[Comparative analysis of LAMP and Real Time PCR methods to detect pathogens of glanders and meliodosis.]

Results of detection of Burkholderia mallei and Burkholderia pseudomallei DNA strains by LAMP (Loop-mediated Isothermal Amplification) and Real Time PCR are shown. It has been revealed that, in Real Time PCR, primers steadily detected DNA of those microorganism for the sequences of which they were designed. The above mentioned primers did not detect DNA of heterologous strains. During LAMP method no set of primers showed high analytical sensitivity and specificity. Primers did not detected DNA of all the strains under research to target genes of which they were not intended, but they were capable of directing the synthesis of fragments of genes of heterologous strains. Furthermore, it was difficult to reach the same results during repeated experiments. Failures during LAMP may occur due to existence of GC-reach regions in Burkholderia mallei and Burkholderia pseudomallei genomes and due to emergence of secondary structures in isothermical conditions. It is recommended to use Real Time PCR in order to detect pathogens, in case of such matrixes as Burkholderia mallei and Burkholderia pseudomallei DNAs which are very complicated for LAMP.

The use of loop-mediated isothermal DNA amplification for the detection and identification of the anthrax pathogen.

The results of detection and identification of Bacillus anthracis strains in loop-mediated isothermal DNA amplification (LAMP) reaction performed under optimized conditions with original primers and thermostable DNA polymerase are presented. Reproducible LAMP-based detection of chromosomal and plasmid DNA targets specific for B. anthracis strains has been demonstrated. No cross reactions with DNA from bacterial strains of other species of the B. cereus group were detected. The development of tests for anthrax-pathogen detection based on the optimized reaction of loop isothermal DNA amplification is planned. These tests will be convenient for clinical studies and field diagnostics due to the absence of requirements for sophisticated equipment.

DNA ligases from thermophilic bacteria enhance PCR amplification of long DNA sequences.

Bacterial NAD-dependent Taq and Tth DNA ligases are capable of significantly increasing the yield of long PCR products when the amplification is carried out using bacterial family A DNA polymerases, e.g. Taq or Tth DNA polymerases, or with enzymatic blends containing these polymerases. We also show that Taq and Tth DNA ligases improve the results of PCR in the absence of NAD and therefore in the absence of DNA ligase activity. These observations suggest that bacterial DNA ligases can interact with these DNA polymerases, presumably as accessory proteins, thereby enhancing the efficiency of DNA polymerization.

Cloning and molecular modeling of duodenase with respect to evolution of substrate specificity within mammalian serine proteases that have lost a conserved active-site disulfide bond.

Mammalian serine proteases such as the chromosome 14 (Homo sapiens, Mus musculus) located granzymes, chymases, cathepsin G, and related enzymes including duodenase collectively represent a special group within the chymotrypsin family which we refer to here as "granases". Enzymes of this group have lost the ancient active-site disulfide bond Cys191-Cys220 (bovine chymotrypsinogen A numbering) which is strongly conserved in classic serine proteases such as pancreatic, blood coagulation, and fibrinolysis proteases and others (granzymes A, M, K and leukocyte elastases). We sequenced the cDNA encoding bovine (Bos taurus) duodenase, a granase with unusual dual trypsin-like and chymotrypsin-like specificity. The sequence revealed a 17-residue signal peptide and two-residue (GlyLys) activation peptide typical for granases. Production of the mature enzyme is apparently accompanied by further proteolytic processing of the C-terminal pentapeptide extension of duodenase. Similar C-terminal processing is known for another dual-specific granase, human cathepsin G. Using phylogenetic analysis based on 39 granases we retraced the evolution of residues 189 and 226 crucial for serine protease primary specificity. The analysis revealed that while there is no obvious link between mutability of residue 189 and the appearance of novel catalytic properties in granases, the mutability of residue 226 evidently gives rise to different specificity subgroups within this enzyme group. The architecture of the extended substrate-binding site of granases and structural basis of duodenase dual specificity based on molecular dynamic method are discussed. We conclude that the marked selectivity of granases that is crucial to their role as regulatory proteases has evolved through the fine-tuning of specificity at three levels--primary, secondary, and conformational.

