The work was oriented to identification of -casein gene polymorphism and analysis of genotype structure in population of Slovak Pinzgau cattle. The material involved 89 cattle. Bovine genomic DNA ...was isolated by fenol-chlorophorm deprotenization and ethanol precipitation and used in order to estimate - casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study there were homozygote genotype A1A1 (27 animals), heterozygote genotype A1A2 (46 animals) and homozygote genotype A2A2 (16 animals). In the total population of cattle heterozygotes A1A2 – 0.5168 were the most frequent, while homozygotes A2A2 – 0.1798 were the least frequent ones. This suggests a slight superiority of allele A1– 0.5618.
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In recent years, the incidence of infections caused by pathogenic fungi has increased globally, especially in immunocompromised patients, transplant patients, or patients undergoing chemotherapy in ...intensive care units. Due to the morbidity and rapid spread of infection to other organs, accurate, rapid diagnosis and treatment of fungal infections is essential. Molecular methods such as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) have significant superiority in sensitivity and specificity compared to conventional methods. The aim of this study is to point out the limitations of common diagnostic methods and factors affecting the sensitivity and specificity of PCR-RFLP molecular diagnostic test. This article is a review study for which subject-related academic papers (before 2023) were collected and studied by searching the keywords of pathogenic fungi, and RFLP molecular diagnostic methods; in Persian databases as well as Latin electronic databases including Scopus, Web of Science, and PubMed . Reviewing numerous articles showed that PCR-RFLP is a rapid, practical, and reliable method, which can be used in laboratories to determine the genotype of specific variants of fungal species isolated from clinical samples. However, there are several limitations for PCR-RFLP method demanding for more attention. The main limitations are the need for specific restriction enzymes and the difficulty of accurately determining single nucleotide polymorphisms (SNPs) in a specific diagnostic locus.
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3.
A family screening of CD19 gene mutation by PCR-RFLP Karaselek, Mehmet Ali; Kapaklı, Hasan; Güner, Şükrü Nail ...
European Journal of Clinical and Experimental Medicine,
2022, Volume:
20, Issue:
2
Journal Article
Peer reviewed
Open access
Introduction and aim. Mutation(s) in the gene encoding the CD19 molecule affect CD19 protein expression and primary immunodeficiency (PID) occurs. The PCR-RFLP method, which is faster and cheaper ...than other mutation detection methods, is rarely used in the diagnosis of PID. The study aimed to genetically identify CD19 deficiency, which is a PID, using the PCR-RFLP method. Material and methods. A total of 8 patients and two healthy controls were included in the study and the relevant region genotypes in the CD19 gene were determined by performing PCR-RFLP analysis. Results. The index case, newborn baby and mother were also included in the study. It was determined that the index case (P6) was homozygous mutant, the newborn baby (P7) and mother (P8) had heterozygous genotype. Based on this situation, one child (P1) was found to be homozygous mutant, mother (P2), father (P3) and other children (P4 and P5) had heterozygous genotype in the family, which was determined to be related to the first case. Conclusion. In our study, it has been shown that PCR-RFLP is a method that can be used in the diagnosis of PID by determining genotypes using PCR-RFLP, and especially in terms of rapid genetic testing of family screenings.
Background: Type 2 diabetes mellitus (T2DM), as a worldwide health challenge, is a multifactorial disease that environmental and genetic factors contribute to its pathogenicity. Gastric Inhibitory ...Polypeptide Receptor (GIPR) is a G-pro receptor that controls the gut hormones release and insulin secretion. The current study aimed to investigate the role of the GIPR rs1800437 gene variant in T2DM susceptibility. Material and methods: A total of 108 confirmed T2DM patients and 100 normal controls were recruited in the study. The GIPR rs1800437 genotypes were determined by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assay. Results: A significant difference was found in genotypes (CC, CG, and GG) frequency of the GIPR rs1800437 variant between T2DM and control groups (P = 0.02). The homozygote CC genotype of the variant significantly decreased the odds ratio (OR) of diabetes mellitus risk, approximately 50 %, in comparison with the heterozygous GC genotype. The frequency of the C allele among cases was considerably lower than controls (P = 0.002, OR = 0.51, CI = 0.33–0.79). Conclusion: The findings of the study show enough evidence that there is a significant association between the rs1800437 GIPR genetic variant and the risk of T2DM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
Despite decreasing costs, generating large-scale, well-replicated and multivariate microbial ecology investigations with sequencing remains an expensive and time-consuming option. As a ...result, many microbial ecology investigations continue to suffer from a lack of appropriate replication. We evaluated two fingerprinting approaches – terminal restriction fragment length polymorphism (T-RFLP) and automated ribosomal intergenic spacer analysis (ARISA) against 454 pyrosequencing, by applying them to 225 polar soil samples from East Antarctica and the high Arctic. By incorporating local and global spatial scales into the dataset, our aim was to determine whether various approaches differed in their ability and hence utility, to identify ecological patterns. Through the reduction in the 454 sequencing data to the most dominant OTUs, we revealed that a surprisingly small proportion of abundant OTUs (< 0.25%) was driving the biological patterns observed. Overall, ARISA and T-RFLP had a similar capacity as sequencing to separate samples according to distance at a local scale, and to correlate environmental variables with microbial community structure. Pyrosequencing had a greater resolution at the global scale but all methods were capable of significantly differentiating the polar sites. We conclude fingerprinting remains a legitimate approach to generating large datasets as well as a cost-effective rapid method to identify samples for elucidating taxonomic information or diversity estimates with sequencing methods.
