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Selective Amplification (selective + amplification)
Selected AbstractsSelective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3, terminus to reduce false-positive signalsLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2000K. Koo A reverse transcription PCR (RT,PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3, terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 °C or 45 °C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 °C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene. [source] Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)ENVIRONMENTAL MICROBIOLOGY, Issue 1 2007Guus Roeselers Summary We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus -like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime. [source] Color responses of the human lateral geniculate nucleus: selective amplification of S-cone signals between the lateral geniculate nucleno and primary visual cortex measured with high-field fMRIEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2008Kathy T. Mullen Abstract The lateral geniculate nucleus (LGN) is the primary thalamic nucleus that relays visual information from the retina to the primary visual cortex (V1) and has been extensively studied in non-human primates. A key feature of the LGN is the segregation of retinal inputs into different cellular layers characterized by their differential responses to red-green (RG) color (L/M opponent), blue-yellow (BY) color (S-cone opponent) and achromatic (Ach) contrast. In this study we use high-field functional magnetic resonance imaging (4 tesla, 3.6 × 3.6 × 3 mm3) to record simultaneously the responses of the human LGN and V1 to chromatic and Ach contrast to investigate the LGN responses to color, and how these are modified as information transfers between LGN and cortex. We find that the LGN has a robust response to RG color contrast, equal to or greater than the Ach response, but a significantly poorer sensitivity to BY contrast. In V1 at low temporal rates (2 Hz), however, the sensitivity of the BY color pathway is selectively enhanced, rising in relation to the RG and Ach responses. We find that this effect generalizes across different stimulus contrasts and spatial stimuli (1-d and 2-d patterns), but is selective for temporal frequency, as it is not found for stimuli at 8 Hz. While the mechanism of this cortical enhancement of BY color vision and its dynamic component is unknown, its role may be to compensate for a weak BY signal originating from the sparse distribution of neurons in the retina and LGN. [source] Single nucleotide polymorphisms in succinate dehydrogenase subunits and citrate synthase genes: association results for impaired spermatogenesisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2007Sandra Bonache Abstract Evaluation of the possible implication of the SDHA, SDHB, SDHC, SDHD and CS genes in non-obstructive male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. blastn searches in the genomic sequence NCBI database evidenced the presence of highly homologous sequences elsewhere on the genome that can interfere with polymerase chain reaction experiments. Therefore, a careful design of the analytical strategy to search for sequence variants was performed. In this report, we provide primer sequences that allowed selective amplification of coding and immediate flanking regions of the five genes. Fifty-five sequence variations in the five genes were identified in infertile and normozoospermic fertile individuals as controls and only one of them (SDHA c.456+32G>A) showed significant genotype association with impairment of sperm production. Moreover, new single nucleotide polymorphisms identified should be useful in future association studies for other human diseases related to nuclear-encoded genes, leading to mitochondrial respiratory chain activity impairment revealing the physiological role of these genes. [source] Inter-laboratory evaluation of three flagellin PCR/RFLP methods for typing Campylobacter jejuni and C. coli: the CAMPYNET experienceJOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2003C.S. Harrington Abstract Aims: To compare typeability, discriminatory ability, and inter-laboratory reproducibility of three flagellin PCR/RFLP (fla typing) methods previously described for Campylobacter. Methods and Results: The sample set (n = 100) was diverse, including both C. jejuni (n = 85) and C. coli (n = 15). Two of the three flaA typing methods amplified flaA alone, whereas one, a multiplex assay, amplified flaB in addition to flaA. DdeI restriction enzyme was employed for all methods, but HinfI was also investigated. 98,100% typeability was obtained for flaA-based methods, but only 93% for the multiplex assay, due to inconsistent amplification of a non-specific product. In addition, there appeared to be selective amplification of flaA over flaB. More DdeI types were generated using a longer flaA PCR amplicon, whilst additional use of HinfI increased the number of types by ca 25%. Inter-laboratory reproducibility for both flaA-based methods was defined at 100%. Conclusions:Fla typing requires standardization with respect to PCR primers and restriction enzymes. This study identified an assay, employing the full flaA gene and DdeI digestion, as an appropriate method on which to standardize. 