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 dia-gnosed 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.

• Klíčová slova
• association, cutaneous melanoma, nucleotide excision repair, single nucleotide polymorphism, single-nucleotide polymorphism,
• MeSH
• DNA vazebné proteiny genetika   MeSH
• dospělí MeSH
• genetická predispozice k nemoci MeSH
• genotyp MeSH
• jednonukleotidový polymorfismus MeSH
• lidé středního věku MeSH
• lidé MeSH
• melanom genetika   MeSH
• nádory kůže genetika   MeSH
• oprava DNA genetika   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH

Recent developments in high-throughput sequencing (HTS) technologies and bioinformatics have drastically changed research in virology, especially for virus discovery. Indeed, proper monitoring of the viral population requires information on the different isolates circulating in the studied area. For this purpose, HTS has greatly facilitated the sequencing of new genomes of detected viruses and their comparison. However, bioinformatics analyses allowing reconstruction of genome sequences and detection of single nucleotide polymorphisms (SNPs) can potentially create bias and has not been widely addressed so far. Therefore, more knowledge is required on the limitations of predicting SNPs based on HTS-generated sequence samples. To address this issue, we compared the ability of 14 plant virology laboratories, each employing a different bioinformatics pipeline, to detect 21 variants of pepino mosaic virus (PepMV) in three samples through large-scale performance testing (PT) using three artificially designed datasets. To evaluate the impact of bioinformatics analyses, they were divided into three key steps: reads pre-processing, virus-isolate identification, and variant calling. Each step was evaluated independently through an original, PT design including discussion and validation between participants at each step. Overall, this work underlines key parameters influencing SNPs detection and proposes recommendations for reliable variant calling for plant viruses. The identification of the closest reference, mapping parameters and manual validation of the detection were recognized as the most impactful analysis steps for the success of the SNPs detections. Strategies to improve the prediction of SNPs are also discussed.

• Klíčová slova
• Bioinformatic, Genomic, Plant, Variant, Virus,
• MeSH
• genom virový genetika   MeSH
• jednonukleotidový polymorfismus * genetika   MeSH
• lidé MeSH
• výpočetní biologie MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• znalosti MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• kineziny * MeSH
• lidé MeSH
• nádorové biomarkery genetika   MeSH
• nádory prsu * patologie   MeSH
• nukleotidy MeSH
• onkogenní proteiny genetika   metabolismus   MeSH
• pilotní projekty MeSH
• prognóza MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• KIF14 protein, human MeSH Prohlížeč
• kineziny * MeSH
• nádorové biomarkery MeSH
• nukleotidy MeSH
• onkogenní proteiny 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
• Názvy látek
• DNA bakterií MeSH
• DNA virů MeSH
• DNA MeSH
• křížová struktura DNA MeSH
• oligonukleotidy MeSH

To facilitate the association studies in complex diseases characterized by hyperhomocysteinemia, we collected structural and frequency data on single-nucleotide polymorphism (SNPs) in 24 genes relating to homocysteine metabolism. Firstly, we scanned approximately 1.2 Mbp of sequence in the NCBI SNP database (dbSNP) build 110 and we detected 1353 putative SNPs with an average in silico genic density of 1:683. Out of 112 putative SNPs in coding regions (cSNPs), we selected a subset of 42 cSNPs and we assessed the applicability of the NCBI dbSNP to the Czech population - a typical representative of European Caucasians - by determining the frequency of the putative cSNPs experimentally by PCR-RFLP or ARMS-PCR in at least 110 control Czech chromosomes. As only 25 of the 42 analyzed cSNPs met the criterion of >/=1% frequency, the positive predictive value of the NCBI data set for our population reached 60%, which is similar to other studies. The correlation of SNP frequency between Czechs and other Caucasians - obtained from NCBI and/or literature - was stronger (r(2)=0.90 for 20 cSNPs) than between Czechs and general NCBI database entries (r(2)=0.73 for 27 cSNPs). Moreover, frequencies of all 20 putative cSNPs, for which data in Caucasians were available, were congruently below or above the 1% frequency criterion both in Czechs and in other Caucasians. In summary, our study shows that the NCBI dbSNP is a useful tool for selecting cSNPs for genetic studies of hyperhomocysteinemia in European populations, although experimental validation of SNPs should be performed, especially if the cSNP entry lacks any frequency data in Caucasians.

