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Addition of Carbopol 934 (carboxypolymethylene, carboxyvinyl polymer) to an in vitro RNA-dependent DNA polymerase assay system resulted in a significant increase in the rate and extent of DNA synthesis. Carbopol may be a valuable tool for increasing the sensitivity of detection of reverse transcriptase activity in oncogenic RNA viruses and malignant tissues.

DNA polymerase B extracted from developing rat brain and subjected to (NH 4 ) 2 SO 4 -fractionation and column chromatography on DEAE-cellulose lost its activity after Sephadex G-200 gel filtration due to loss of an activating factor. The partially inactivated DNA polymerase was markedly stimulated by the addition of this factor. The ability of the activating factor to enhance DNA polymerase B was destroyed by its treatment with a proteolytic enzyme as well as by heating. The effect of the activating factor on polymerase B does not appear to be a consequence of either nuclease or “nickase” activity.

Evidence is presented that DNA polymerase of avian myeloblastosis virus has an obligatory zinc requirement for activity. Previous studies indicate that the purified polymerase contains zinc in a stoichiometry of about 1 g-atom/mole. We now find that the enzyme-bound zinc is exchangeable with radioactive 65 Zn; after isoelectric focusing, the radioactive 65 Zn is coincident with polymerase activity. Dialysis of the 65 Zn-labeled polymerase against the chelator, 1,10-phenanthroline, results in a progressive loss of radioactive 65 Zn and polymerase activity. Thereupon, incubation of the inactivated enzyme with Zn 2+ fully restores activity. Thus, the DNA polymerase present in an oncogenic RNA virus, like animal DNA polymerases, can be rigorously classified as a zinc metalloenzyme. DNA polymerase of avian myeloblastosis virus is inactivated by 1,10-phenanthroline at a much faster rate than the bacterial and animal DNA polymerases that have been tested. It may, therefore, be possible to inactivate selectively DNA polymerases from animal tumor viruses by brief exposure to appropriate metal chelators.

RNA-directed DNA polymerase was isolated from milk-borne B-type murine mammary tumor virus of the RIII mouse strain. The several hundred-fold-purified enzyme sediments at 5.5 to 5.7 S with an average molecular weight of approximately 100,000. The purified enzyme is completely template dependent and responds to RNA, DNA, and synthetic templates. Stability studies indicate differential lability dependent on the exogenous template used to monitor activity.

We have purified the small nuclear DNA polymerase from cultured KB cells to ≥95% homogeneity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel slabs demonstrates that the polymerase is comprised of a single polypeptide chain of molecular weight 43,000, and this size is in agreement with that of the polymerase activity as determined by gel filtration. Although the enzyme carries out a conventional polymerization reaction, incorporating complementary deoxynucleoside triphosphates at 3'-hydroxyl termini with concomitant stoichiometric release of PPi, it cannot be demonstrated to carry out PPi exchange. The polymerase is devoid of exonuclease activities, particularly the 3'→5'-exonuclease activity, as tested by the most sensitive available assays. However, the enzyme does exhibit prominent primer-template-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate. We propose that this reaction represents an independent polymerase-associated activity that is unrelated to 3'→5'-exonuclease, in contrast to the model that has been developed from studies with prokaryotic polymerases. Finally, the KB polymerase is unable to excise mismatched primer termini from a synthetic homopolymer primer-template, but rather can utilize such termini as functional initiation sites for polymerization.

Normal adult rat liver contains a high level of a synthetic RNA-dependent DNA polymerase activity, which is distinct from cellular DNA-dependent polymerases. It uses poly(dT).poly(rA) and poly(rA).poly(rU) as templates but has little or no response to DNA or several single-stranded RNAs.

The excitement aroused by the idea that transformation of cells by RNA tumour viruses might depend on the activity of a reverse transcriptase enzyme(s) specific to the virus has stimulated much research since the enzyme was discovered in 1970. But the questions of the physical identity and catalytic activities of the enzyme(s), its function in transformation and whether it is unique to RNA tumour viruses remain unanswered.

Infection of HeLa cells with herpes simplex virus leads to a large increase in a new DNA polymerase activity in the nucleus of infected cells. This herpes simplex virus-induced DNA polymerase has been purified 400- to 1300-fold from infected cells and shown to be different in its properties from the normal host cell DNA polymerases. The herpes simplex virus-induced DNA polymerase can be eluted as two peaks of activity on DEAE-cellulose in the presence of glycerol. However, these two DNA polymerase activities seem to have the same molecular weight (1.8 x 105) and exhibit similar properties after further purification on phosphocellulose. The herpes simplex virus-induced DNA polymerase is markedly different from the host DNA polymerases in its high salt requirement for maximal activity. It shows a strong preference for an oligomer-homopolymer template containing deoxyguanylate and deoxycytidylate. A nuclease activity which degrades the polydeoxyadenylate-deoxythymidylate copolymer and the deoxyguanylate-deoxycytidylate homopolymer pair is found to co-chromatograph with the herpes simplex virus-induced DNA polymerase.

