Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intact RNA from various rat organs was isolated by an efficient and rapid method. This method of RNA isolation is a modification of an earlier method that uses guanidinium isothiocynate followed by extraction in the presence of sarcosyl, acetate and phenol. The RNA obtained by the method reported here was comparable with the RNA prepared by the CsCl2 ultracentrifugation method and the commercially available kit based on published methods. The quality of RNA was found suitable for Northern blotting analysis, RNase protection assays and reverse transcriptase-polymerase chain reaction (RT-PCR). Since reverse transcriptase is active in the buffer used for Taq DNA polymerase, only one reaction needs to be set up. We also found that the use of aurintricarboxylic acid in the RNA preparation prevents the degradation of RNA during storage. Expression of low density lipoprotein (LDL) receptor, apolipoprotein (apo) AI, AII and AIV mRNAs were quantified in various rat organs. Our results indicated that rat LDL receptor mRNA is expressed in several organs whereas apoAI and AIV mRNAs were expressed mainly in the liver and intestine. However, apo AII mRNA is expressed mainly in the liver. Unlike mice and some species of monkeys, in the rat apoAI mRNA is expressed at 5-6 times higher levels in the intestine compared to liver. Apo AIV mRNA abundance was also found to be several fold higher in intestine compared to hepatic tissues. We present here, for the first time, data on the absolute amounts of LDL receptor, apoAI, AII and AIV mRNA in various rat organs which were quantified by a novel RNase protection/solution hybridization assay.
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PMID:Expression of low density lipoprotein receptor, apolipoprotein AI, AII and AIV in various rat organs utilizing an efficient and rapid method for RNA isolation. 137 76

Eight Belgian AIDS Reference Laboratories established a multicentre quality control to evaluate the performance of their diagnostic human immunodeficiency virus type 1 (HIV-1) DNA polymerase chain reaction (PCR). A set of Belgian and African HIV-1 seropositive and seronegative patient samples, collected in Belgium, and the British Medical Research Council (MRC) HIV-1 PCR reference reagent kit, containing plasmid HIV-1 DNA at several dilutions in human carrier DNA with appropriate negative controls, were tested by the laboratories. No false positive results were reported. All laboratories were able to detect one to two copies of HIV-1 DNA. Among the 17 Belgian and African HIV-1 seropositives, some laboratories reported up to four indeterminate results, mainly due to failure of the SK38-39, SK68-69 (Ou et al. (1988) Science 239, 295-297) and/or gag881-882 (Simmonds et al. (1990) J. Virol. 64, 864-872) primers and a poorly performing algorithm. Only the H1POL4235-4538 nested pol primer set, developed by one of the laboratories, correctly identified all the tested HIV-1 positive and negative samples. Consequently, the laboratories decided to evaluate these pol primers as a reference primer set and to standardise the testing algorithm. All laboratories achieved a sensitivity and specificity of 100% on testing 10 additional Belgian and African patient samples, when adapting a standardised algorithm based on three HIV-1 primer sets, one of which is the H1POL4235-4538 primer set.
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PMID:Standardisation of primers and an algorithm for HIV-1 diagnostic PCR evaluated in patients harbouring strains of diverse geographical origin. The Belgian AIDS Reference Laboratories. 773 51

The TaqMan LS-50B PCR Detection System facilitates the automated and direct detection of polymerase chain reaction (PCR) products. The system employs the 5' nuclease activity of Taq DNA polymerase to hydrolyse a Salmonella specific internal fluorogenic probe for monitoring the amplification of a 287-bp region of the Salmonella invA gene. Using the fluorogenic 5' nuclease assay, 164 Salmonella strains representing all the subspecies of Salmonella enterica were detected while over 50 non-Salmonella strains were not detected. The detection limit of the assay was two colony forming units (cfu) per PCR reaction when a pure culture of S. typhimurium was used. Six protocols for the isolation of PCR-amplifiable DNA were evaluated using chicken carcass rinses, ground beef, ground pork and raw milk contaminated with Salmonella. Of the six DNA isolation protocols, a modified sample preparation protocol using the EnviroAmp kit was chosen for subsequent studies because it was reliable, easy to use and efficient for the isolation of PCR-amplifiable DNA from foods. A detection limit of 3-7 cfu per PCR reaction was obtained using food samples that were pre-enriched overnight and then inoculated with Salmonella. The detection limit was below 3 cfu/25 g or 25 ml when foods inoculated with Salmonella were pre-enriched overnight. Naturally contaminated foods (50 chicken carcass rinses and 60 raw milk samples) were examined using both the fluorogenic 5' nuclease assay and a modified semi-solid rappaport vassiliadis (MSRV) culture method. Thirty four of the 110 samples tested were Salmonella-positive and 74 were Salmonella-negative by both the 5' nuclease assay and the MSRV method. Two samples were Salmonella-positive by the 5' nuclease assay, but negative by the MSRV method. The correlation between the 5' nuclease assay and the MSRV method was over 98%.
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PMID:The evaluation of a fluorogenic polymerase chain reaction assay for the detection of Salmonella species in food commodities. 910 33

Amplification of random regions of genomic DNA using 10-base primers in the random-amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) was used to differentiate Anopheles minimus A and An. minimus C. Genomic DNA was extracted from individual mosquitoes of An. minimus A and An. minimus C and amplified in PCR reactions using single primers of arbitrary nucleotide sequence. Fifteen different commercially available primers (Operon oligonucleotides kit M from Operon Technologies, Inc.), six primers were selected on the basis of presence or absence of the bands of A and C. They gave 8 different amplified DNA fragments of these two species of An. minimus. The primers revealed only species A are OPM8 and OPM13 at the 0.8 and 2.15 and 2, while both species A and C were in OPM17 at 0.8 Kb revealed A, at the 0.55 and 1.5 Kb revealed C. OPM 12 gave a 0.5 Kb DNA fragment in A while 0.4 Kb in C. OPM15 showed C at 0.7 Kb. These findings indicated that An. minimus species A and C can be differentiated by RAPD-PCR technique.
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PMID:Differentiation of Anopheles minimus species complex by RAPD-PCR technique. 934 74

