nucleotide validation
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Východiska: Účinky jednonukleotidových polymorfizmů (single nucleotide polymorphisms – SNPs) genů nukleotidové excizní reparace (nucleotide excision repair – NER) na náchylnost ke kožnímu melanomu (cutaneous melanoma – CM) jsou předmětem velkého zájmu. V současné době je v několika epidemiologických studiích hodnoceno, zda polymorfizmy XPC, XPD, XPG a XPF souvisí s CM. Výsledky těchto studií jsou ale kontroverzní nebo nevedou k jednoznačnému závěru. Proto jsme provedli studii s cílem zhodnotit vztah mezi sedmi často zkoumanými polymorfizmy dráhy NER a rizikem CM. Metody: Do studie bylo zařazeno celkem 150 patients s diagnózou CM a 150 zdravých kontrol. Sedm SNPs dráhy NER vč. XPC (Lys939Gln a Ala499Val), XPD (Lys157Gln, Asp272Asn a Arg751Arg), XPG (Asp1104His) a XPF (Arg415Gln) bylo analyzováno stanovením polymorfizmu délky štěpných fragmentů pomocí polymerázové řetězové reakce. Výsledky: Mezi polymorfizmy XPC Lys939Gln, Ala499Val, XPD Asp272Asn, Arg751Arg, Arg751Arg, XPF Arg415Gln a XPG Asp1104His a zvýšeným rizikem CM nebyl zjištěn významný vztah. Závěry: Tato studie odhalila, že polymorfizmy XPC, XPD, XPG a XPF nebyly pro náchylnost k CM rizikovým faktorem. Pro další hodnocení a validaci našich výsledků je třeba více studií s dobrým designem a vyšším počtem subjektů v různých populacích. Přesnější důkazy a další objasnění vlastního mechanizmu CM přinesou v budoucnosti studie, které budou brát v úvahu interakce mezi geny jako takovými a mezi geny a prostředím.
Background: The effects of single nucleotide polymorphisms (SNPs) at nucleotide excision repair (NER) pathway on susceptibility to cutaneous melanoma (CM) are of great interest. To date, several epidemiological studies have evaluated whether the XPC, XPD, XPG and XPF polymorphisms are associated with CM. However, those studies results are controversial or inconclusive. Therefore, we conducted a study to evaluate the association of seven frequently investigated NER pathway polymorphisms with CM risk. Methods: A total of 150 patients diagnosed with CM and 150 healthy controls were enrolled in the study. Seven SNPs in the NER pathway including XPC (Lys939Gln and Ala499Val), XPD (Lys157Gln, Asp272Asn, and Arg751Arg), XPG (Asp1104His) and XPF (Arg415Gln) were analyzed by polymerase chain reaction restriction fragment length polymorphism assay. Results: There was no a significant association between XPC Lys939Gln, Ala499Val; XPD Asp272Asn, Arg751Arg, Arg751Arg; XPF Arg415Gln; and XPG Asp1104His polymorphisms and an increased risk of CM. Conclusions: This study results revealed that the XPC, XPD, XPG and XPF polymorphisms were not risk factor for susceptibility to CM. However, more well-designed with larger sample size studies in different populations are necessary to further evaluate and validate our results. Future studies which take into account gene-gene and gene-environment interactions are warranted for more precise evidence and further elucidation of the underlying mechanism of CM.
- MeSH
- jednonukleotidový polymorfismus MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- melanom * genetika MeSH
- oprava DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
DNA cruciform structures play an important role in the regulation of natural processes including gene replication and expression, as well as nucleosome structure and recombination. They have also been implicated in the evolution and development of diseases such as cancer and neurodegenerative disorders. Cruciform structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling and protein binding. They have received broad attention because of their important roles in biology. Computational approaches to study inverted repeats have allowed detailed analysis of genomes. However, currently there are no easily accessible and user-friendly tools that can analyse inverted repeats, especially among long nucleotide sequences. We have developed a web-based server, Palindrome analyser, which is a user-friendly application for analysing inverted repeats in various DNA (or RNA) sequences including genome sequences and oligonucleotides. It allows users to search and retrieve desired gene/nucleotide sequence entries from the NCBI databases, and provides data on length, sequence, locations and energy required for cruciform formation. Palindrome analyser also features an interactive graphical data representation of the distribution of the inverted repeats, with options for sorting according to the length of inverted repeat, length of loop, and number of mismatches. Palindrome analyser can be accessed at http://bioinformatics.ibp.cz.
