V. M. Kramarov

Bio: V. M. Kramarov is an academic researcher from Russian Academy of Sciences. The author has contributed to research in topics: Hot start PCR & DNA polymerase. The author has an hindex of 4, co-authored 6 publications receiving 90 citations.

A New Approach to Enhanced PCR Specificity

Abstract: A new approach to enhanced specificity and product yield of polymerase chain reaction is proposed. It is based on control of DNA polymerase activity during PCR by changing the magnesium ion concentration, which depends on the temperature of the reaction mixture. A slightly soluble magnesium salt, magnesium oxalate, whose solubility depends on temperature, was used as a source of magnesium ions. During PCR, magnesium oxalate was maintained at saturating concentration by the presence of an insoluble excess of this salt, and the concentration of magnesium ions depended on the salt solubility: binding of magnesium ions at lower temperatures and their release at higher temperatures was shown to affect the DNA polymerase activity and to favor the specific PCR amplification of the target DNA fragment.

[DNA polymerase mediated amplification of DNA fragments using primers with mismatches in the 3'-region].

Abstract: The ability of three thermostable enzymes, Tth, Taq, and Klentaq DNA polymerases, to amplify DNA with primers containing mismatches in the 3'-terminal region was studied. It is shown that Tth polymerase, in contrast to the Taq and Klentaq enzymes, synthesizes equally well DNA with primers perfectly complementary to the template and with those containing mismatches next the 3'-end. The use of Tth DNA polymerase in the polymerase chain reaction was shown to result, in some cases, in a great number of additional, nonspecific DNA fragments as compared with Taq DNA polymerase. This may be due to the ability of Tth polymerase for DNA primer extension even if the 3'-terminal region of the primer contains nucleotides non-complementary to the template. Tth DNA polymerase and a Klentaq/Tth mixture (100:1) can be efficiently used in the amplification of DNA with degenerated primers and primers forming nonperfect duplexes with the template.

Structure of ribosomal rna

Abstract: PERSPECTIVES AND SUMMARy............................................................................................................. 119 rRNA Species-Nomenclature .. ... ... ... ... .... .. ............................ ... ... ..... .. ... .... ... .. ... .... .... . 120 PRIMARY STRUCTURE............................................................................................................................ 121 SECONDARY STRUCTURE....................................................................................................................... 124 General Approaches ....................... ... .. .. ... .. ... .. . . . .. ........................ .... ... .. .... .. .... ... . .. ... .. 124 Description of Secondary-Structure M ode/s......... ... ....... ... ........... ... .. . ....... ..................... .. 129 Phylogenetic Comparison ............ . .. . .... ... .. ... .. . .... ... .. .................... ... ... ... ... .. . ... .... ..... .. .. 136 Experimental Tests.... . . .. .. ....... ........ ... .... .. . .. ... ................ ... .. ... ........ .... .. .... ............... .. . ... 136

Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin

Abstract: A photo-activatable analogue of biotin, N-(4-azido-2-nitrophenyl)-N'-(N-d-biotinyl-3-aminopropyl)-N'-methyl-1,3- propanediamine (photobiotin), has been synthesized and used for the rapid and reliable preparation of large amounts of stable, non-radioactive, biotin-labelled DNA and RNA hybridization probes. Upon brief irradiation with visible light, photobiotin formed stable linkages with single- and double-stranded nucleic acids yielding probes which were purified from excess reagent by 2-butanol extraction and ethanol precipitation. Using single-stranded phage M13 DNA probes chemically labelled with one biotin per 100-400 residues and dot-blot hybridization reactions on nitrocellulose, as little as 0.5 pg (6 X 10(-18) mol) of target DNA was detected colorimetrically by avidin or streptavidin complexes with acid or alkaline phosphatase from three commercial sources. The sensitivity of detection of target RNA in dot-blots and Northern blots was equivalent to that obtained with 32p-labelled DNA probes. Photobiotin was also used for the labelling of proteins with biotin.

