Efficiency of Peptide Nucleic Acid-Directed PCR Clamping and Its Application in the Investigation of Natural Diets of the Japanese Eel Leptocephali

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source

Detection of Ki-ras mutations in tissue and plasma samples of patients with pancreatic cancer using PNA-mediated PCR clamping and hybridisation probes

In the present study, we combined the PCR-clamping approach with melting curve analysis using mutant specific hybridisation probes and wild-type specific peptide nucleic acids (PNAs) to determine the genotypes of the most frequent point mutation in codon 12 of the proto-oncogene Ki-ras in tissue and plasma samples of patients with pancreatic cancer. The sensitivity of our assay was 1–5 × 10−5. The melting curve analysis of tissue samples of four patients revealed two valine mutations, one none-valine mutation and one wild-type sequence. Ki-ras alterations were found in 28% of DNAs (18 out of 64) of nonrelated plasma samples of 10 patients with ductal adenocarcinoma of the pancreas. The valine mutation was the predominantly detected gene alteration (83%). Out of ten patients investigated, four patients (40%) became positive during clinical observation with respect to Ki-ras mutation. All four patients exhibited progressive disease and high levels of tumour marker CA 19-9. In conclusion, the one-step procedure discribed may be a useful clinical tool for analysing Ki-ras point mutations in tissue and plasmas samples. In addition, this method can be adapted for simultanous detection of multiple mutations and quantitation

At the intersection of globalization and "civilizational originality' : cultural production in Putin's Russia

This special issue originates from a transnational collaboration of scholars in philology, comparative literature, social theory, sociology, anthropology, ethnography, and media studies. The collection strives to advance a research agenda built on the nexus of three intellectual and academic domains: post-Soviet Russian cultural studies', the research paradigm put forward by Cultural Studies, as well as empirical methods developed in sociology. The collection illustrates the importance of expanding the experience of Cultural Studies beyond its established spheres of national investigation, while it also speaks to the necessity to re-evaluate the hegemony of the English-language academic and cultural production on the global scale. The collection offers insights into the gamut of cultural practices and institutional environments in which Russian cultural production happens today. It shows how cultural industries and institutions in Russia are integrated into the global marketplace and transnational communities, while they also draw on and contribute to local lives and experiences by trying to create an autonomous space for symbolic production at personal and collective levels. Through diverse topics, the issue sheds light on the agency, i.e. practitioners and participants, creators and consumers, of Russian cultural production and the neoliberal practices implemented on creative work and cultural administration in Russia today. The Introduction outlines the development of academic studies on Russian cultural practices since 1991; describes main political developments shaping the cultural field in Putin's Russia; and, finally, identifies the Cultural Studies debates the editors of the collection find most productive for investigations of Russia, i.e. the instrumentalization of culture and culture as resource. Relocated in an analysis of a post-socialist society, these conceptualisations seem increasingly problematic in a situation where local and federal policies governing cultural and creative work focus simultaneously on marketization and on nationalism as the main tools of legitimizing the federal government.Peer reviewe

A simple model for gene targeting.

Sequence-specific binding to genomic-size DNA sequences by artificial agents is of major interest for the development of gene-targeting strategies, gene-diagnostic applications, and biotechnical tools. The binding of one such agent, peptide nucleic acid (PNA), to a randomized human genome has been modeled with statistical mass action calculations. With the length of the PNA probe, the average per-base binding constant k(0), and the binding affinity loss of a mismatched base pair as main parameters, the specificity was gauged as a "therapeutic ratio" G = maximum safe [PNA](tot)/minimal efficient [PNA](tot). This general, though simple, model suggests that, above a certain threshold length of the PNA, the microscopic binding constant k(0) is the primary determinant for optimal discrimination, and that only a narrow range of rather low k(0) values gives a high therapeutic ratio G. For diagnostic purposes, the value of k(0) could readily be modulated by changing the temperature, due to the substantial Delta H degrees associated with the binding equilibrium. Applied to gene therapy, our results stress the need for appropriate control of the binding constant and added amount of the gene-targeting agent, to meet the varying conditions (ionic strength, presence of competing DNA-binding molecules) found in the cell