- MeSH
- DNA bakterií analýza genetika MeSH
- DNA virů analýza genetika MeSH
- DNA analýza genetika MeSH
- Escherichia coli genetika MeSH
- genom bakteriální genetika MeSH
- genom virový genetika MeSH
- internet * MeSH
- křížová struktura DNA analýza genetika MeSH
- obrácené repetice genetika MeSH
- oligonukleotidy analýza genetika MeSH
- reprodukovatelnost výsledků MeSH
- sekvence nukleotidů MeSH
- viry klasifikace genetika MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
Increasing importance of single-nucleotide polymorphisms (SNPs) in determination of disease susceptibility or in prediction of therapy response brings attention of many molecular diagnostic laboratories to simple and low-cost SNP genotyping methodologies. We have recently introduced a mutation detection technique based on analysis of homo- and heteroduplex PCR fragments resolved in cycling temperature gradient conditions on a conventional multicapillary-array DNA sequencer. The main advantage of this technique is in its simplicity with no requirement for sample cleanup prior to the analysis. In this report we present a practical application of the technology for genotyping of SNP markers in two separate clinical projects resulting in a combined set of 44 markers screened in over 500 patients. Initially, a design of PCR primers and conditions was performed for each SNP marker. Then, optimization of CE running conditions (limited just to the proper selection of temperature cycling) was performed on pools of 20 DNA samples to increase the probability of having each of the two allele types represented in the sample. After selecting the optimum conditions, screening of markers in patients was performed using a multiple-injection approach for further acceleration of the sample throughput. The rate of successful optimization of experimental conditions without any pre-selection based on the SNP sequence or melting characteristics was 80% from the initial SNP marker candidates. By studying the failed markers, we attempt to identify critical factors enabling successful typing. The presented technique is very useful for low to medium sized SNP genotyping projects mostly applied in pharmacogenomic research as well as in clinical diagnostics. The main advantages include low cost, simple setup and validation of SNP markers.
- MeSH
- elektroforéza kapilární * metody MeSH
- genetická predispozice k nemoci * genetika MeSH
- genetické markery genetika MeSH
- genetické testování * metody MeSH
- genotyp MeSH
- jednonukleotidový polymorfismus * MeSH
- lidé MeSH
- průtoková injekční analýza MeSH
- schizofrenie genetika MeSH
- sekvenční analýza DNA * metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- validační studie MeSH
Previously reported DNA aptamers developed against surface proteins extracted from Campylobacter jejuni were further characterized by aptamer-based Western blotting and shown to bind epitopes on proteins weighing ~16 and 60 kD from reduced C. jejuni and Campylobacter coli lysates. Proteins of these approximate weights have also been identified in traditional antibody-based Western blots of Campylobacter spp. Specificity of the capture and reporter aptamers from the previous report was further validated by aptamer-based ELISA-like (ELASA) colorimetric microplate assay. Finally, the limit of detection of the previously reported plastic-adherent aptamer-magnetic bead and aptamer-quantum dot sandwich assay (PASA) was validated by an independent food safety testing laboratory to lie between 5 and 10 C. jejuni cells per milliliter in phosphate buffered saline and repeatedly frozen and thawed chicken rinsate. Such ultrasensitive and rapid (30 min) aptamer-based assays could provide alternative or additional screening tools to enhance food safety testing for Campylobacter and other foodborne pathogens.
- MeSH
- aptamery nukleotidové genetika MeSH
- biotest přístrojové vybavení metody MeSH
- Campylobacter genetika izolace a purifikace MeSH
- kontaminace potravin analýza MeSH
- kur domácí MeSH
- kvantové tečky chemie MeSH
- magnetismus metody MeSH
- maso mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- validační studie MeSH
Although the analysis of length polymorphism at STR loci has become a method of choice for grape cultivar identification, the standardization of methods for this purpose lags behind that of methods for DNA profiling in human and animal forensic genetics. The aim of this study was thus to design and validate a grapevine STR protocol with a practically useful level of multiplexing. Using free bioinformatics tools, published primer sequences, and nucleotide databases, we constructed and optimized a primer set for the simultaneous analysis of six STR loci (VVIi51, scu08vv, scu05vv, VVMD17, VrZAG47, and VrZAG83) by multiplex PCR and CE with laser-induced fluorescence, and tested it on 90 grape cultivars. The new protocol requires subnanogram quantities of the DNA template and enables automated, high-throughput genetic analysis with reasonable discriminatory power. As such, it represents a step toward further standardization of grape DNA profiling.