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The identification of different fish species by molecular methods has become necessary to avoid both the incorrect labelling of individuals involved in repopulation programmes and the commercial ...frauds on the fish market. Different fish species of great economical importance, like the salmonids, which are very much requested for their meat, can be identified using molecular techniques such as PCR-RFLP. The method is based on the amplification of a target region from the genome by PCR reaction followed by endonucleases digestion to detect the polymorphism of restriction fragments. In our study we analysed the following salmonid species from Romania: Salmo trutta fario, Salmo labrax, Salvelinus fontinalis, Onchorhynchus mykiss, Thymallus thymallus and Hucho hucho. In order to discriminate between the analysed species we amplified a fragment of mitochondrial genome comprising tRNAGlu/ cytochrome b/ tRNAThr/ tRNAPro/ D-loop/ tRNAPhe, followed by digestion with a specific restriction enzyme. The direct digestion of unpurified PCR products generated species-specific restriction patterns and proved to be a simple, reliable, inexpensive and fast method. Thus, it may be successfully utilized in specialized laboratories for the correct identification of the fish species for multiple purposes, including the traceability of fish food products.
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The work was oriented to identification of -s1 casein gene polymorphism and analysis of genotype structure in population of Slovak Pinzgau cattle. The material involved 39 cattle. Bovine genomic DNA ...was isolated by commercial kit NucleoSpin Blood (Macherey-Nagel) and ethanol precipitation and used in order to estimate -s1 casein genotypes by means of PCR-RFLP method. The PCR products were digested with MaeIII restriction enzyme. In the population included in the study, homozygote genotype BB (39 animals) and heterozygote genotype BC (3 animals). Homozygote genotype CC has not been observed. In the total population of cattle homozygotes BB – 0.9231 were the most frequent, while BC – 0.0769 were the least frequent ones. This suggests a superiority of allele B – 0.9615.
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Aims
The present study aimed at gaining an insight into the abundance and genetic diversity of culturable N‐fixing epiphyte bacteria on the phyllosphere of maize in arid and semi‐arid regions of ...Iran.
Methods and Results
Leaf samples of the maize variety, ‘single cross 704’ (Zea mays L.) were collected from different locations in Iran. The community of culturable N‐fixing epiphyte bacteria present was examined by 16S rRNA sequencing, BOXAIR‐polymerase chain reaction (PCR) and restricted fragment length polymorphisms analysis of 16S rRNA gene (16S‐RFLP). Approximately, 31·82% of the 242 isolates were identified as N‐fixers by cultivation of bacteria in Rennie medium and detection of their nifH gene. The N‐fixers were affiliated with four bacterial phyla: Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. 16S rRNA sequencing detected 16 genera and 24 different species in the identified phyla. The most dominant genus was Bacillus and the species identified were B. pumilus, B. amyloliquefaciens, B. subtilis, B. paralicheniformis, B. licheniformis, B. niabensis and B. megaterium. In total, 22 RFLP groups were present among the isolates originally identified as N‐fixing bacteria. BOXAIR‐PCR showed that there was a low similarity level among the N‐fixing bacteria isolates, and genetic differentiation of individual strains was relatively great.
Conclusions
Our findings suggest that nitrogen‐fixing epiphyte bacteria on the phyllosphere of maize may provide significant nitrogen input into arid and semi‐arid ecosystem.
Significance and Impact of the Study
This research implies that phyllosphere epiphyte diazotrophs have much to offer in sustainable agriculture and can be an alternative to chemical N‐fertilizers for providing nitrogen to crops arid and semi‐arid regions.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The work was oriented to identification of -s1 casein gene polymorphism and analysis of genotype structure in population of Slovak Pinzgau cattle. The material involved 93 cattle. Bovine genomic DNA ...was isolated by fenol-chlorophorm deprotenization and ethanol precipitation and used in order to estimate -s1 casein genotypes by means of PCR-RFLP method. The PCR products were digested with MaeIII restriction enzyme. In the population included in the study there were homozygote genotype BB (81 animals) and heterozygote genotype BC (12 animals). Homozygote genotype CC has not been observed. In the total population of cattle homozygotes BB – 0.871 were the most frequent, while BC – 0.129 were the least frequent ones. This suggests a superiority of allele B – 0.9355.
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Little is known about the response of arbuscular mycorrhizal fungal communities to ecosystem development. We use a long‐term soil chronosequence that includes ecosystem progression and retrogression ...to quantify the importance of host plant identity as a factor driving fungal community composition during ecosystem development. We identified arbuscular mycorrhizal fungi and plant species from 50 individual roots from each of 10 sites spanning 5–120 000 yr of ecosystem age using terminal restriction fragment length polymorphism (T‐RFLP), Sanger sequencing and pyrosequencing. Arbuscular mycorrhizal fungal communities were highly structured by ecosystem age. There was strong niche differentiation, with different groups of operational taxonomic units (OTUs) being characteristic of early succession, ecosystem progression and ecosystem retrogression. Fungal alpha diversity decreased with ecosystem age, whereas beta diversity was high at early stages and lower in subsequent stages. A total of 39% of the variance in fungal communities was explained by host plant and site age, 29% of which was attributed to host and the interaction between host and site (24% and 5%, respectively). The strong response of arbuscular mycorrhizal fungi to ecosystem development appears to be largely driven by plant host identity, supporting the concept that plant and fungal communities are tightly coupled rather than independently responding to habitat.
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