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.

[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.

[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.

[Isolation of site-specific endonucleases and methylases from Bacillus cereus 83]. / Vydelenie sait-spetsificheskikh éndonukleazy i metilazy iz Bacillus cereus 83.

The site-specific endonuclease R Bce83I and methylase M Bce83I were isolated from Bacillus cereus 83 by three consecutive chromatographies on blue agarose, hydroxyapatite and heparin-Sepharose. R Bce83I recognizes [formula: see text] sequences on the DNA and cleaves the DNA as indicated by the arrows. The endonuclease is stimulated by S-adenosyl-L-methionine and may consequently be referred to type IV restriction enzymes.

[New site site-specific endonuclease and methylase from Bacillus licheniformis 736]. / Novye sait-spetsificheskie éndonukleaza i metilaza iz Bacillus licheniformis 736.

The site-specific endonuclease R Bli736I and methylase M Bli736I have been isolated from the Bacillus licheniformis strain 736 by blue-agarose, hydroxyapatite-Ultragel and heparin-Sepharose chromatography. The enzymes are free from interfering impurities. R Bli736I recognizes the 5'-GGTCTCN-3' decreases and decreases 5'-NNNNNGAGACC-3' sequences on the DNA and cleaves the DNA as indicated by arrows to form single-stranded 4-nucleotide 5'-protruding termini. This enzyme is an isoschizomer of Eco3II isolated from E. coli.

[Amplification of the phage lambda DNA sequence by polymerase chain reaction using thermostable DNA polymerase]. / Amplifikatsiia posledovatel'nostei DNK faga lambda metodom polimeraznoi tsepnoi reaktsii s ispol'zovaniem termostabil'noi DNK-polimerazy.

The efficiency of phage DNA amplification by the method of polymerase chain reaction (PCR) with Tth DNA-polymerase was studied for optimization of PCR conditions. The effect on amplification efficiency of medium ionic strength and pH, the presence of univalent cations, detergents, gelatin, ATP, pyrophosphate, SH-reagents and ratio of concentrations of Mg and dNTPs, primers and template was studied. It has been found that a pH optimum for PCR with Tth DNA-polymerase varies from 8.5 to 9.0. An ionic strength optimum for PCR is about 0.08. The influence of univalent cations on the activity of Tth DNA-polymerase can be expressed as NH4+ greater than Na+ greater than K+. 0.01% Tween-20 significantly increases the efficiency of PCR and 0.01% gelatin inhibits it. Addition of ATP, pyrophosphate, SH-reagents to the reaction mixture did not increase the yield of PCR product. It has been also shown that for the given PCR-system an optimum Mg/dNTPs molar ratio is within the range of 1.5-2.0. An optimum concentration of each of the pair of primers for this PCR-system is about 0.3 microM. The possibility of PCR-amplification of 500-8500 b.p. DNA fragments has been demonstrated.

Amplification of DNA sequences of Epstein-Barr and human immunodeficiency viruses using DNA-polymerase from Thermus thermophilus.

Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). DNA-polymerase from Thermus thermophilus (molecular mass of 80-86 kDa) differs in its physico-chemical properties from DNA-polymerase from Thermus aquaticus (molecular mass of 62-68 kDa). To amplify the specific EBV DNA sequence, oligonucleotide primers for the virus replicon region (oriP region) were used. As a result of amplification, a specific 405-bp DNA fragment was produced.

[The site-specific endonucleases of bacteria in the genus Bacillus]. / Sait-spetsificheskie éndonukleazy bakterii roda Bacillus.

Production regularities of site-specific endonucleases by aerobic spore-forming bacteria, separated from different ecological sources have been studied. It is shown that more than 1/3 of all the studied cultures produce site-specific endonucleases. A dependence of occurrence frequency of bacteria producers on the econiche of their separation has been noticed. The data on production of species-specific restrictases are obtained which can serve additional characteristics for differentiation of close species of Bacillus.

[Amplification of DNA sequences in Epstein-Barr virus and human immunodeficiency virus using DNA polymerase from Thermus thermophilus]. / Amplifikatsiia posledovatel'nostei DNK virusa Epshtein-Barr i virusa immunodefitsita cheloveka s ispol'zovaniem DNK-polimerazy iz Thermus thermophilus.