- MeSH
- algoritmy MeSH
- DNA rostlinná analýza genetika MeSH
- genetické markery genetika MeSH
- mikrosatelitní repetice genetika MeSH
- multiplexová polymerázová řetězová reakce metody MeSH
- reprodukovatelnost výsledků MeSH
- víno MeSH
- Vitis klasifikace genetika MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
During a survey on grapevine yellows disease complex in vineyards of Lombardy region (northern Italy), phytoplasmas associated with Flavescence dorée disease were identified in symptomatic grapevines. Polymerase chain reaction and restriction fragment length polymorphism (RFLP) analyses of 16S rDNA revealed the prevalence of phytoplasmal subgroup 16SrV-D. Bioinformatic analyses of nucleotide sequences of rplV and rpsC genes, amplified from 16SrV-D phytoplasma infected grapevines and cloned, underscored the presence of five confirmed rpsC single nucleotide polymorphism (SNP) lineages, determined by different combination of SNPs at nucleotide positions 29, 365, 680, and 720 of rpsC gene. Virtual and actual RFLP analyses with the enzyme TaqI validated the presence of these SNPs. Co-infections by up to four distinct rpsC SNP lineages of 16SrV-D phytoplasma were found in grapevines. These results could open new perspectives for the study of the ecology and the epidemiology of Flavescence dorée.
- MeSH
- bakteriální proteiny genetika MeSH
- DNA bakterií genetika chemie MeSH
- DNA fingerprinting MeSH
- financování organizované MeSH
- fylogeneze MeSH
- jednonukleotidový polymorfismus MeSH
- molekulární sekvence - údaje MeSH
- nemoci rostlin mikrobiologie MeSH
- Phytoplasma genetika izolace a purifikace klasifikace MeSH
- polymerázová řetězová reakce MeSH
- polymorfismus délky restrikčních fragmentů MeSH
- ribozomální DNA genetika MeSH
- ribozomální proteiny genetika MeSH
- RNA ribozomální 16S genetika MeSH
- sekvenční analýza DNA MeSH
- sekvenční homologie MeSH
- shluková analýza MeSH
- Vitis mikrobiologie MeSH
- Geografické názvy
- Itálie MeSH
Routinely used typing methods including MLST, rep-PCR and whole genome sequencing (WGS) are time-consuming, costly, and often low throughput. Here, we describe a novel mini-MLST scheme for Eschericha coli as an alternative method for rapid genotyping. Using the proposed mini-MLST scheme, 10,946 existing STs were converted into 1,038 Melting Types (MelTs). To validate the new mini-MLST scheme, in silico analysis was performed on 73,704 strains retrieved from EnteroBase resulting in discriminatory power D = 0.9465 (CI 95% 0.9726-0.9736) for mini-MLST and D = 0.9731 (CI 95% 0.9726-0.9736) for MLST. Moreover, validation on clinical isolates was conducted with a significant concordance between MLST, rep-PCR and WGS. To conclude, the great portability, efficient processing, cost-effectiveness, and high throughput of mini-MLST represents immense benefits, even when accompanied with a slightly lower discriminatory power than other typing methods. This study proved mini-MLST is an ideal method to screen and subgroup large sets of isolates and/or quick strain typing during outbreaks. In addition, our results clearly showed its suitability for prospective surveillance monitoring of emergent and high-risk E. coli clones'.