[A new approach to enhance PCR specificity]. / Novyi podkhod k uvelicheniiu spetsifichnosti PTsR.

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.

Mutation S543N in the thumb subdomain of the Taq DNA polymerase large fragment suppresses pausing associated with the template structure.

Substitution of Asn for the conserved Ser543 in the thumb subdomain of the Taq DNA polymerase large fragment (Klentaq DNA polymerase) prevents pausing during DNA synthesis and allows the enzyme to circumvent template regions with a complex structure. The mutant enzyme (KlentaqN DNA polymerase) provides specific PCR amplification and sequencing of difficult templates, e.g. those with a high GC% content or strong secondary structure.

Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase increases the efficiency of synthesis of long DNA molecules.

Substitution of Asn for Ser543 in the large fragment of Taq DNA polymerase (Klentaq) increases several times the efficiency of synthesis of long (over 2 kbp) DNA molecules. The difference in the DNA synthesis efficiencies by the mutant and native enzymes increased with the increase in the DNA fragment length.

[Integration of IS186 element into the -10-region of the promoter of heat shock lon-gene in Escherichia coli]. / Integratsiia élementa IS186 v -10-oblast' promotora teplovogo shoka lon-gena Escherichiia coli.

The insertion element IS186 was found to be incorporated into the -10 region of the promoter of the E. coli lon gene. The integration represses lon gene expression. Homology between the integration sites of IS186, the -10 region of E. coli heat shock promoters, and the terminal inverted repeat of IS186 was revealed.

[DNA polymerase mediated amplification of DNA fragments using primers with mismatches in the 3'-region]. / Amplifikatsiya Tth-DNK-polimerazoi fragmentov DNK s praimerami, soderzhashchimi nekomplementarnye matritse nukleotidy v 3'-kontsevoi oblasti.

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.

[The demonstration of the genes controlling enterotoxigenicity in Staphylococcus aureus strains by using the polymerase chain reaction]. / Vyiavlenie genov, kontroliruiushchikh énterotoksigennost' u shtammov Staphylococcus aureus, s pomoshch'iu polimeraznoi tsepnoi reaktsii.

The selection and subsequent synthesis, according to the nucleotide sequences of S. aureus genes responsible for the expression of enterotoxins A and B, of highly specific primers for polymerase chain reaction Pcr were carried out with the use of the program "Primer". The optimum temperature conditions of polymerase chain reaction for all pairs of primers were selected. The method for the rapid determination of the enterotoxigenic properties of S. aureus strains by means of Pcr was proposed. The enterotoxigenic properties of several S. aureus strains were determined, which revealed that 3 clinical isolates had the gene of enterotoxin A in their genome, while laboratory strain FRI-722(H) carried the genes of enterotoxins A and B.

[Cloning the Staphylococcus aureus enterotoxin B gene, obtained by polymerase chain reaction, and its expression in Escherichia coli cells]. / Klonirovanie gena énterotoksina B Staphylococcus aureus, poluchennogo metodom polimeraznoi tsepnoi reaktsii, i ego ékspressiia v kletkakh Escherichia coli.

To determine the Staphylococcus aureus enterotoxigenicity, we have developed an approach based on polymerase chain reaction (PCR). Using this method several S. aureus strains have been screened for the presence of the enterotoxin B gene. A DNA fragment of the selected strain (FRI 722H) containing enterotoxin B gene has been obtained by the PCR method and cloned in the pUC19 vector. It is shown that enterotoxin B with the leader peptide forms insoluble complexes in E. coli cells, whereas the mature toxin is present in cytoplasmic fraction in a soluble form. The recombinant toxin made up for 1.7% of the total cellular protein in E. coli JM 109 cells.