Derivatives of rifamycin-SV with substituted cyclic-amine side chains in position 3 of the ansa ring are strong inhibitors of RNA-directed DNA and DNA-directed DNA polymerase activity of RNA tumor viruses of murine, feline, and avian origin. Among 37 3-amine derivatives of rifamycin-SV that were tested, 29 3-cyclic amine derivatives were good inhibitors of the viral polymerases. Especially active were 3-piperidyl derivatives of rifamycin-SV with cyclohexyl and cyclohexylalkyl substituents. Derivatives that were effective against the viral polymerase also blocked cell transformation by the murine sarcoma virus. A DNA-directed DNA polymerase preparation from human KB cells was less sensitive to inhibition by these derivatives than the virion polymerase.

Introduction: Drug resistant tuberculosis is a signifi cant threat to tuberculosis control because only a few effective drugs are available against M. tuberculosis . The aims of this study were to compare multi drug resistant (MDR) strains of tuberculosis by phenotypic and genotypic method and determine type, location and frequency of rpoB and KatG gene mutations. Methodology: Anti-tuberculosis drugs susceptibility test of M. tuberculosis grown on Lowenstein Jensen medium was performed by proportion method. MDR cases were analyzed for mutation of rpoB and KatG genes. The regions of these genes were amplifi ed by polymerase chain reaction (PCR) and sequenced. Results: Two different mutations were identifi ed in rifampicin resistant strains. The most common point mutations were in codons TCG 531→TTG (85%) and GAC 516→TTC (15%) of the rpoB gene. Two different mutations in KatG gene were detected. The most common KatG point mutations were AGC 315 ACC (Ser→Thr) (85%) and CGG 463 CTG (Arg→Leu) (10%). In this study DNA sequencing analysis did not fi nd mutation on KatG gene of one of the strain tested. Male and female were equally affected by MDR tuberculosis and majorities (35%) of them were found in 21-30 years age group. Conclusion: The present investigation agrees that genetic mutation is responsible for change in phenotypic characteristics of M. Tuberculosis. DOI: http://dx.doi.org/10.3126/saarctb.v8i2.5896 SAARCTB 2011; 8(2): 17-22

Background: By amplifying small amount of DNA, polymerase chain reaction (PCR) can be used for the detection of very small amount of microbial agent, and may be especially useful in certain cases which are difficult to be diagnosed microbiologically or serologically. Tuberculous pleurisy is a disease that can be diagnosed in only 70% of cases by conventional diagnostic tools, and PCR would be a very rapid, easy, and sensitive diagnostic method. Method: The specificity and sensitivity of PCR to detect Mycobacterium tuberculosis DNA were evaluated using various strains of Mycobacteria. To evaluate the diagnostic usefulness of PCR in tuberculous pleurisy, we used PCR to detect Mycobacterium tuberculosis DNA in pleural fluid. The amplification target was 123 base pair DNA, a part of IS6110 fragment, 10~16 copies of which are known to exist per genome. The diagnostic yield of PCR was compared with conventional methods, including pleural fluid adenosine deaminase (ADA) activity. Also, the significance of PCR in undiagnosed pleural effusion was evaluated prospectively with antituberculosis treatment. Results: 1) Using cultured Mycobacterium tuberculosis and other strains, PCR could detect upto 1 fg DNA and specific for only Mycobacterium tuberculosis and Mycobacterium bovis. 2) Using pleural effusions of proven tuberculosis cases, the sensitivity of PCR was 80.0% (16/20), and the specificity 95.0% (19/20). 3) Among 13 undiagnosed, but suspected tuberculous effusion, the positive rate was 60% in 10 improved cases after antituberculosis medications, and 0% in 3 cases of proven malignancy later. 4) Adenosine deaminase level of proven and clinically diagnosed tuberculous pleurisy patients was significantly higher than that of excluded patients, and correlated well with PCR results. Conclusion: We can conclude that PCR detection of Mycobacterium tuberculosis in pleural effusion has acceptable sensitivity and specificity, and could be an additional diagnostic tool for the diagnosis of tuberculous pleurisy.

