rich region of ORF73 IRD, compared with ORF73 of BC-1 KSHV. Similar sequence variations of ORF73 were also identified in two other isolates. None of the sequence variations caused any translational frame-shift in these four KSHV isolates examined, suggesting that LNA has a conservative function in virus latent infection. The frequent sequence variations in repeat region 2 of ORF73 IRD were also identified by PCR-RFLP genotyping in 26 KSHV isolates, suggesting that this region is a 'hot-spot' for genetic variations. Each Kaposi's sarcoma lesion sample contained one virus genotype with a unique RFLP pattern, indicating that in vivo KSHV infection was established with single predominate genotypes, which was further supported by the presence of invariable genotypes in multifocal lesions from individual KS patients. Four KSHV subtypes were classified based on the RFLP patterns that represent the patterns of DNA sequence variations in the ORF73 IRD. PCR-RFLP genotyping is capable of identifying LNA genetic variations and differentiating individual KSHV isolates, and thus may be useful for KSHV molecular epidemiology studies.

Antigens, Viral↗

PubMed2000

Genetic diversity of Ralstonia solanacearum as assessed by PCR-RFLP of the hrp gene region, AFLP and 16S rRNA sequence analysis, and identification of an African subdivision.

The genetic diversity among strains in a worldwide collection of Ralstonia solanacearum, causal agent of bacterial wilt, was assessed by using three different molecular methods. PCR-RFLP analysis of the hrp gene region was extended from previous studies to include additional strains and showed that five amplicons were produced not only with all R. solanacearum strains but also with strains of the closely related bacteria Pseudomonas syzygii and the blood disease bacterium (BDB). However, the three bacterial taxa could be discriminated by specific restriction profiles. The PCR-RFLP clustering, which agreed with the biovar classification and the geographical origin of strains, was confirmed by AFLP. Moreover, AFLP permitted very fine discrimination between different isolates and was able to differentiate strains that were not distinguishable by PCR-RFLP. AFLP and PCR-RFLP analyses confirmed the results of previous investigations which split the species into two divisions, but revealed a further subdivision. This observation was further supported by 16S rRNA sequence data, which grouped biovar 1 strains originating from the southern part of Africa.

Africa↗

PubMed1997

Comparison of three restriction endonucleases in IS1245-based RFLP typing of Mycobacterium avium.

IS1245-based restriction fragment length polymorphism (RFLP) analysis has been proposed recently for molecular typing of Mycobacterium avium isolates. As there is no standardised method with respect to the optimal restriction enzyme, three restriction endonucleases were tested for analysis of 17 human isolates. The restriction endonucleases, selected on the basis of the physical maps of IS1245 and of the highly homologous IS1311, were BsaAI, that cleaves IS1245, PvuII, that cleaves IS1311, and NruI, that cleaves both IS1245 and IS1311. All the restriction endonucleases yielded polymorphic and complex RFLP patterns. However, BsaAI- and NruI-generated bands were more evenly distributed and easier to detect than PvuII-generated bands, most of which clustered in a narrow zone of the fingerprint. In some cases, DNA digestion with BsaAI or NruI yielded probe-specific restriction fragments of molecular size lower than expected. Moreover, digestion with NruI, which was expected to generate the highest numbers of bands in all the isolates, yielded fewer bands than were obtained with BsaAI or PvuII in 14 and 5 isolates, respectively. These findings might suggest the existence of unidentified IS1245-related insertion element(s) in M. avium isolates. Computer analysis of the IS1245-based RFLP patterns of M. avium isolates showed that the restriction endonucleases were capable, although with minor differences, of defining distinct banding patterns and clusters of identical or highly related isolates, thus confirming IS1245-based RFLP analysis as a useful technique for epidemiological studies.

DNA Transposable Elements↗

PubMed2004

Second-line treatment of Helicobacter pylori infection after dilution agar methods and PCR-RFLP analysis.

BACKGROUND: After unsuccessful first-line treatment of Helicobacter pylori infection, the percentage of clarithromycin-resistant strains has been reported as between 30% and 70% in Japan and other countries. A high prevalence of clarithromycin-resistant strains is reported to be associated with eradication failure. AIM: We examined antibiotic susceptibility testing using a combination of dilution agar methods with PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS: We enrolled 41 patients in whom first-line treatment with LAC (lansoprazole, amoxycillin and clarithromycin) was unsuccessful. Endoscopic biopsied specimens were used to examine antibiotic susceptibility to clarithromycin by dilution agar methods. PCR-RFLP analysis was performed to determine the presence of point mutations, which are primarily responsible for resistance to clarithromycin. RESULTS: Clarithromycin-resistance rate after failure of the LAC regimen was 73.2%. Drug susceptibilities of three strains obtained by PCR-RFLP analysis were different from those by dilution agar methods. One strain with MIC values to clarithromycin of 0.05 micro g/mL had a point mutation, A2144G. This strain was not eradicated by repeating LAC, but was eradicated by substituting metronidazole for clarithromycin. CONCLUSIONS: Dilution agar methods should be combined with PCR-RFLP analysis before second-line eradication to increase the accuracy of clarithromycin-susceptibility testing and to improve eradication efficacy.