[8] Photoaffinity labeling

Abstract: Publisher Summary This chapter discusses the photoaffinity labeling which could be used as a method that allows to unleash the reagent at a particular time and place, when the chemical affinity labeling restricts it. The chapter notes that the possibility that a labile group of appropriate reactivity cannot be incorporated into the ligand molecule without excessive disturbance of the recognition process, there are two limitations to the affinity labeling approach. The first challenge; the range of chemical reactivity of groups that can be incorporated into the ligand is limited by the fact that these groups must not react so rapidly with water that they are destroyed hydrolytically before the ligand that carries them can reach the binding site. And secondly, it is becoming clear that some biological problems require a reagent whose reactivity remains masked until the experimenter chooses to activate it. Both of the two limitations of classical chemical affinity labeling discussed above can in principle be circumvented by the use of a photogenerated reagent.

Predicting Stability of DNA Duplexes in Solutions Containing Magnesium and Monovalent Cations

Abstract: Accurate predictions of DNA stability in physiological and enzyme buffers are important for the design of many biological and biochemical assays. We therefore investigated the effects of magnesium, potassium, sodium, Tris ions, and deoxynucleoside triphosphates on melting profiles of duplex DNA oligomers and collected large melting data sets. An empirical correction function was developed that predicts melting temperatures, transition enthalpies, entropies, and free energies in buffers containing magnesium and monovalent cations. The new correction function significantly improves the accuracy of predictions and accounts for ion concentration, G-C base pair content, and length of the oligonucleotides. The competitive effects of potassium and magnesium ions were characterized. If the concentration ratio of [Mg2+]0.5/[Mon+] is less than 0.22 M−1/2, monovalent ions (K+, Na+) are dominant. Effects of magnesium ions dominate and determine duplex stability at higher ratios. Typical reaction conditions for PCR an...

Peptidyl transferase : protein, ribonucleoprotein, or RNA ?

Abstract: Peptide bonds in proteins are formed on the ribosome by an enzymatic activity called peptidyl transferase (11). The first era of peptidyl transferase research began with the demonstration that this enzyme is an integral part of the ribosome itself (11). Monro and collaborators (18) went on to devise the fragment reaction, a simple peptidyl transferase assay in which the P-site tRNA is replaced by an oligonucleotide, such as CAACCA-(fMet), and the A-site tRNA is replaced by the antibiotic puromycin (Fig. 1). Puromycin contains an aminoacyl-adenosine analog resembling the 3' terminus of an aminoacylated tRNA and donates its amino group to the carbonyl radical of the peptidyl substrate to form a product containing a peptide bond, in this case fMet-puromycin. Although the fragment reaction requires the presence of 33% ethanol or methanol, the authenticity of the fragment reaction is established by the fact that in Escherichia coli ribosomes, it is specifically inhibited by antibiotics, such as chloramphenicol, carbomycin, and lincomycin, which are known to inhibit peptide bond formation specifically under physiological conditions in vitro and in vivo (6). The fragment reaction has very simple requirements; besides the Aand P-site substrates and alcohol, it needs only the 50S ribosomal subunit and magnesium and potassium ions. No mRNA, 30S subunits, translational factors, GTP, ATP, or even intact tRNAs are required. Thus, this system allows one to separate the relatively simple task of peptide bond formation from the highly complex process of translation. More importantly, it is reasonable to expect that catalysis of this model reaction could eventually be carried out with only a small substructure of the 50S ribosomal subunit. Experiments by Staehelin and Monro in the late 1960s showed that even complete 50S particles were not required. By banding subunits in cesium chloride density gradients, Staehelin et al. (24) were able to show that core particles lacking several ribosomal proteins retained high levels of peptidyl transferase activity. Removal of additional proteins, however, eventually resulted in loss of activity, which could be restored by reconstitution of the cores with the split proteins. Similar results were obtained by other groups, stripping proteins with LiCl (17, 19). It was shown that a single split protein, L16, was responsible for restoration of the activity to the protein-depleted cores (17). Moreover, nearly half of the 30-odd large-subunit proteins were completely absent in the resulting active reconstituted particles. Excitement over the possible catalytic role of L16 diminished when it was shown that this protein was required for maintaining the correct three-dimensional folding of the particles (28). In fact, no isolated protein or mixture of RNA-free ribosomal proteins has ever been shown to catalyze the peptidyl transferase reaction. By reconstitution of rRNA with various partial mixtures of 50S proteins, Nierhaus and coworkers (10) narrowed the essential proteins down to a relatively small cadre that included L2, L3, L4, L15, and L16.