primer specificity
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HIV-1 reverse transcriptase (RT) possesses both DNA polymerase activity and RNase H activity that act in concert to convert single-stranded RNA of the viral genome to double-stranded DNA that is then integrated into the DNA of the infected cell. Reverse transcriptase-catalyzed reverse transcription critically relies on the proper generation of a polypurine tract (PPT) primer. However, the mechanism of PPT primer generation and the features of the PPT sequence that are critical for its recognition by HIV-1 RT remain unclear. Here, we used a chemical cross-linking method together with molecular dynamics simulations and single-molecule assays to study the mechanism of PPT primer generation. We found that the PPT was specifically and properly recognized within covalently tethered HIV-1 RT-nucleic acid complexes. These findings indicated that recognition of the PPT occurs within a stable catalytic complex after its formation. We found that this unique recognition is based on two complementary elements that rely on the PPT sequence: RNase H sequence preference and incompatibility of the poly(rA/dT) tract of the PPT with the nucleic acid conformation that is required for RNase H cleavage. The latter results from rigidity of the poly(rA/dT) tract and leads to base-pair slippage of this sequence upon deformation into a catalytically relevant geometry. In summary, our results reveal an unexpected mechanism of PPT primer generation based on specific dynamic properties of the poly(rA/dT) segment and help advance our understanding of the mechanisms in viral RNA reverse transcription.
- MeSH
- DNA primery biosyntéza chemie MeSH
- DNA virů MeSH
- HIV reverzní transkriptasa metabolismus fyziologie MeSH
- HIV-1 genetika MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová metody MeSH
- nukleové kyseliny MeSH
- poly A MeSH
- poly U MeSH
- polynukleotidy MeSH
- puriny chemie MeSH
- ribonukleasa H metabolismus MeSH
- RNA virová chemie MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
Detection of Clavibacter michiganensis subsp. michiganensis (Cmm), causing bacterial canker of tomato, was verified using PTA-ELISA and IFAS with PAbs of Neogen Europe Ltd. (UK), and with published and also laboratory-generated PCR primers from the Cmm tomatinase gene. The specificity of this technique was determined with 15 plant-pathogenic and 4 common, saprophytic bacteria. With IFAS, crossreactions were found for Pantoea dispersa, P. agglomerans and Rahnella aquatilis, and with PTA-ELISA for Curtobacterium flaccumfaciens, Pectobacterium atrosepticum and Dickeya sp. Cross-reactions with subspecies other than michiganensis were also found using both methods. Molecular methods were optimized by verification of annealing temperatures and times for both primers. Conditions were finally adjusted to 30 s at 65 degrees C for Dreier's and 10 s at 69 degrees C for our primer set. After this optimization, both primer pairs produced positive reaction only with Cmm. By means of PTA-ELISA and IFAS, Cmm strains were detected at a concentration up to 10(5) CFU/mL and 10(3) CFU/mL, respectively. The PCR test with bacterial cell suspensions reached a sensitivity of 10(3) CFU/mL with our designed primers and 104 CFU/mL with Dreier's primer pair.