Md. Sayeedur Rahman 11.6568 Normal 0 false false false MicrosoftInternetExplorer4 /* Style Definitions */ table.MsoNormalTable {mso-style-name:"Table Normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} In this study, for the detection of distinct genotype of E. histolytica of human, a nested Real-Time PCR amplification of SREHP gene using SYBR Green I and melting curve analysis was done. A total of 60 specimens (stool and liver aspirate specimens), which were found Entamoeba histolytica positive by E. histolytica specific ELISA and ssrRNA gene PCR, were selected and the experiment was conducted during the period of July 2003 to June 2004. After melting curve analysis of amplified PCR products from these isolates of stool and liver aspirate specimens, 5 genotypes were found belonging to the melting temperatures 84°C, 83°C, 82°C, 81°C and 79°C. All these 5 genotypes were present in intestinal amoebiasis patients and when the genotypes from intestinal amoebiasis patients were compared with the genotypes of amoebic liver amoebiasis patients, the genotype 84°C melting temperature was found to be absent in amoebic liver amoebiasis patients. For both the cases of intestinal and amoebic liver amoebiasis patients the genotype belonging to 83°C melting temperature was more prevalent than the other genotypes which suggest that this genotype is more responsible for the development of amoebiasis. In comparison to conventional PCR method where we found 23 different banding patterns, the Real-Time PCR and melting curve analysis method was found to be more reliable for the detection of distinct genotypes of E. histolytica because with this method we found only 5 genotypes. In conclusion, this Real-Time PCR using SYBR Green I and melting curve analysis for the genotyping of E. histolytica , excludes the need of post PCR manipulations and would be helpful for the rapid detection and screening of E. histolytica genotypes among endemic population and also for the epidemiological study. Key words: Entamoeba histolytica , genotype, diarrhoea, Real-Time PCR, melting curve analysis doi:10.3329/bjvm.v4i1.1526 Bangl. J. Vet. Med. (2006). 4 (1): 53-60

Polymerase Chain Reaction (PCR) based technology RAPD was tested for its applicability and suitability for molecular systematics of terrestrial bladderworts. PCR was carried out and the resultant products were subjected to agarose gel electrophoresis. The banding patterns were compared among ten species of Utricularia. Out of 40 random oligonucleotide primers examined, 16 primers generated distinguishable bands. Cluster analysis, unweighted pair group method with arithmetic mean (UPGMA) and ordination approach Scatter diagram using Matrix plot were performed based on RAPD fingerprints. The UPGMA and ordination analysis revealed a clear portrayal of systematic relationships in bladderwort species which were concordant with previous studies based on morphology and molecular approaches, indicating the reliability of RAPD markers for estimation of genetic variation and species relationships in Utricularia. Key words: Utricularia; RAPD; PCR; Cluster analysis; Molecular taxonomy DOI: 10.3329/bjb.v39i1.5532Bangladesh J. Bot. 39(1): 97-102, 2010 (June)

