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Taq Polymerase (taq + polymerase)
Selected AbstractsUse of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4AJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 3 2001Kimberly B. Bleicher Abstract Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan®) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan® probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3, end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families. © 2001 John Wiley & Sons, Inc. J Biochem Mol Toxicol 15:133,142, 2001 [source] Evaluation and optimisation of five different extraction methods for soy DNA in chocolate and biscuits.JOURNAL OF THE SCIENCE OF FOOD AND AGRICULTURE, Issue 11 2004Extraction of DNA as a first step in GMO analysis Abstract A method is described to discriminate between genetically modified (GM) and non-modified foodstuffs by detecting the presence of newly introduced genes at the protein or DNA level. Currently available methods operate almost exclusively at the DNA level and are based on the polymerase chain reaction (PCR). The first and most crucial step in this process is the isolation of DNA. In this study, five different methods for the isolation of DNA from chocolate and biscuits were evaluated, using four commercially available extraction kits and a non-commercial method for amplification of the soybean-specific lectin gene. The latter method involves the use of hot-start Taq polymerase, to prevent the formation of non-specific amplification products, and an increase in the number of cycles from 35 to 41. The performance of the non-commercial cetyl trimethylammonium bromide (CTAB)-based method was the best, taking into consideration the adaptations of the extraction procedure, although this method was more time-consuming than the others. Chocolate (white, milk and dark) and several biscuits generated positive amplification results using this PCR approach. Copyright © 2004 Society of Chemical Industry [source] Identification of Lactobacillus crispatus by polymerase chain reaction targeting S-layer protein geneLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2002M. Horie Aims: ,This study aimed to develop a polymerase chain reaction (PCR) method to identify Lactobacillus crispatus . Methods and Results: ,A primer set (CbsA2F,CbsA2R) for amplifying conserved regions of S-layer genes was designed to identify Lact. crispatus and the specificity of this set was compared with that of another primer set (Cri 16SI,Cri 16SII) which has been reported as a species-specific primer set targeting the 16S rRNA gene. Among species in the Lact. acidophilus A1,A4 groups, when KOD polymerase was used for amplification, the primer set CbsA2F,CbsA2R gave PCR products with Lact. crispatus strains only. However, when Taq polymerase was used, this primer set gave products with one Lact. amylovorus strain as well as with Lact. crispatus strains. The primer set Cri 16SI,Cri 16SII gave PCR products with Lact. crispatus strains and two Lact. acidophilus strains, regardless of whether the polymerase used was KOD or Taq . Conclusions: ,A PCR targeting the S-layer gene and amplified with KOD polymerase can identify Lact. crispatus accurately and rapidly. Significance and Impact of the Study: ,To the authors' knowledge, this is the first paper to provide a PCR method for the specific identification of Lact. crispatus . [source] Purification and characterization of Taq polymerase: A 9-week biochemistry laboratory project for undergraduate studentsBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2010Robert M. Bellin Abstract We have developed a 9-week undergraduate laboratory series focused on the purification and characterization of Thermus aquaticus DNA polymerase (Taq). Our aim was to provide undergraduate biochemistry students with a full-semester continuing project simulating a research-like experience, while having each week's procedure focus on a single learning goal. The laboratory series has been taught for the past 7 years, and survey-based assessment of the effectiveness of the laboratory series was completed during the 2006 and 2007 fall semesters. Statistical analysis of the survey results demonstrate that the laboratory series is very effective in teaching students the theory and practice of protein purification and analysis while also demonstrating positive results in more broad areas of scientific skill and knowledge. Amongst the findings, the largest reported increases in knowledge were related to students' understanding of how patent law relates to laboratory science, a topic of great importance to modern researchers that is readily discussed in relation to Taq polymerase. Overall, this laboratory series proves to be a very effective component in the curricula of undergraduate biology and chemistry majors and may be an appropriate laboratory experience for undergraduates. [source] Estrogen regulation of the expression of a cytochrome P450 isoenzyme,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 6 2004Jozsef Szeberenyi Terms to be familiar with before you start to solve the test: cytochrome P450 enzymes, xenobiotics, estrogens, steroid receptors, gene induction, SDS, chromatin, immunoprecipitation, immunoglobulin G (IgG), agarose gel electrophoresis, promoter, transcription factors, enhancer, histones, nonhistone proteins, PCR, Taq polymerase, primers, oligonucleotides, radioactive labeling, nondenaturing PAGE. [source] Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosusBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2006Pongpan Laksanalamai Abstract Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 100°C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts. © 2005 Wiley Periodicals, Inc. [source] Controlling DNA Polymerization with a Switchable AptamerCHEMBIOCHEM, Issue 14 2007Eike Friedrichs Controllable biochemical reactions. DNA polymerization by Taq polymerase can be controlled by switching an aptamer for Taq Pol between a binding and a nonbinding form. [source] | |||||||||||||