- MeSH
- Actinomycetales MeSH
- bakteriologické techniky metody MeSH
- časové faktory MeSH
- DNA primery genetika MeSH
- ELISA metody MeSH
- financování organizované MeSH
- hodnotící studie jako téma MeSH
- hybridizace nukleových kyselin metody MeSH
- nemoci rostlin mikrobiologie MeSH
- polymerázová řetězová reakce metody MeSH
- senzitivita a specificita MeSH
- Solanum lycopersicum mikrobiologie MeSH
- teplota MeSH
- zkřížené reakce MeSH
- Publikační typ
- srovnávací studie MeSH
BACKGROUND: Recently we proposed efficient method to exclude undesirable primers at any stage of amplification reaction, here termed NOPE (NOnsense-mediated Primer Exclusion). According to this method, added oligonucleotide overlapping with the 3'-end of unwanted amplification primer (NOPE oligo) simultaneously provides a template for its elongation. This elongation disrupts specificity of unwanted primer, preventing its further participation in PCR. The suggested approach allows to rationally manage the course of PCR reactions in order to facilitate analysis of complex DNA mixtures as well as to perform multistage PCR bypassing intermediate purification steps. RESULTS: Here we apply NOPE method to DNA library preparation for the high-throughput sequencing (HTS) with the PCR-based introduction of unique molecular identifiers (UMI). We show that NOPE oligo efficiently neutralizes UMI-containing oligonucleotides after introduction of UMI into sample DNA molecules, thus allowing to proceed with further amplification steps without purification and associated loss of starting material. At the same time, NOPE oligo does not affect the efficiency of target PCR amplification. CONCLUSION: We describe a simple, robust and cheap modification of UMI-labeled HTS libraries preparation procedure, that allows to bypass purification step and thus to preserve starting material which may be limited, e.g. circulating tumor DNA, circulating fetal DNA, or small amounts of isolated cells of interest. Furthermore, demonstrated simplicity and robustness of NOPE method should make it popular in various PCR protocols.
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
3rd ed. xxiv, 532 s.
- MeSH
- hypertenze MeSH
- Publikační typ
- monografie MeSH
- Konspekt
- Patologie. Klinická medicína
- NLK Obory
- kardiologie
Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.
- MeSH
- Ascomycota klasifikace genetika izolace a purifikace MeSH
- DNA primery genetika MeSH
- molekulární sekvence - údaje MeSH
- mykorhiza klasifikace genetika izolace a purifikace MeSH
- polymerázová řetězová reakce přístrojové vybavení metody MeSH
- půdní mikrobiologie MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
AIMS: In recent years, beer-spoilage cases from strictly anaerobic bacteria have risen in frequency, in connection with the production of non-pasteurized, non-alcohol and low-alcoholic beers and with the lowering of dissolved oxygen in the packaged beer. Selenomonas lacticifex, found in brewer's yeast and in biofilms covering some surfaces in brewery bottling area, is considered to be a beer-spoilage organism. This study aims to develop S. lacticifex-specific PCR assay. The objective of this study was also evaluation of the specificity and reproducibility of the developed PCR assay in real brewery samples. METHODS AND RESULTS: Three primers (one forward and two reverse) were designed for identification of the strictly anaerobic bacterium S. lacticifex on the basis of the species-specific sequences of the 16S rDNA region. The specificity of the primers was tested against 44 brewery-related non-target micro-organisms that could potentially occur in the same brewery specimens. None of the primer pairs amplified DNA from any of the non-S. lacticifex strains tested including genera from the same family (Pectinatus, Megasphaera, Zymophilus) and the closely related species Selenomonas ruminantium, showing thus 100% specificity. CONCLUSIONS: The PCR assay developed in this study enables the detection of the strictly anaerobic bacterium S. lacticifex in real brewery samples including pitching yeast. SIGNIFICANCE AND IMPACT OF THE STUDY: Selenomonas lacticifex-specific PCR assay developed in this study allows for the extension of the spectra of detected beer-spoilage micro-organisms in brewing laboratories and thus lowering the risk of contamination of the final product.
In the biosynthesis of diverse natural bioactive products the adenylation domains (ADs) of nonribosomal peptide synthetases select specific precursors from the cellular pool and activate them for further incorporation into the scaffold of the final compound. Therefore, the drug discovery programs employing PCR-based screening studies of microbial collections or metagenomic libraries often use AD-coding genes as markers of relevant biosynthetic gene clusters. However, due to significant sequence diversity of ADs, the conventional approach using only one primer pair in a single screening experiment could be insufficient for maximal coverage of AD abundance. In this study, the widely used primer pair A3F/A7R was compared with the newly designed aa194F/aa413R one by 454 pyrosequencing of two sets of actinomycete strains from highly dissimilar environments: subseafloor sediments and forest soil. Individually, none of the primer pairs was able to cover the overall diversity of ADs. However, due to slightly shifted specificity of the primer pairs, the total number and diversity of identified ADs were noticeably extended when both primer pairs were used in a single assay. Additionally, the efficiency of AD detection by different primer combinations was confirmed on the model of Salinispora tropica genomic DNA of known sequence.