연구배경 : 결핵은 아직도 한국에서는 중요한 질병의 하나이지만 결핵을 진단하기 위한 보편적인 방법들이 단점인 예민도와 검사기간등이 문제가 되고 있다. 최근 분자유전학에 PCR 기법이 도입된 이래 결핵균의 DNA를 추출하여 짧은 시간내에 결핵을 진단하고자 하는 많은 보고들이 있어 왔다. 그러나 보고된 방법들의 다양성과 각 방법에 따른 소요시간등이 매우 다른 것이 문제가 되고 있고 임상에서 빠른 시간내에 비교적 간단한 방법으로 결핵율의 DNA를 분리한다면 보다 빨리 결핵을 진단하여 치료를 할 수 있다고 생각되어 결핵균의 DNA를 분리하는 방법중 가장 좋은 방법을 확인하고자 하였다. 방법 : 결핵율의 DNA를 추출하는 방법은 총 6가지의 방법으로서 SDS-microwave oven method, NaOH lysis method, Triton X-100-proteinase K method, Lysis buffer method, SDS-proteinase K method 및 Bead beater method를 사용하여 비교하였으며 사용된 균주는 Mycobacterium tuberculosis $H_{37}Rv$ strain 이었고 임상가검물중 도말양성 객담 5예 및 결핵성 뇌막염이 의심되고 도말음성이었으나 항결핵제 투여로 완치판정을 받은 환자의 뇌척수액 5예를 대상으로 하였다. 사용된 primer는 IS6110의 123bp를 target DNA를 하였고 PCR반응은 각각 $94^{\circ}C,\;68^{\circ}C$ 및 $72^{\circ}C$ 로 각각 2분씩 총 30주기를 시행하였다. 결과 : $H_{37}Rv$ strain에서 각 방법으로 추출된 DNA를 10배씩 serial dilution하여 PCR을 시행한 결과 SDS-microwave oven법과 NaOH lysis법은 50 fg까지 양성이었으며 나머지 4가지 방법에서는 5fg까지 양성이었다. 또한 각각의 방법으로 도말양성 환자의 객담에서 PCR을 시행한 결과 전예에서 양성이었고 결핵성 뇌막염이 의심스러운 환자의 뇌척수액에서는 SDS-microwave법과 NaOH lysis법으로는 5예중 3예에서 양성이었고 나머지 4가지 방법으로는 5예중 4예에서 양성이었다. 결론 : 이상의 실험으로 경제적이고 간편한 측면에서 볼 때 SDS-proteinase K법이 가장 좋은 방법임을 알 수 있었다. 【Background: The polymerase chain reaction (PCR) is a very sensitive method for the detecting of mycobacterial DNA. There are many reports revealing the efficacy of PCR for the diagnosis of M. tuberculosis, but there are many different methods for DNA extraction from Mycobacterium tuberculosis. Bead beater method is a very useful method for DNA extraction from clinical spectimens, but its procedures are relatively complicated and time-consuming. So we studied other methods for the DNA extraction from Mycobacterium tuberculosis $H_{37}Rv$ and some clinical specimens (5 smear positive sputa and 5 smear negative CSF). Method: We extracted the mycobacterial DNA with 6 different methods from H37Rv strain and clinical specimens. The methods included SDS-microwave oven method, NaOH lysis method, Triton X-100-Proteinase K method, Lysis buffer method, SDS-proteinase K method and bead beater method. The target DNA was 123bp of IS6110 and was detected by examination of ethidium bromide-stained agarose gels. Results: Among 6 methods, SDS-proteinase K method, bead beater method, lysis buffer method and triton X-100-proteinase K method were excellent, but SDS-proteinase K method was the best method in the aspect of simplicity and cost-effectiveness. Conclusion: We suggest that SDS-porteinase K method is a simple and convinient method and might be the best method for the extraction of mycobacterial DNA.】

A multiplex polymerase chain reaction (PCR) method was evaluated for simultaneous detection of Escherichia coli O157:H7, Listeria monocytogenes and Salmonella enteritidis in shrimp samples. The sensitivity of DNA amplification by PCR in this method was found to be 103 cfu/ml for each pathogen. When this protocol was adopted for the detection of each of the above mentioned pathogen in spiked shrimp extract culture, similar sensitivity was observed. However, this method detected 1 bacterial cell for E. coli O157:H7 and S. enteritidis and 100 for L. monocytogenes per 25 g spiked shrimp samples after overnight enrichment. In the commercially imported shrimp samples, none was found to contain any of the three pathogens by multiplex PCR or by conventional method, which suggests that the multiplex PCR is a reliable and useful for rapid screening of shrimp samples for E. coli O157:H7, L. monocytogenes and S. enteritidis. This will save time and increase our ability to assure food safety. Keywords: Multiples PCR, Shrimp extract, Spiked sample, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidisDOI: http://dx.doi.org/10.3329/bjm.v24i1.1236 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 42-46

A membrane filter assay has been devised to study the binding of Escherichia coli RNA polymerase to DNA. RNA polymerase is quantitatively retained on nitrocellulose filters, and DNA bound in complexes between RNA polymerase and labeled T7 DNA is retained with an efficiency of up to 70%. The attachment of a single RNA polymerase molecule to a molecule of T7 DNA is sufficient to cause retention of the DNA molecule on the filter. Using the filter assay, it has been shown that both RNA polymerase holoenzyme and core polymerase bind to T7 DNA. The interaction of RNA polymerase holoenzyme with T7 DNA leads to two classes of complex. Approximately eight polymerase molecules can bind to each T7 DNA molecule to form a highly stable complex (Kassoc. = 1012 to 1014 m−1). In addition, there are many other sites on the DNA molecule at which a weak complex can be formed (Kassoc. = 108 to 108 m−1). The majority of holoenzyme molecules bound in the highly stable complex are thought to be bound at or near the T7 early promoter region, while the weak binding sites are thought to reflect a low, general affinity of holoenzyme for other regions of the DNA helix. In contrast, only a single major class of binding sites is found on T7 DNA for core polymerase (Kassoc. = 2 × 1011 m−1) and there are many such sites on the T7 genome. These sites are interpreted as reflecting a high general affinity of core polymerase for all regions of the DNA helix. It is concluded that RNA polymerase can exist in two conformational states, one programmed for promoter binding and initiation (holoenzyme) and the other designed for RNA chain elongation (core polymerase). Sigma subunit may function in site selection primarily to switch the enzyme reversibly between these two states.