- MeSH
- bakteriální geny * MeSH
- denaturace nukleových kyselin MeSH
- DNA bakterií chemie genetika MeSH
- DNA primery MeSH
- epidemický výskyt choroby MeSH
- Escherichia coli klasifikace genetika izolace a purifikace MeSH
- genom bakteriální MeSH
- genotypizační techniky * MeSH
- infekce vyvolané Escherichia coli mikrobiologie MeSH
- jednonukleotidový polymorfismus * MeSH
- multilokusová sekvenční typizace metody MeSH
- počítačová simulace MeSH
- polymerázová řetězová reakce metody MeSH
- repetitivní sekvence nukleových kyselin MeSH
- sekvenování celého genomu MeSH
- surveillance populace MeSH
- techniky typizace bakterií * MeSH
- zastoupení bazí MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
Amplicon-based next-generation sequencing (NGS) of immunoglobulin (IG) and T-cell receptor (TR) gene rearrangements for clonality assessment, marker identification and quantification of minimal residual disease (MRD) in lymphoid neoplasms has been the focus of intense research, development and application. However, standardization and validation in a scientifically controlled multicentre setting is still lacking. Therefore, IG/TR assay development and design, including bioinformatics, was performed within the EuroClonality-NGS working group and validated for MRD marker identification in acute lymphoblastic leukaemia (ALL). Five EuroMRD ALL reference laboratories performed IG/TR NGS in 50 diagnostic ALL samples, and compared results with those generated through routine IG/TR Sanger sequencing. A central polytarget quality control (cPT-QC) was used to monitor primer performance, and a central in-tube quality control (cIT-QC) was spiked into each sample as a library-specific quality control and calibrator. NGS identified 259 (average 5.2/sample, range 0-14) clonal sequences vs. Sanger-sequencing 248 (average 5.0/sample, range 0-14). NGS primers covered possible IG/TR rearrangement types more completely compared with local multiplex PCR sets and enabled sequencing of bi-allelic rearrangements and weak PCR products. The cPT-QC showed high reproducibility across all laboratories. These validated and reproducible quality-controlled EuroClonality-NGS assays can be used for standardized NGS-based identification of IG/TR markers in lymphoid malignancies.
- MeSH
- akutní lymfatická leukemie genetika MeSH
- genetické markery genetika MeSH
- genová přestavba T-lymfocytů genetika MeSH
- geny pro imunoglobuliny genetika MeSH
- geny TcR genetika MeSH
- imunoglobuliny genetika MeSH
- lidé MeSH
- receptory antigenů T-buněk genetika MeSH
- referenční standardy MeSH
- rekombinace genetická genetika MeSH
- reprodukovatelnost výsledků MeSH
- reziduální nádor genetika MeSH
- výpočetní biologie metody MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. RESULTS: In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. CONCLUSIONS: Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.
- MeSH
- cystická fibróza genetika MeSH
- lidé MeSH
- plazmidy MeSH
- protein CFTR genetika MeSH
- sekvenční analýza DNA metody MeSH
- tandemové repetitivní sekvence * MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
INTRODUCTION: Human kinesin 14 (KIF14) is one of the 70 prognostic marker genes (so-called Amsterdam profile) previously identified by the microarray of breast carcinomas, and its high transcript expression in tumor specimens indicates a poor prognosis for patients. We performed a pilot study to explore the prognostic and predictive meaning of KIF14 germline genetic variability in breast cancer patients. METHODS: KIF14 coding sequence, including 5' and 3' untranslated regions and overlaps to introns for identification of splicing sites, was analyzed using next-generation sequencing in the testing set of blood DNA samples from 105 breast cancer patients with clinical follow-up. After rigorous evaluation of major allele frequency, haplotype blocks, in silico predicted functional aspects, expression quantitative trait loci, and clinical associations, eight single nucleotide variants were subsequently validated in the evaluation set of 808 patients. RESULTS: Carriers of minor alleles G (rs17448931) or T (rs3806362) had significantly shorter overall survival than wild type homozygotes (p = 0.010 and p = 0.023, respectively) thus successfully replicating the results of the testing set. Both associations remained significant in the multivariate Cox regression analysis, including molecular subtype and stage as covariates (hazard ratio, HR = 1.7, 95% confidence interval (CI) = 1.1-2.8 for rs17448931 and HR = 1.9, CI 1.2-3.0 for rs3806362). DISCUSSION: In conclusion, our preliminary data suggest that minor alleles in rs17448931 and rs3806362 of KIF14 represent candidate biomarkers of poor prognosis of breast cancer patients. After pending validation in independent populations and eventual functional characterization, these candidates might become useful biomarkers in the clinics.
