High resolution melting (HRM) is a convenient method for gene scanning as well as genotyping of individual and multiple single nucleotide polymorphisms (SNPs). This rapid, simple, closed-tube, homogenous, and cost-efficient approach has the capacity for high specificity and sensitivity, while allowing easy transition to high-throughput scale. In this paper, we provide examples from our laboratory practice of some problematic issues which can affect the performance and data analysis of HRM results, especially with regard to reference curve-based targeted genotyping. We present those examples in order of the typical experimental workflow, and discuss the crucial significance of the respective experimental errors and limitations for the quality and analysis of results. The experimental details which have a decisive impact on correct execution of a HRM genotyping experiment include type and quality of DNA source material, reproducibility of isolation method and template DNA preparation, primer and amplicon design, automation-derived preparation and pipetting inconsistencies, as well as physical limitations in melting curve distinction for alternative variants and careful selection of samples for validation by sequencing. We provide a case-by-case analysis and discussion of actual problems we encountered and solutions that should be taken into account by researchers newly attempting HRM genotyping, especially in a high-throughput setup.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

No abstract

Background/ObjectivesGenome-wide association studies (GWAS) have identified many loci associated with body mass index (BMI) in many different populations. Variants in the FTO locus are reported to be one of the strongest genetic predictors of obesity. Recent publications pointed also to a topologically associated domain (TAD) which is identified as a novel region affecting BMI. The TAD area encompasses the IRXB cluster (IRX3, IRX5, IRX6), FTO and RPGRIP1L genes.Subjects/MethodsIn this study, we investigated the relationship between variation of the FTO and IRX genes and obesity in Poles. We presented a case—control association analysis (normal versus overweight and/or obesity group) of Polish adult individuals (N = 5418). We determined whether or not the chromosomal region 16:53 500 000–55 500 000 contains polymorphic variants which are correlated with BMI in Polish population, including sex and age stratified analysis.ResultsThe obtained results showed that the problem of weight-height abnormalities differently affects populations of Polish women and men (χ2 = 187.1; p<0.0001). From 353 SNPs enrolled to this study, 86 were statistically significant (highest χ2 = 15.72; p = 7.35E-05 observed for rs1558902). Linkage disequilibrium (LD) analysis revealed 61 blocks in the tested region of chromosome 16, with 24 SNPs located within the same block (block 8) of approximately 40 kb, in almost complete LD (|D’|>0.98, r2>0.80). We confirmed presence of the genetic susceptibility loci located in intron 1 of the FTO gene, which were correlated with BMI in our study group. For the first time, our analyses revealed strong association of FTO intronic variants (block 8) with overweight in group of men only. We have also identified association of the IRX region with overweight and/or obesity in Polish individuals.ConclusionOur study demonstrated how tested SNPs make differential contributions to obesity and overweight risk. We revealed sex dependent differences in the distribution of tested loci which are associated with BMI in the population of Poles.

The aim of the present study was to define the mtDNA variability of Polish population and to visualize the genetic relations between Poles. For the first time, the study of Polish population was conducted on such a large number of individuals (5852) representing administrative units of both levels of local administration in Poland (voivodeships and counties). Additionally, clustering was used as a method of population subdivision. Performed genetic analysis, included FST, MDS plot, AMOVA and SAMOVA. Haplogroups were classified and their geographical distribution was visualized using surface interpolation maps. Results of the present study showed that Poles are characterized by the main West Eurasian mtDNA haplogroups. Furthermore, the level of differentiation within the Polish population was quite low but the existing genetic differences could be explained well with geographic distances. This may lead to a conclusion that Poles can be considered as genetically homogenous but with slight differences, highlighted at the regional level. Some patterns of variability were observed and could be explained by the history of demographic processes in Poland such as resettlements and migrations of women or relatively weaker urbanisation and higher rural population retention of some regions.

No abstract

No abstract

BackgroundMultidrug resistance-associated protein 1 (MRP1), encoded by the ABCC1 gene, is an ATP-binding cassette transporter mediating efflux of organic anions and xenobiotics; its overexpression leads to multidrug resistance. In this study, 30 exons (from 31 in total) of the ABCC1 gene as well as and their flanking intron sequences were screened for genetic variation, using the High Resolution Melting (HRM) method, for 190 healthy volunteers representing the Polish population. Polymorphism screening is an indispensable step in personalized patient therapy. An additional targeted SNP verification study for ten variants was performed to verify sensitivity of the scanning method.ResultsDuring scanning, 46 polymorphisms, including seven novel ones, were found: one in 3’ UTR, 21 in exons (11 of them non-synonymous) and 24 in introns, including one deletion variant. These results revealed some ethnic differences in frequency of several polymorphisms when compared to literature data for other populations. Based on linkage disequilibrium analysis, 4 haplotype blocks were determined for 9 detected polymorphisms and 12 haplotypes were defined. To capture the common haplotypes, haplotype-tagging single nucleotide polymorphisms were identified.ConclusionsTargeted genotyping results correlated well with scanning results; thus, HRM is a suitable method to study genetic variation in this model. HRM is an efficient and sensitive method for scanning and genotyping polymorphic variants. Ethnic differences were found for frequency of some variants in the Polish population compared to others.Thus, this study may be useful for pharmacogenetics of drugs affected by MRP1-mediated efflux.Electronic supplementary materialThe online version of this article (doi:10.1186/s12863-015-0271-3) contains supplementary material, which is available to authorized users.