We describe a patient with early onset severe axonal Charcot-Marie-Tooth disease (CMT2) with dominant inheritance, in whom Sanger sequencing failed to detect a mutation in the mitofusin 2 (MFN2) gene because of a single nucleotide polymorphism (rs2236057) under the PCR primer sequence. The severe early onset phenotype and the family history with severely affected mother (died after delivery) was very suggestive of CMT2A and this suspicion was finally confirmed by a MFN2 mutation. The mutation p.His361Tyr was later detected in the patient by massively parallel sequencing with a gene panel for hereditary neuropathies. According to this information, new primers for amplification and sequencing were designed which bind away from the polymorphic sites of the patient's DNA. Sanger sequencing with these new primers then confirmed the heterozygous mutation in the MFN2 gene in this patient. This case report shows that massively parallel sequencing may in some rare cases be more sensitive than Sanger sequencing and highlights the importance of accurate primer design which requires special attention.

• Klíčová slova
• Massively parallel sequencing (MPS), mitofusin 2 (MFN2), polymerase chain reaction (PCR), primer mismatch, single nucleotide polymorphism (SNP),
• MeSH
• Charcotova-Marieova-Toothova nemoc diagnóza   genetika   MeSH
• DNA primery MeSH
• GTP-fosfohydrolasy genetika   MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé MeSH
• mitochondriální proteiny genetika   MeSH
• mutace * MeSH
• mutační analýza DNA MeSH
• předškolní dítě MeSH
• rodokmen MeSH
• vysoce účinné nukleotidové sekvenování MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA primery MeSH
• GTP-fosfohydrolasy MeSH
• MFN2 protein, human MeSH Prohlížeč
• mitochondriální proteiny MeSH

In the present study, we have developed an allele-specific primer-polymerase chain reaction (ASP-PCR) for genotyping a single nucleotide polymorphism (SNP) of swine Toll-like receptor 2 (TLR2) (C406G), which is related to the prevalence of pneumonia caused by Mycoplasma hyopneumoniae. We also compared the allele frequency among several pig breeds of Japan and the Czech Republic. Allele-specific primers were constructed by introducing 1-base mismatch sequence before the SNP site. The swine TLR2 C406G mutation was successfully determined by the ASP-PCR using genomic DNA samples in Japan as previously genotyped by a sequencing method. Using the PCR condition determined, genomic DNA samples from pig blood obtained from 110 pigs from 7 different breeds in the Czech Republic were genotyped by the ASP-PCR. The genotyping results from the ASP-PCR were completely matched with the results from the sequencing method. The allele frequency of the swine TLR2 C406G mutation was 27.5% in the Czech Republic and 3.6% in Japan. The C406G mutation was only found in the Landrace breed in Japan, and was almost exclusively found in the Landrace breed in the Czech Republic as well. These results indicated the usefulness of ASP-PCR for detecting a specific SNP for swine TLR2.

• MeSH
• alely * MeSH
• chov MeSH
• DNA primery genetika   MeSH
• DNA genetika   MeSH
• genomika MeSH
• jednonukleotidový polymorfismus * MeSH
• polymerázová řetězová reakce metody   veterinární   MeSH
• prasata genetika   MeSH
• toll-like receptor 2 genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Japonsko MeSH
• Názvy látek
• DNA primery MeSH
• DNA MeSH
• toll-like receptor 2 MeSH

The 18S rRNA gene is a widely used molecular marker for haemogregarines. In recent decades, many primers more or less specific to various haemogregarine genera have been designed. This study applied five commonly used primers targeting the 18S rRNA gene of haemogregarines to blood samples from 168 individuals of nine turtle species captured in Northern Vietnam. Three haemogregarine genera, Haemogregarina, Hemolivia, and Hepatozoon, were detected. Selective specificity of primers EF/ER, HemoFN/HemoRN, and Hemo1/Hemo2 to haemogregarine genera was observed and elucidated by primer-template mismatches. In total, 13 out of 168 turtles (prevalence 7.7%) were both microscopically and PCR positive for haemogregarines. Additionally, a single Heosemys grandis turtle was PCR positive but microscopically negative. Numerous turtles carried mixed infections by various haemogregarines; a single turtle was even coinfected by haemogregarines of all three studied genera: Haemogregarina, Hemolivia, and Hepatozoon. Among the detected haemogregarines, some provided sufficient molecular and morphological data for completing their species diagnosis. Two were described as new species: Haemogregarina cyclemydis sp. nov. from Cyclemys pulchristriata and Hemolivia cruciata sp. nov. from Cuora galbinifrons, so far the first Hemolivia from Southeast Asia. Haemogregarina cuorae Chai and Chen, 1990, required a redescription with reassignment to the genus Hepatozoon Miller, 1908.