Using thermophilic DNA-polymerase from Thermus thermophilus we have amplified by polymerase chain reaction (PCR) specific DNA sequences of Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). DNA-polymerase from Thermus thermophilus (the molecular mass of 80 to 86 kDa) differs in its physico-chemical properties from DNA-polymerase from the Thermus acquaticus (the molecular mass of 62 to 68 kDa). To amplify the specific EBV DNA sequence oligonucleotide primers for the virus replicon region (oriP-region) were used. As a result of amplification, a specific 405 b.p. DNA fragment was produced. Primers for the virus Gag region were used for amplification of HIV DNA. The possibility to conduct amplification cycles under two temperature conditions was demonstrated.

[New producers of site-specific endonucleases from microorganisms of the Bacillus genus]. / Novye produtsenty saitspetsificheskikh éndonukleaz iz mikroorganizmov roda Bacillus.

52 strains of Bacillus generum have been tested for production of site-specific endonucleases. The sequence recognized by the enzyme was determined for 23 enzymes, the cleavage site inside the sequence was determined for 5 enzymes. All the enzymes under study were found to be isomers of the known enzymes. The selected strains are peculiar for the high level of site-specific endonucleases content and may be used as producents of the enzymes.

[A new type of cleavage of the recognition site by the site-specific endonuclease Bst 4.4I from Bacillus stearothermophilus 4.4]. / Novyi tip rasshchepleniia uznavaemoi posledovatel'nosti nukleotidov sait-spetsificheskoi éndonukleazoi Bst 4.4I iz Bacillus stearothermophilus 4.4.

A site-specific endonuclease Bst 4.4I was isolated from the cell extract of Bacillus stearothermophilus 4.4 and partially purified by chromatography on Ultragel AcA-44 and heparin-Sepharose. It was shown that the endonuclease cleaves lambda and M13 DNA yielding distinct fragments just as endonucleases of II and III types but, in contrast to them can produce two two-strand cuts separated with 30 to 32 nucleotides in the region of the recognition site.

[Restriction endonucleases from Bifidobacteria]. / Restriktsionnye éndonukleazy iz bifidobakterii.

The sitespecific restriction endonucleases were found in four strains among the twelve strains of anaerobic bacteria of generum Bifidobacterium. Two of the restriction endonucleases studied, BadI from B. adolescentis LVA1 and BbfI from B. bifidum LVA3, are isoshizomers of XhoI and recognize the nucleotide sequence CTCGAG. The restriction endonucleases Bbf7411I from B. bifidum 7411 and Bla7920I from B. lactentis 7920 recognize and hydrolize the nucleotide sequence TCCGGA having the specifity analogous to the one of restriction endonuclease CauB3I. Like CauB3I, these restriction endonucleases are unable to hydrolyize DNA if the adenine residues in the recognition site are methylated.

[Site-specific endonuclease CauB31 from Chloroflexus aurantiacus B3]. / Sait-spetsificheskaia éndonukleaza CauB31 iz Chloroflexus aurantiacus B3.

A sequence-specific endonuclease CauB3I has been isolated from cell extracts of Chloroflexus aurantiacus and partially purified by chromatography on heparin-sepharose; the yield was 3000 units per 1 g of cells. The final preparation is free of non-specific nucleases. It is shown that endonuclease CauB3I recognizes 5' T decreases CCGGA 3' sequence in double-stranded DNA and cleaves it as shown by an arrow. Methylation of adenine in the recognition sequence makes it resistant to CauB3I.

Isolation and some properties of the site-specific endonuclease and methylase Bme2161 from Bacillus megaterium 216.

The site-specific endonuclease Bme2161 was isolated as a homogeneous preparation by chromatography on phosphocellulose, hydroxyapatite and heparin-agarose. The molecular mass of the enzyme, determined by gel filtration and by electrophoresis under denaturing conditions, was found to be 60 kDa and 30 kDa respectively. These data indicate that the native enzyme consists of two identical subunits. The enzyme recognized the decreases pentanucleotide sequence 5'-GGACC-3' X 3'-CCTGG-5' and cleaves the sequence as indicated by arrows. The increases optimal concentration for endonuclease reaction is 6-7 mM Mg2+. The endonuclease relaxes its specificity in the presence of glycerol or dimethyl sulfoxide at low Mg2+ concentration (1-3 mM). Methylase Bme2161, which protects DNA against endonuclease Bme2161 action by DNA methylation, was isolated from the same bacterial strain.