The burrowing nematode, Radopholus similis, is an obligate migratory endoparasite. Currently detection of this nematode is carried out mostly by physically extracting them from soil and then observing under a light microscope. To identify this nematode, a thorough knowledge about their morphological features is quite indispensable. Developing a DNA based detection technique makes it more convenient and accurate in detection. Though PCR based methods have been reported by earlier workers, developing a Real-Time PCR based method will enable estimating their population in field samples. In this study, Real-Time PCR primers were designed using the DNA sequences from the ITS region of R. similis. It can detect R. similis up to the limit of 100 fg μL-1 DNA. The real time PCR based detection serves as an efficient tool for the detection and estimation of this nematode from soil samples.

This study was carried out to assess the occurrence of Escherichia coli (E. coli) in the bats, birds and rodents as representative of wildlife from different habitats in Sibu and Kapit, Sarawak, Malaysia. A total of 682 swab samples were collected from wildlife hosts and screened for the bacteria E. coli and E. coli O157:H7 using standard microbiological methods and molecular techniques. The overall occurrence rates of E. coli among these hosts were 14%, 17% and 54% for bats, birds and rodents, respectively. The occurrence of E. coli was the highest in rodents regardless of the habitats. Isolated E. coli were then screened for E. coli O157:H7 by using a multiplex PCR with four primer pairs targeting for Shiga toxin producing genes (slt-I and slt-II), and the genes involved in biosynthesis of O157 antigen (rfbE) and H7 antigen (fliCH7 ). slt-I, slt-II and rfbE genes were not detected in any of the E. coli isolates. However, the gene encoding for H7 antigen was detected in 23 E. coli isolates. This indicated that E. coli O157:H7 strain was not detected in the wildlife studied. Absence of E. coli O157:H7 in the wildlife studied indicated these wild animals do not serve as an important reservoir of E. coli O157:H7. However, precautions have to be taken as other group of pathogenic E. coli may pose a zoonotic risk for humans and other animals.

Under standard assay conditions, the replicative DNA polymerase partially purified from rat liver non-histone chromosomal proteins exhibited a marked preference for native DNA over heat-denatured DNA as the template. However, work reported in the present paper demonstrates that this template preference could be reversed by changing the enzyme to template ratio in the assay system. This change in template preference was observed when either the concentration of DNA polymerase or of DNA was varied. Furthermore, it is shown that this alteration in template specificity is not the direct result of the action of an alkaline endonuclease present in the DNA polymerase preparation.

SummaryThe presence of DNA polymerase in normal human serum and its possible association with Australia antigen was investigated. DNA polymerase activity was determined in 135 samples of normal serum. Only 3 samples revealed appreciable amounts of activity. The addition of a variety of polynucleotide templates did not stimulate polymerase activity.Australia antigen is a particle associated with hepatitis and is reported to contain RNA. A DNA polymerase, “reverse transcriptase” might be required for replication of this RNA. We found DNA polymerase activity in only one of five preparations of purified Australia antigen. A similar activity was also found to be present in serum from certain normal volunteers. In either case, polymerase activity does not require nor is it stimulated by added polynucleotides suggesting that the enzyme may already be associated with a template such as RNA or DNA. A comparison between the amount of DNA polymerase activity in serum and the presence of Australia antigen in three se...

Summary A technique has been developed which solubilizes RNA polymerase from soybean chromatin. Dissociation of RNA polymerase from chromatin DNA is achieved by incubation in the presence of 0.5 M (NH 4 ) 2 SO 4 . Basic proteins and DNA are removed by centrifugation while the inhibitory levels of (NH 4 ) 2 SO 4 are removed by Sephadex G-50 chromatography. The polymerase preparation so obtained is dependent on added DNA, actinomycin-D sensitive, rifampin insensitive and partially inhibited by α -amanitin. The polymerase has a Mg 2+ optimum of 10.0 mM and a Mn 2+ optimum of 1.25 mM. No differences were noted in template activity of several native DNA's tested. Denatured calf thymus DNA was transcribed more efficiently than the native DNA's examined.