• MeSH
• běloši genetika   MeSH
• databáze genetické MeSH
• exprimované sekvenční adresy MeSH
• frekvence genu genetika   MeSH
• genetické markery genetika   MeSH
• genom lidský * MeSH
• homocystein metabolismus   MeSH
• jednonukleotidový polymorfismus genetika   MeSH
• komplementární DNA chemie   MeSH
• lidé MeSH
• veřejný sektor MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Československo MeSH
• Názvy látek
• genetické markery MeSH
• homocystein MeSH
• komplementární DNA MeSH

Corneal ectasias, among which keratoconus (KC) is the single most common entity, are one of the most frequent reasons for corneal grafting in developed countries and a threatening complication of laser in situ keratomileusis. Genome-wide association studies have previously found lysyl oxidase (LOX) and hepatocyte growth factor (HGF) associated with susceptibility to KC development. The aim of our study was to validate the effects of seven single-nucleotide polymorphisms (SNPs) within LOX and HGF over KC. Unrelated Czech cases with KC of European descent (108 males and 57 females, 165 cases in total) and 193 population and gender-matched controls were genotyped using Kompetitive Allele Specific PCR assays. Fisher's exact tests were used to assess the strength of associations. Evidence for association was found for both of the tested loci. It was strongest for rs3735520:G>A near HGF (allelic test odds ratio (OR)=1.45; 95% confidence interval (CI), 1.06-1.98; P=0.018) with A allele being a risk factor and rs2956540:G>C (OR=0.69; 95% CI, 0.50-0.96; P=0.024) within LOX with C allele having a protective effect. This first independent association validation of rs2956540:G>C and rs3735520:G>A suggests that these SNPs may serve as genetic risk markers for KC in individuals of European descent.

• MeSH
• alely MeSH
• běloši MeSH
• genetická predispozice k nemoci MeSH
• genetické asociační studie * MeSH
• genotyp MeSH
• hepatocytární růstový faktor genetika   MeSH
• jednonukleotidový polymorfismus MeSH
• keratokonus genetika   patologie   MeSH
• lidé MeSH
• rohovka patologie   MeSH
• scavengerové receptory - třída E genetika   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• hepatocytární růstový faktor MeSH
• HGF protein, human MeSH Prohlížeč
• OLR1 protein, human MeSH Prohlížeč
• scavengerové receptory - třída E 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
• časopisecké články MeSH
• práce podpořená grantem MeSH
• validační studie MeSH
• Názvy látek
• genetické markery 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 chemie   genetika   MeSH
• DNA fingerprinting MeSH
• fylogeneze MeSH
• jednonukleotidový polymorfismus * MeSH
• molekulární sekvence - údaje MeSH
• nemoci rostlin mikrobiologie   MeSH
• Phytoplasma klasifikace   genetika   izolace a purifikace   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
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Itálie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• ribozomální DNA MeSH
• ribozomální proteiny MeSH
• RNA ribozomální 16S 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.

• Klíčová slova
• External Magnet, Foodborne Pathogen, Sandwich Assay, Small Detection Area, Sterile Buffer,
• 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
• Názvy látek
• aptamery nukleotidové 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.

• Klíčová slova
• Grapevine DNA analysis, Multiplex PCR, STRs, Vitis vinifera L,
• 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
• práce podpořená grantem MeSH
• Názvy látek
• DNA rostlinná MeSH
• genetické markery MeSH