• Klíčová slova
• 18S rDNA, Haemogregarina, Hemolivia, Hepatozoon, Vietnam, primer specificity,
• MeSH
• Asijci MeSH
• Eucoccidiida * genetika   MeSH
• fylogeneze MeSH
• lidé MeSH
• RNA ribozomální 18S genetika   MeSH
• sladká voda MeSH
• želvy * genetika   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Vietnam MeSH
• Názvy látek
• RNA ribozomální 18S MeSH

UNLABELLED: The BsmI polymorphism in the VDR gene has been extensively investigated by PCR and restriction digestion in bone genetics. A SNP within the corresponding region for the previously published reverse primer was observed and confirmed by DNA sequencing. BsmI mis-genotyping caused by this SNP could confound genetic findings. INTRODUCTION: By analyzing the FokI, BsmI, ApaI, and TaqI polymorphisms in the vitamin D receptor (VDR) gene, we observed a significantly different genotype distribution in the BsmI polymorphic locus with a deviation from Hardy-Weinberg equilibrium. One of the reasons for polymerase chain reaction (PCR) non-amplification may be a mismatched base at the primer binding region. Therefore, the aim of this study was to analyse whether a single nucleotide polymorphism (SNP), which has been recently described as TruI, is responsible for the discrepancy between expected and observed genotype frequencies. MATERIALS AND METHODS: The VDR genotypes were identified in a cohort of 165 peri- and postmenopausal women of white origin. PCR amplification was carried out using the originally published primers and followed by restriction cleavage. The BsmI genotypes were further verified with a reverse primer external to the original binding site. The presence of the TruI polymorphism under the previously published reverse primer was confirmed by a restriction digestion and DNA sequencing. In Bb subjects, the colocalization of b allele with the TruI restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both BsmI and TruI restriction enzymes. RESULTS: The BsmI reanalysis with an external primer provided a higher number of heterozygous subjects with a proportionally smaller number of BB subjects, and the changed genotype distribution was under Hardy-Weinberg equilibrium (BB, 31; Bb, 80; bb, 54; r = 0.0203; p = 0.90). In our primary analysis, the presence of the TruI polymorphism led to a drop out of b allele during PCR amplification and thus to the false prevalence of BB genotypes (BB, 50; Bb, 61; bb, 54; r = 11.17; p = 0.01). CONCLUSION: The SNP in the region corresponding to the reverse primer may lead to BsmI mis-genotyping, which may have confounded some previous genetic studies.

• MeSH
• alely MeSH
• genotyp MeSH
• heterozygot MeSH
• homozygot MeSH
• jednonukleotidový polymorfismus * MeSH
• lidé MeSH
• modely genetické MeSH
• polymorfismus genetický MeSH
• receptory kalcitriolu genetika   MeSH
• restrikční endonukleasy typu II metabolismus   MeSH
• sekvenční analýza DNA MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• endodeoxyribonuclease BsmI MeSH Prohlížeč
• receptory kalcitriolu MeSH
• restrikční endonukleasy typu II MeSH

Four 6-substituted 4-amino-pyrimido[4,5-b]indole 2'-deoxyribonucleoside triphosphates (dA(BX)TPs) were prepared by glycosylation of 4,6-dichloropyrimidoindole followed by ammonolysis, cross-coupling and triphosphorylation. They were found to be moderate to good substrates for DNA polymerases in primer extension. They also exerted fluorescence with emission maxima 335-378nm. When incorporated to oligonucleotide probes, they did not show significant mismatch discrimination but the 6-benzofuryl 4-amino-pyrimido[4,5-b]indole nucleotide displayed a useful sensitivity to protein binding in experiment with SSB protein.