Monospecific antiserum was prepared against purified deoxyribonucleic acid (DNA) polymerase from avian myeloblastosis virus (AMV). Immunodiffusion assay with purified DNA polymerase revealed that the anti-DNA polymerase serum formed one precipitation band, whereas no reaction with any of the seven major structural proteins of AMV was observed. The antiserum also demonstrated enzyme-neutralizing antibody activity that was associated with the immunoglobulin G fraction. There was no difference in the neutralization of DNA polymerase activity directed by ribonucleic acid (RNA), DNA, or RNA-DNA hybrid templates.

The T4 DNA polymerase catalyzes extensive exonucleolytic degradation of linear duplex DNA starting at the 3' ends. However, the enzyme will release only a few nucleotides from each DNA molecule if the degradation is run in the presence of a single deoxynucleoside triphosphate. Several experiments suggested that the polymerase degrades a chain only until a nucleotide is released which can be replaced by transfer from the triphosphate. Subsequent reaction consists only of alternating removal and replacement of that nucleotide, without net degradation of the DNA. Because only a few nucleotides are released in the presence of a single triphosphate, it seemed possible that the polymerase could be used for analysis of nucleotide sequences at 3' termini of duplex DNA. This possibility was verified by the analysis of the 3'-terminal sequence of fragments of bacteriophage T7 DNA produced by endonuclease R, the Hemophilus influenzae restriction endonuclease. This terminal sequence had been previously identified by Kelly and Smith (J. Mol. Biol., 51, 393 (1970)). The method used in this analysis, which should also be applicable to other DNAs, was to degrade 32P-labeled DNA by the T4 polymerase in the presence of each of the four triphosphates. The nucleotides released in each case were identified and quantitatively determined by thin layer chromatography, and were consistent with the previously determined terminal sequence.

Cholera caused by toxigenic Vibrio cholerae is a major public health problem confronting developing countries, where outbreaks occur in a regular seasonal pattern and are particularly associated with poverty and poor sanitation. It is generally accepted that seven distinct pandemics of cholera have occurred since the onset of the first pandemic in 1817. Again Vibrio cholerae is capable of surviving in aquatic environments for extended periods and is considered as autochthonous species in estuarine and brackish waters. Therefore, the present study was designed to isolate V. cholerae from natural environmental samples subsequently identified by conventional and molecular biological techniques. A total number of 10 isolates were included randomly in this study based on their initial identification. The serotypes of the isolates were determined by serological test (slide agglutination) and the number of serotypes O1, O139 and non-O1/O139 were 3, 2 and 5 respectively which were reconfirmed by PCR method. Finally, the toxigenicity of the isolates was analyzed by multiplex PCR method and five (5) isolates were found to contain the ctx gene, the major virulence factor of V. cholerae. Key Words: Vibrio cholerae, Simplex PCR, Multiplex PCR, Serotypes, Toxigenicity. Â doi:10.3329/sjps.v1i1.1811 S. J. Pharm. Sci. 1(1&2): 69-75

A total of 28 local rice ( Oryza sativa L.) varieties of Bangladesh were selected for DNA fingerprinting with seven microsatellite DNA markers. Upon PCR amplification the alleles were separated on Agarose gel using a sequencing gel electrophoresis system. The loci were polymorphic (P 95 ) in all of the varieties. Variation was found in number of alleles, allele frequency, observed and expected heterozygosity. The primer, RM335 having motif (CTT) 20 also yielded highest number of alleles (15) and highest PIC value (0.909). Genetic differentiation (F st ) values were found in the ranges 0.84 to 1.00 with an average of 0.92 and gene flow (N m ) values ranged from 0.047 to 0.00 with an average of 0.02. High level genetic differentiation and low level gene flow values in 28 rice ( Oryza sativa L.) varieties which were indicated of diversity among the varieties as most of these varieties were of landraces. Over all Nei’s genetic distance value (D) ranged from nil to 2.56 among 378 varietals pairs resulting as a means of permutation combination of 28 rice varieties. The UPGMA dendrograme based on Nei's genetic distance placed the varieties into different clusters. All of the varieties were identified with at least one and/or combination of 7 primers. Keywords: Microsatellites; DNA fingerprinting; Oryza sativa ; Bangladesh DOI: 10.3329/jbau.v8i1.6391 J. Bangladesh Agril. Univ. 8(1): 7-17, 2010

DNA polymerase activity was found in chick embryo cells (CEC) that were infected with a tumor virus, strain MC29 (myelocytomatosis) virus. The enzyme activity in homogenates of virus infected cells was greater than in homogenates from noninfected control cells. DNA polymerase activity from noninfected CEC was not able to use the homopolymer duplex, poly dC:poly rG as a template. However, polymerase in cells infected with tumor virus was able to utilize this homopolymer duplex as template, as was the DNA polymerase associated with MC29 virus. The results demonstrated that DNA polymerase activity found in MC29 virus was detectable in the infected-cell homogenate and was distinguished from the cell DNA polymerase by its ability to utilize dC:rG as template.