- MeSH
- Actinobacteria klasifikace genetika izolace a purifikace MeSH
- DNA primery * MeSH
- interakční proteinové domény a motivy genetika MeSH
- konsenzuální sekvence MeSH
- peptidsynthasy chemie genetika MeSH
- polymerázová řetězová reakce MeSH
- pozičně specifické skórovací matice MeSH
- půdní mikrobiologie MeSH
- rychlé screeningové testy * MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Borrelial specific DNA was examined in a group of 62 patients with different forms of Lyme borreliosis (LB) (32 patients suffered from neuroborreliosis, 19 manifested erythema migrans, and 11 joint involvement). METHODS: Nested-PCR system with five newly derived primers was used in parallel. The study was organized prospectively, the presence of DNA was tested for plasma, CSF, joint fluid and urine before treatment, and plasma, joint fluid and urine were examined after treatment. RESULTS: Before therapy, 36 patients (58.1%) were DNA positive on the whole; 21 positive patients (65.6%) were found in the group of neuroborreliosis, 8 (42.1%) showed signs of skin involvement, and 7 (63.6%) were positive in arthritis. After treatment, 11 patients (36.7%) were positive in neuroborreliosis, 3 (17.6%) in skin form, and 6 (54.5%) in joint form of LB. Among 97 positive amplifications the most frequent target was found in primer corresponding with 16S rDNA (50 samples, 51.5%). Lower but very similar results were obtained with primers for OspA (18 positive amplifications; 18.6%), OspC (13 positive amplifications; 13.4%), and flagellin (13 positive amplifications; 13.4%). There were 11 patients in whom only DNA and no specific antibodies were found. CONCLUSIONS: Specific DNA was found in all clinical groups of LB with similar sensitivity. Examination of the borrelial DNA in urine displayed the same sensitivity as in CSF and had a two times higher sensitivity than in plasma.
- MeSH
- Borrelia burgdorferi komplex genetika MeSH
- DNA bakterií analýza genetika MeSH
- DNA primery MeSH
- infekční artritida mikrobiologie MeSH
- kůže mikrobiologie MeSH
- lidé MeSH
- lymeská nemoc genetika imunologie MeSH
- polymerázová řetězová reakce MeSH
- prospektivní studie MeSH
- protilátky bakteriální analýza MeSH
- senzitivita a specificita MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
Závěrečná zpráva o řešení grantu Interní grantové agentury MZ ČR
Přeruš. str. : tab., il. ; 30 cm
Studie je zaměřena na PCR diagnostiku akutních a chronických forem lymeské boreliózy. Sleduje ověření senzitivity a specifity chromozomálních a plazmidových primerů.; Study is focused on PCR diagnostic of acute and chronic forms of lyme borreliosis. The sensitivity and specificity of chromosomal and plasmide primers will be approved and correlated with a clinical symptoms.
- MeSH
- akutní nemoc MeSH
- chronická nemoc MeSH
- diagnostické techniky molekulární MeSH
- DNA primery analýza MeSH
- infekce bakteriemi rodu Borrelia MeSH
- kožní nemoci etiologie MeSH
- lymeská nemoc diagnóza MeSH
- nemoci kloubů etiologie MeSH
- nemoci nervového systému etiologie MeSH
- plazmidy analýza MeSH
- polymerázová řetězová reakce metody využití MeSH
- senzitivita a specificita MeSH
- Konspekt
- Biochemie. Molekulární biologie. Biofyzika
- NLK Obory
- infekční lékařství
- biologie
- NLK Publikační typ
- závěrečné zprávy o řešení grantu IGA MZ ČR