A selected number of PCR protocols were evaluated to determine if they could serve as a universal protocol for detecting and identifying all arboviruses. In this study, four parameters that affect the efficacy of RT-PCR (RNA extraction method, choice of reverse transcriptase, choice of DNA polymerase and thermocycling program) were evaluated in combination. The most optimal combination of those parameters employed use of silica gel membrane spin column, RAV-2 reverse transcriptase, Tth DNA polymerase, and a simple modification of a published thermocycling program. By this modified protocol, viral RNA could be amplified satisfactorily with more than 50 pairs of primers designed for diagnosis of arboviruses representing five families. The sensitivity and specificity obtained by this universal protocol were comparable to those obtained by the original protocol for each primer pair tested; and for some primers, improved sensitivity was observed. It was also found that a simple modification of a suggested protocol of a commercial RT-PCR kit could produce nearly identical results and serve as another universal protocol. With the use of a universal diagnostic reverse transcriptase-polymerase chain reaction (RT-PCR) protocol, simultaneous screening of clinical or biological specimens against a large number of RNA viruses belonging to many families can be performed more efficiently for etiologic determination in the situations complicated by the difficulty of differential diagnosis. Furthermore, such a universal protocol facilitates reducing the cost of PCR-based diagnostic operation and standardizing the qualities of PCR-based diagnosis within an institution or among collaborating institutions. A logical strategy is to conduct diagnosis in two stages by using broadly group-reactive primers in the first stage to narrow the range of possible etiologic agents and using virus-specific primers in the second stage for identification. Before such a strategy is employed, however, more group-reactive primers for a large number of arboviruses, for which no such primers currently exist, must be made available. Furthermore, the best pair or pairs of primers need to be selected for each virus for the second stage of the strategy.
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PMID:Universal diagnostic RT-PCR protocol for arboviruses. 967 30

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.
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PMID:Evaluation of TaqMan PCR assay for detecting Salmonella in raw meat and shrimp. 1041 4

A fast and efficient cDNA cloning procedure for plant RNA viruses was developed. In this procedure, double-stranded RNA (dsRNA) was used as a template source. Standard cDNA synthesis reagents and random hexamers were then used for making cDNAs. Taq DNA polymerase was used to add additional (A) at the ends of cDNAs, a TA cloning kit to ligate the cDNAs to vectors, and an electroporator to transform the DNAs to E. coli cells. dsRNAs were extracted from grapevine tissues infected with four different viruses and used for cloning. These viruses included grapevine rupestris stem pitting associated virus, grapevine leafroll associated virus 5, and two uncharacterized grapevine viruses, one each closely related to marafivirus and vitivirus groups. Selected cDNA clones were sequenced and PCR primers were developed for RT-PCR detection of these viruses in host plants.
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PMID:A strategy for rapid cDNA cloning from double-stranded RNA templates isolated from plants infected with RNA viruses by using Taq DNA polymerase. 1064 87

We evaluated the TaqMan PCR Salmonella amplification/detection kit (PE Applied Biosystems) for rapid detection of Salmonella from a variety of meat samples. This system uses the 5' nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The detection sensitivity of the kit, using 2 kinds of DNA extraction protocols, was compared with that obtained with 4 protocols of official culture methods. A total of 98 meat samples (16 raw beef, 31 pork and 51 chicken) were tested. The results of the TaqMan PCR method and the combined results of the 4 cultural protocols showed excellent agreement. However, no single culture protocol showed optimal recovery of Salmonella comparable to the PCR method. These results suggest that the TaqMan PCR method is a reliable and rapid method useful for detecting Salmonella in meat products.
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PMID:Comparison of TaqMan Salmonella amplification/detection kit with standard culture procedure for detection of Salmonella in meat samples. 1138 54

Under certain conditions, T4 gene 32 protein is known to increase the efficiency of different enzymes, such as Taq DNA polymerase, reverse transcriptase, and telomerase. In this study, we compared the efficiency of the SMART PCR cDNA synthesis kit with and without the T4 gene 32 protein. The use of this cDNA synthesis procedure, in combination with T4 gene 32 protein, increases the yield of RT-PCR products from approximately 90% to 150%. This effect is even observed for long mRNA templates and low concentrations of total RNA (25 ng). Therefore, we suggest the addition of T4 gene 32 protein in the RT-PCR mixture to increase the efficiency of cDNA synthesis, particularly in cases when low amounts of tissue are used.
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PMID:Increased yield of PCR products by addition of T4 gene 32 protein to the SMART PCR cDNA synthesis system. 1146 24

Nineteen strains from bovine abscesses identified as Fusobacterium necrophorum by the VPI method were examined by other methods. The API 20A test kit characterized all 19 strains as F. necrophorum. Seven of the strains had haemagglutinating activity and were classified as F. necrophorum subspecies necrophorum, and the remaining, 12 nonhaemagglutinating strains, were classified as F. necrophorum subspecies funduliforme. We used RAPD-PCR with a 10-mer oligonucleotide primer, W1L-2, to confirm this differentiation of the two subspecies. These results suggest that random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) with a suitable primer can be used as a new tool for the differentiation of F. necrophorum subspecies isolated from bovine pathological lesions.
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PMID:Differentiation of Fusobacterium necrophorum subspecies from bovine pathological lesions by RAPD-PCR. 1150 31


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