• Klíčová slova
• DNA polymerase, Deazapurines, Fluorescence, Nucleosides, Nucleotides, Pyrimidoindoles,
• MeSH
• chybné párování bází MeSH
• deoxyadeninnukleotidy chemická syntéza   chemie   metabolismus   MeSH
• deoxyribonukleosidy chemická syntéza   chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• Escherichia coli chemie   metabolismus   MeSH
• fluorescenční barviva chemická syntéza   chemie   metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• indoly chemická syntéza   chemie   metabolismus   MeSH
• oligonukleotidové sondy chemická syntéza   chemie   metabolismus   MeSH
• proteiny z Escherichia coli chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 2'-deoxyadenosine triphosphate MeSH Prohlížeč
• deoxyadeninnukleotidy MeSH
• deoxyribonukleosidy MeSH
• DNA vazebné proteiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• fluorescenční barviva MeSH
• indoly MeSH
• oligonukleotidové sondy MeSH
• proteiny z Escherichia coli MeSH
• SSB protein, E coli MeSH Prohlížeč

Differential scanning calorimetry (DSC) was used to measure the thermodynamic changes associated with translesion synthesis across major lesion induced in DNA by antitumor oxaliplatin [1,2-d(GG) intrastrand cross-link]. Insertion of matched nucleotides dC at the primer terminus (across unique 3'- or 5'-dG in the unplatinated template) and subsequent extensions resulted in an incremental increase in thermodynamic parameters. In contrast, incorporation of dC opposite either platinated dG in the intrastrand cross-link formed in the template strand and subsequent extensions by one nucleotide resulted only in little changes in thermodynamics. A similar thermodynamic delay was observed for a control template primer containing a dG:dT mismatch across 3'- or 5'-dG in the template and subsequent Watson-Crick primer extensions. The thermodynamic scarcity generated by either the lesion or mismatches was not localized but extended to the 5'-downstream sites, which may be connected with the phenomenon termed "short-term memory" of replication errors retained by some DNA polymerases responding to DNA damages or mismatches. Interestingly, formation of the 1,2-d(GG) intrastrand cross-link of oxaliplatin altered the overall DSC profiles of the dG:dT mismatch template/primers only in a very small extent. While addition of matched nucleotide dC across either dG in the template strand was thermodynamically favored over the presence of a mismatched dT (ΔΔG(0)(310) was 7.6 or 6.8 kJ mol(-1), ΔΔH was 14 or 49 kJ mol(-1)), no such thermodynamic advantage was observed with the 1,2-d(GG) intrastrand cross-link of oxaliplatin at these positions (ΔΔG(0)(310) was 2.8 or -0.3 kJ mol(-1), ΔΔH was 4 or 9 kJ mol(-1)). The equilibrium thermodynamic data also provide insight into the processes associated with misincorporation of incorrect nucleotides during replication bypass across major cross-links of antitumor oxaliplatin. On the other hand, besides thermodynamic effects also kinetic factors play an important role in the processing of the cross-links of antitumor platinum drugs. The impact of the two effects in overall processing DNA adducts by a particular DNA polymerase will depend on its nature.

• MeSH
• chemické modely MeSH
• diferenciální skenovací kalorimetrie metody   MeSH
• molekulární struktura MeSH
• organoplatinové sloučeniny chemická syntéza   chemie   farmakologie   MeSH
• oxaliplatin MeSH
• protinádorové látky chemická syntéza   chemie   farmakologie   MeSH
• sekvence nukleotidů MeSH
• termodynamika MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• organoplatinové sloučeniny MeSH
• oxaliplatin MeSH
• protinádorové látky MeSH

SUMMARY: PRIMEX (PRImer Match EXtractor) can detect oligonucleotide sequences in whole genomes, allowing for mismatches. Using a word lookup table and server functionality, PRIMEX accepts queries from client software and returns matches rapidly. We find it faster and more sensitive than currently available tools. AVAILABILITY: Running applications and source code have been made available at http://bioinformatics.cribi.unipd.it/primex

• MeSH
• algoritmy * MeSH
• genom * MeSH
• oligonukleotidy genetika   MeSH
• sekvenční analýza DNA metody   MeSH
• sekvenční seřazení metody   MeSH
• software * MeSH
• uživatelské rozhraní počítače MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• srovnávací studie MeSH
• validační studie MeSH
• Názvy látek
• oligonukleotidy MeSH

Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.

• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• DNA metabolismus   MeSH
• fluorescenční spektrometrie MeSH
• hybridizace nukleových kyselin * MeSH
• kationty MeSH
• lidé MeSH
• lipidy chemie   MeSH
• nádorové buněčné linie MeSH
• nukleotidy chemická syntéza   chemie   MeSH
• oligonukleotidové sondy metabolismus   MeSH
• rozpouštědla chemie   MeSH
• sekvence nukleotidů MeSH
• sloučeniny boru chemie   MeSH
• teplota MeSH
• thiofeny chemie   MeSH
• vazba proteinů MeSH
• viskozita MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA-dependentní DNA-polymerasy MeSH
• DNA MeSH
• kationty MeSH
• lipidy MeSH
• nukleotidy MeSH
• oligonukleotidové sondy MeSH
• rozpouštědla MeSH
• sloučeniny boru MeSH
• thiofeny MeSH

The introduction of primed in situ labeling (PRINS) into plant cytogenetics provided a novel means for fast and highly specific visualization of DNA sequences on chromosomes and in interphase nuclei. Although the technique does not reach the sensitivity of fluorescence in situ hybridization that is needed for detection of single-copy targets, it is superior in its speed and simplicity. Thus, the main applications of PRINS include fluorescent labeling of repeated DNA sequences, such as ribosomal DNA and satellite repeats, which are used to discriminate individual chromosome types within karyotypes and to assess the purity of chromosome fractions separated by flow-sorting. Recently, an application of PRINS for the discrimination of sequence subfamilies of satellite repeats has been developed that takes advantage of the sensitivity of Taq polymerase to mismatches between 3'-end of the primer and the template sequences. This approach allows the distinguishing of sequences that differ in only a few nucleotides and has proved to be valuable for studies of satellite DNA evolution in plants.

• MeSH
• chromozomy rostlin genetika   metabolismus   MeSH
• in situ značení DNA s primerem metody   MeSH
• vikev genetika   MeSH
• Publikační typ
• časopisecké články MeSH

The use of HLA-DRB1 and -DQB1 polymerase chain reaction-sequence-specific primer (PCR-SSP) typing at different levels of resolution for MLR prediction was assessed in 54 HLA-A and -B matched donor/recipient unrelated pairs and 89 HLA-A and -B identical siblings. Graft-versus-host (GvH) direction one-way MLR was evaluated unless stated otherwise. The typing of DRB1 alleles satisfactory for MLR prediction in HLA identical siblings (P = 0.0015) does not appear to be sufficient in matched unrelated pairs (P = 0.2407). Using more discriminatory PCR-SSP typing, the disparity in DRB1 allelic subtypes was predominantly found in the category of DRB1 allele compatible, MLR positive unrelated pairs. Besides, DRB1 allelic subtype mismatches were revealed in five of the forty-one DRB1 allele compatible, MLR negative unrelated pairs. More discriminatory typing made the correlation between DRB1 compatibility and MLR negativity extremely significant (P = 0.0001). As for these five exceptional cases, the reciprocal host-versus-graft (HvG) direction MLR was considered, too. This allowed HLA-D disparity to be disclosed in two of them. An uninterpretable result reflecting defective MLR reactivity occurred in one case. Negative reciprocal MLR in the last two DRB1 allelic subtype incompatible pairs is hardly to explain without postulation of MLR silent DRB1 allelic subtype mismatches. An analysis in unrelated pairs showed a role of some DQB1 gene products in the proliferative response too. GvH direction positive MLR was found in two HLA identical siblings among the 89 tested. The DPB1 incompatibility detected in one of them could be a potential cause of proliferative response but MLR reactivity in the other, DPB1 identical, pair cannot be interpreted easily.

• MeSH
• alely MeSH
• dárci tkání MeSH
• HLA-A antigeny genetika   MeSH
• HLA-B antigeny genetika   MeSH
• HLA-D antigeny genetika   MeSH
• HLA-DQ antigeny genetika   MeSH
• HLA-DQ beta řetězec MeSH
• HLA-DR antigeny genetika   MeSH
• HLA-DRB1 řetězec MeSH
• hodnotící studie jako téma MeSH
• lidé MeSH
• nukleární rodina MeSH
• polymerázová řetězová reakce metody   MeSH
• prediktivní hodnota testů MeSH
• test smíšené lymfocytární kultury * MeSH
• testování histokompatibility metody   MeSH
• transplantace kostní dřeně imunologie   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• HLA-A antigeny MeSH
• HLA-B antigeny MeSH
• HLA-D antigeny MeSH
• HLA-DQ antigeny MeSH
• HLA-DQ beta řetězec MeSH
• HLA-DQB1 antigen MeSH Prohlížeč
• HLA-DR antigeny MeSH
• HLA-DRB1 řetězec MeSH