100% inter-laboratory reproducibility was demonstrated using that method. Significance and Impact of the Study: This work should facilitate progress towards inter-laboratory standardization of fla typing. [source] Identification of Novel Target Genes of the Bone-Specific Transcription Factor Runx2,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2004Michael Stock Abstract Fifteen putative transcriptional target genes regulated by the osteogenic transcription factor Runx2 were identified by cDNA microarray and differential hybridization techniques. Expression pattern and regulation of one gene, Pttg1ip, was analyzed in detail. Introduction: The transcription factor Runx2 is a key regulator of osteoblast development and plays a role in chondrocyte maturation. The identification of transcriptional target genes of Runx2 may yield insight into how osteoblastic differentiation is achieved on a molecular level. Materials and Methods: Using a differential hybridization technique (selective amplification through biotin and restriction-mediated enrichment [SABRE]) and cDNA microarray analysis, 15 differentially expressed genes were identified using mRNA from C3H 10T1/2 cells with constitutive and inducible overexpression of Runx2. Results and Conclusions: Among the 15 genes identified, 4 encode the extracellular matrix proteins Ecm1, Mgp, Fbn5, and Osf-2, three represent the transcription factors Esx1, Osr1, and Sox9, whereas others were Ptn, Npdc-1, Hig1, and Tem1. The gene for Pttg1ip was upregulated in Runx2-expressing cells. Pttg1ip is widely expressed during development, but at highest levels in limbs and gonads. The Pttg1ip promoter binds Runx2 in a sequence specific manner, and Runx2 is able to transactivate the Pttg1ip promoter in MC3T3-E1 cells. Therefore, Pttg1ip is likely to be a novel direct transcriptional target gene of Runx2. In conclusion, the genes identified in this study are important candidates for mediating Runx2 induced cellular differentiation. [source] Selective amplification of bacterial RNA: use of a DNA primer containing mismatched bases near its 3, terminus to reduce false-positive signalsLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2000K. Koo A reverse transcription PCR (RT,PCR) method designed to reduce false-positive results due to the co-amplification of contaminating genomic DNA is reported. Feasibility of the method was evaluated using 16S rRNA sequences specific to Bacillus cereus. A DNA oligonucleotide primer, consisting of 22-bases containing three consecutive mismatched bases near its 3, terminus (primer B16RT), was used for reverse transcription and in subsequent cDNA amplification. Specific rRNA was reverse transcribed at low temperature (40 °C or 45 °C) in the presence of primer B16RT. As subsequent PCR using primer B16RT at high (62 °C) annealing temperatures is not nearly as efficient as amplification using the specific primer, amplification of genomic DNA was hindered relative to the amplification of cDNA. The method was readily adapted to the selective amplification of mRNA of the Listeria monocytogenes listeriolysin O (hly) gene. [source] Estimating Genetic Diversity in Durum and Bread Wheat Cultivars from Turkey using AFLP and SAMPL MarkersPLANT BREEDING, Issue 1 2008S. Alt Abstract Since 1925, more than 100 wheat varieties were developed and released in Turkey, and many more were introduced from abroad, but no systematic analysis of their genetic diversity has been performed yet. In this research, a total of 34 domestic and foreign cultivars (12 durum and 22 bread wheats), released in Turkey between 1936 and 2000, were fingerprinted by means of five amplified fragment length polymorphism and three selective amplification of microsatellite polymorphic loci (SAMPL) primer combinations, to evaluate their genetic variation and to determine the existence of cultivar-specific bands. Among the 344 amplicons scored, 214 were polymorphic. The primer combination EACG/MAGG yielded the highest number and the primer combination SAMPL,6/M AGA produced the lowest number of polymorphic bands. Most cultivars were molecularly very similar, although a few distinct ones (the durum wheat ,Kunduru,1149' and the bread wheat ,,kizce,96') were also identified. Seven cultivar-specific markers for different bread wheat cultivars (,Golia', ,Seri,82', ,Adana,99', ,Pandas' and ,Sertak,52') and six cultivar-specific markers for durum wheat cv ,Kunduru' were observed. Our results show that genetic diversity among old and present,day wheat cultivar commonly grown in Turkey is limited. [source] Clone differentiation and varietal identification by means of SSR, AFLP, SAMPL and M-AFLP in order to assess the clonal selection of grapevine: the case study of Manto Negro, Callet and Moll, autochthonous cultivars of MajorcaANNALS OF APPLIED BIOLOGY, Issue 2 2010E. Cretazzo Clonal selection is the most worldwide spreading method to improve the performance of wine grapevine (Vitis vinifera) cultivars. In the special case of autochthonous varieties with only local interest, such as Manto Negro, Callet and Moll in Majorca (Spain), good knowledge of their genotypic resources is helpful to assess the development of viticultural and enological potentialities. In this study, 94 vines (including Manto Negro, Callet, Moll and wrongly identified samples) were analysed by means of genetic markers. Several varietal identification mistakes related to the clonal selection in Majorca were detected by the amplification of 33 simple sequence repeats (SSRs) or microsatellite loci, mainly because of the close genetic relationships between Manto Negro, Callet, Moll and other varieties. A very low degree of intravarietal genetic diversity, possibly related to high incidence of virus infections, was shown in all three varieties. However, analysis by amplified fragment length polymorphism (AFLP), selective amplification of microsatellite loci (SAMPL) and microsatellite-amplified fragment length polymorphism (M-AFLP) was suitable for clone genetic discrimination. More than 900 scorable bands were obtained by nine primer combinations. The most efficient system to detect intravarietal genetic differences was M-AFLP, which generated the highest number of polymorphic bands. The use of these markers allowed clustering vines in homogeneous groups, providing essential information about sanitation strategies in order to obtain certified propagation material. [source] | |||||||||||||