Chicken embryo cells normally contain, in addition to deoxyribonucleic acid (DNA)-dependent DNA (D-DNA) polymerases, a novel “R-DNA-polymerase” which specifically copies polyriboadenylic acid strands. This R-DNA polymerase cannot copy natural ribonucleic acid or polyribocytidylic acid strands to a significant extent. Infection of cells with the leukovirus RAV-2 leads to the intracellular formation of large amounts of the viral RNA-dependent DNA polymerase whose properties differ from the cell R-DNA polymerase. Chicken cells transformed by a Rous sarcoma virus mutant which produce noninfectious alpha-type Rous sarcoma virus (f), a leukovirus known to be deficient in the viral RNA-dependent DNA polymerase, do not contain detectable viral RNA-dependent DNA polymerase, whereas the cellular R-DNA polymerase is found in normal amounts. There seems to be no relationship between the cellular R-DNA polymerase and the RNA-dependent DNA polymerase of the avian leukoviruses.

Survival study of toxigenic Vibrio cholerae O1 Classical Inaba and El Tor Ogawa biotypes was carried out in artificial microcosm without any nutrients under stressed physiochemical conditions. The organisms were found to undergo viable but non-culturable (VBNC) state, which was detected by polymerase chain reaction (PCR) technique using primers that flank a specific fragment of the cholera toxin gene (ctx). The findings of the study showed that about 89% of samples gave positive result although the organisms were in nonculturable state. Depending upon the conditions the Classical biotype of vibrios had undergone non-culturable state within 2-15 days and 55-92 days were subjected to PCR assay after 45 and 120 days of incubation respectively. A similar tend was also observed in case of El Tor biotype. With non-culturable cell only 5 of 18 microcosms (of both Classical and El Tor biotypes) at pH 4.5 showed no viability. Since the PCR assay demonstrated positive results after 90 (in case of El Tor biotype) and 120 days (in case of Classical biotype), the total survival time thus ranges over 3 and 4 months. Thus PCR has an important advantage over the techniques those are used in routine laboratories for the identification of pathogenic organisms. Keywords: Physicochemical stresses, Survivality, Vibrio cholerae, Artificial microcosms, Polymerase chain reaction (PCR), Non-culturable state, Viable but non-culturable (VBNC)DOI: http://dx.doi.org/10.3329/bjm.v24i2.1258 Bangladesh J Microbiol, Volume 24, Number 2, December 2007, pp 133-136

Bacterial numbers in surface water samples, collected randomly from six different water bodies, were estimated by acridine orange direct counting (AODC) and conventional culture-based heterotrophic plate counting (HPC). Bacterial genomic DNA was prepared from water samples by employing methods used for stool samples, including the population dynamics, were determined by primer extension of the 16S rDNA (V6/V8 region) using polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE), a metagenomic tool that is capable of separating unrelated DNAs based on the differences in their sequences and GC contents. The bacterial numbers in water samples ranged from 10 3 – 10 6 CFU/ mL for HPC and 10 4 – 10 7 cells/ mL for AODC, showing that a great majority of bacteria prevail as uncultivable which do not respond to culture methods that are used widely for tracking bacterial pathogens. The acridine orange-stained bacteria varied in sizes and shapes, and appeared either as planktonic (solitary) cells or as clusters of biofilms, showing the presence of diverse community under the epifluorescence microscope. The DGGE of the ca. 457 bp amplicons, as confirmed by agarose gel electrophoresis, produced bands that ranged in intensities and numbers from 18 to 31, with each band possibly indicating the presence of one or more closely related bacterial species. The enrichment of pathogenic bacteria in the aquatic ecosystem is known to precede the seasonal diarrhoeal outbreaks; therefore, bacterial community dynamics determined by Metagenomic 16S PCR-DGGE during pre-epidemic enrichment appears promising in predicting the upcoming diarrheal outbreaks. Key words: Metagenomics; 16S rDNA; PCR; DGGE; bacterial community dynamics DOI: http://dx.doi.org/10.3329/bjm.v27i2.9171 BJM 2010; 27(2): 46-50