2-Pyridinylmethylsulfinylbenzimidazoles↗

PubMed1994

Expression pattern, genomic cloning and RFLP analyses of the swine PIT-1 gene.

The swine PIT-1 POU domain cDNA was used to study PIT-1 expression and to clone the PIT-1 genomic region to identify additional PIT-1 polymorphisms. PIT-1 was expressed only in the pituitary, confirming the swine cDNA identity. To study the PIT-1 genomic region, a clone containing 13.7 kb DNA was isolated. EcoRI fragments hybridizing to PIT-1 were sequenced, and the 3' portion of PIT-1 gene identified. Primers for polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis were then designed. Restriction analysis of PCR products identified a RsaI RFLP. In contrast to other PIT-1 RFLPs, the RsaI PCR-RFLP was widely distributed in American breeds. Thus, the RsaI RFLP and the previously reported PIT-1 RFLPs allow detection of PIT-1 alleles in most, if not all, breeds currently used in the US and European reference/resource pig mapping families.

Animals↗

PubMed1995

Sequence and PCR-RFLP analysis of 14 novel BoLA-DRB3 alleles.

The genetic diversity of the bovine class II DRB3 locus was investigated by polymerase chain reaction (PCR) amplification and DNA sequencing of the first domain exon. Studying 34 animals of various cattle breeds, 14 previously unrecognized DRB3 alleles were identified. In three alleles, amino acid substitutions were observed that had not been previously found in bovine DRB3, but occurred at the same position in bovine DQB and in the DRB alleles of other mammals. For all newly identified alleles, the restriction fragment length polymorphism (RFLP) patterns of PCR products obtained with the enzymes RsaI, BstYI, and HaeIII were compared with patterns of 38 previously described alleles. Altogether, eleven novel PCR-RFLP types were defined. Twelve out of the 42 PCR-RFLP types identified so far were not found to be fully informative because they corresponded to more than one allelic sequence. PCR-RFLP may therefore be a rapid and useful method for DRB3 typing in cattle families, but for studies on outbred populations, sequencing and hybridization techniques are required.

Alleles↗

PubMed1993

An MseI RFLP in the 5' flanking region of the factor IX gene: its use for haemophilia B carrier detection in Caucasian and Thai populations.

We have identified an MseI restriction fragment length polymorphism (RFLP) in the 5' flanking region of the factor IX gene. RFLPs in the factor IX locus are routinely used as linkage markers to track defective factor IX genes through affected pedigrees, allowing diagnosis of the haemophilia B carrier state in female members of the kindred. Currently, seven intragenic polymorphic loci have been characterized allowing carrier diagnosis in 89% of cases in the Caucasian population. Additional screening with the MseI RFLP reported in this publication increases this figure to 94% of cases. In Asian populations only one of these RFLPs (HhaI) is present at any significant frequency. Hence, carrier detection rates are much reduced in comparison to the corresponding figure of 89% in Caucasians. We report that this MseI RFLP is also present in the Thai population. Indeed, when used in combination with the HhaI polymorphism, the MseI RFLP should significantly improve the carrier detection rate in the Thai population from 11% to 40% of cases.

Alleles↗

PubMed1995

Genomic RFLP typing of human platelet alloantigens Zw(PlA), Ko, Bak and Br (HPA-1, 2, 3, 5).

The diallelic human platelet alloantigen systems 1-5 have been found to result from single base pair substitutions in the encoding genes of platelet membrane glycoproteins IIIa, Ib, IIb and Ia. This is the basis of DNA methods for determination of platelet alloantigens. In this study, 98 blood donors were typed in the HPA-1, 2, 3 systems and, for the first time, in the HPA-5 system. Serologically obtained data (MAIPA and platelet agglutination) were compared with results from analysis of restriction fragment length polymorphisms (RFLP). Discordances were found in the HPA-2 and 3 systems and can be ascribed to false typing results in both the serological and genomic methods. In the HPA-1, 2 and 5 systems, all samples were typed correctly with RFLP analysis. Serologically, two donors were falsely typed positive with anti-HPA-2b in platelet agglutination and one donor with anti-HPA-3a in MAIPA assay. In the HPA-3 system, another four donors were misinterpreted to be HPA-3a negative in RFLP analysis. Possible technical problems in PCR-RFLP-typing are discussed and another strategy of HPA-1 typing using the restriction enzyme Scr FI is evaluated.

Antigens, Human Platelet↗

PubMed1996

Characterization of Rhizobium 'hedysari' by RFLP analysis of PCR amplified rDNA and by genomic PCR fingerprinting.

The taxonomic and discriminatory power of RFLP analysis of PCR amplified parts of rhizobial rrn operons was compared to those of genomic PCR fingerprinting with arbitrary and repetitive primers. For this purpose, the two methods were applied for characterization of a group of bacterial isolates referred to as Rhizobium 'hedysari'. As outgroups, representatives of the family Rhizobiaceae, belonging to the Rhizobium galegae, Rhizobium meliloti, Rhizobium leguminosarum and Agrobacterium tumefaciens species were used. By the RFLP analysis of the PCR products corresponding to the variable 5'-half of the 23S rRNA gene and of the amplified spacer region between the 16S and 23S rRNA genes all Rh. 'hedysari' strains studied were tightly clustered together while the outgroups were placed in an outer position. The PCR products of the 3' end parts of the 23S rDNA did not show significant RFL polymorphism and no species differentiation on their basis was possible. In parallel, analysis of the same strains was performed by PCR amplification of their DNA with 19, 18 and 10 bp long arbitrary primers (AP-PCR) as well as with single primers corresponding to several bacterial repetitive sequences (rep-PCR). By both AP and rep-PCR an identification of every particular strain was achieved. In general, all primers provided taxonomic results that are in agreement with the species and group assignments based on the RFLP analysis of the rrn operons. On the basis of the results presented here it can be concluded that AP and rep-PCR are more informative and discriminative than rDNA and RFLP analysis of the rhizobial strains studied.

Base Sequence↗

PubMed2004

Identification of extracellular enzyme producing thermophilic bacilli from Balcova (Agamemnon) geothermal site by ITS rDNA RFLP.

AIMS: Molecular characterization of extracellular enzyme producing thermophilic bacilli from Balcova geothermal site. METHODS AND RESULTS: Three types of geothermal samples were collected: mud, re-injection water, and samples from uncontrolled hydrothermal vents. Isolates grown at 55 degrees C in culture media prepared in sterilized re-injection water, were screened for extracellular enzyme activity by using eight different substrates: casein, carboxymethylcellulose, pectin, polygalacturonic acid (PGA), soluble starch, Tween 20 and 80, and xylan. In total, 109 thermoaerophilic isolates were selected. All of the isolates could hydrolyse Tween 20 (100%) but not Tween 80. Soluble starch was hydrolysed by 96%, casein by 55%, xylan and carboxymethylcellulose by 9%, and pectin and PGA by 2% of the isolates. The isolates were grouped into 14 different homology groups by the restriction pattern analysis of 16S-internal transcribed spacer (ITS) rDNA RFLP. Each of the RFLP groups was also studied by 16S rRNA gene partial sequence analysis. Plasmid DNA profiles revealed that 15 of the isolated strains contained small plasmid DNA molecules ranging in size from 12 000 to 35 000 bp. CONCLUSIONS: Combined analysis of 16S-ITS rDNA RFLP and 16S rRNA gene partial sequence results indicated the presence of novel or existing species of Anoxybacillus (nine species) and Geobacillus (three species). SIGNIFICANCE AND IMPACT OF THE STUDY: In this study 16S-ITS rDNA RFLP was applied for the first time to differentiate thermophilic bacilli. It was also the first study on thermophilic bacilli of Balcova geothermal site.

Bacillus↗

PubMed2004

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP.

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.

DNA, Bacterial↗

PubMed1995

Association of HincII RFLP of low density lipoprotein receptor gene with obesity in essential hypertensives.

Obese (BMI > or = 26 kg/m2; n = 51) and lean (BMI < 26 kg/m2; n = 61) Caucasian patients with severe, familial essential hypertension, were compared with respect to genotype and allele frequencies of a HincII RFLP of the low density lipoprotein receptor gene (LDLR). A similar analysis was performed in obese (n = 28) and lean (n = 68) normotensives. A significant association of the C allele of the T-->C variant responsible for this RFLP was seen with obesity (chi 2 = 4.6, P = 0.029) in the hypertensive, but not in the normotensive, group (odds ratio = 3.0 for the CC genotype and 2.7 for CT). Furthermore, BMI tracked with genotypes of this allele in the hypertensives (P = 0.046). No significant genotypic relationship was apparent for plasma lipids. Significant linkage disequilibrium was, moreover, noted between the HincII RFLP and an ApaLI RFLP (chi 2 = 33, P < 0.0005) that has previously shown even stronger association with obesity (odds ratio 19.6 for cases homozygous for the susceptibility allele and 15.2 for heterozygotes). The present study therefore adds to our previous evidence implicating LDLR as a locus for obesity in patients with essential hypertension.

Alleles↗