Polymerase chain reaction (PCR) assay was performed to detect and identify the genetically modified maize ( Bt11 ). Primer pair Bt11 1-5´ and Cry1Ab 1-3´ detected the region of the insect resistant Cry1Ab gene sequence inserted in GM Bt11 maize and a primer pair ZE01-ZE02 were used to detect the maize intrinsic zein ( Ze1 ) gene in maize DNA. The presence of the corresponding DNA segments was specifically detected in GM maize by the designed primers. It was concluded that this method is useful for fast and easy screening of Bt gene in the food products and GM Bt crops. Key words: Detection, Cry1Ab gene, Maize intrinsic gene, GM maize DOI = 10.3329/ptcb.v17i1.1127 Plant Tissue Cult. & Biotech. 17(1): 103-108, 2007 (June)

Infectious laryngotracheitis is an acute upper respiratory tract infection of chickens caused by infectious laryngotracheitis virus. The study was conducted to standardize the polymerase chain reaction targeting a relatively conserved region of the thymidine kinase gene for the rapid detection of infectious laryngotracheitis virus. The vaccine samples were collected from two renowned company of Bangladesh. DNA was extracted from diluted vaccine samples by using Wizard® Genomic DNA purification kit and thymidine kinase gene was amplified by using PCR system 9600 Thermocycler. Two vaccine samples were positively amplified by polymerase chain reaction. A procedure was developed for rapid detection of infectious laryngotracheitis virus by polymerase chain reaction of the conserved region of viral thymidine kinase gene containing DNA fragments. The results obtained in this study suggested that the polymerase chain reaction procedure could serve as a fast and sensitive method for the detection of vaccine strains of infectious laryngotracheitis viruses. Key words: Infectious laryngotracheitis virus; viral thymidine kinase (TK) gene; polymerase chain reaction DOI: http://dx.doi.org/10.3329/ujzru.v29i1.9468 UJZRU 2010; 29(1): 61-64

Culex quinquefasciatus Say is the major vector of the filarial parasite Wuchereria bancrofti (Cobbold) which causes lymphatic filariasis in humans. A repetitive DNA sequence from the genome of C. quinquefasciatus has been cloned and completely sequenced. The 693 bp cloned fragment had an A+T content of 72%. Dot matrix analysis of the fragment did not reveal any direct or inverted repeats within it. Southern blot analysis using a variety of restriction enzymes appeared to indicate that the cloned fragment was interspersed within the genome with a copy number of approximately 30,000. A search of the GenBank database did not reveal significant homologies to any previously cloned sequences. Although the probe was sensitive enough to detect picogram quantities of DNA, it was not specific for C. quinquefasciatus, as it hybridized with DNA from other mosquito species, Culex pseudovishnui Colless, Culex gelidus Theobald, Culex tritaeniorhynchus Giles, Anopheles vagus Donitz and Mansonia uniformis (Theobald). However PCR primers derived from the cloned sequence, IpC, were found to be specific and amplified only C. quinquefasciatus DNA. The optimized PCR assay was found to be very sensitive and was capable of detecting DNA from all stages of C. quinquefasciatus thus making it an ideal diagnostic tool.

The objective of this review article was to highlight briefly the recent advances made relevant to applications of free circulating nucleic acids (FcNAs) in clinical medicine. Detection of FcNAs in plasma, serum and other body fluids from healthy subjects as well as in patients has opened up the possibility of diagnosis and monitoring of diseases. With the rapid developments in molecular biology techniques such as real-time quantitative polymerase chain reaction (rt-qPCR), quantitative methylation specific PCR (qMS-PCR), matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry, quantitative fluorescent PCR (QF-PCR), single allele primer extension reaction (SAPER) method and other techniques the applications in clinical medicine have increased. The recent discovery of epigenetic changes in placental/fetal DNA and the detection of fetal/placental-specific RNAs have made FcNAs to be used for diagnosis of genetic disorders in all pregnancies irrespective of the gender of the fetus in early intrauterine life.  It is now possible to detect very small amounts of, and specific mutations in, fetal DNA in the presence of excess non-specific maternal DNA. In oncol­ogy, detection and monitoring of tumors are now possible by the detection of tumor-derived FcNAs. In other conditions, such as diabetes mellitus, trauma and stroke FcNAs have been shown to be useful also. In spite of these advances questions re­garding the origin and biologic significance of FcNAs remain to be answered. Standardization of methodologies including pre-analytical and analytical aspects will revolutionize the applications of FcNAs in the diagnosis and monitoring of diseases in clinical medicine in the next few years.Keywords: Circulating nuclic acid, MedicineDOI: 10.3329/bmrcb.v34i1.1168Bangladesh Med Res Counc Bull 2008; 34: 26-32