Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Differential display (DD) has been used extensively to detect differentially expressed genes. However, the low reproducibility of displayed bands makes verification by Northern blot difficult and the technique is labor-intensive. This report describes a fluorescent DD with a ROX (carboxy-X-rhodamine)-labeled anchor primer and a revised RT-PCR, utilizing AMV reverse transcriptase, a more thermostable reverse transcriptase than Mu-MLV, and optimized concentrations of dNTPs and of MgCl2. Our technique yielded clear fingerprints with high reproducibility. Further, we have developed a method of rapid screening to select the cDNA fragments of interest in excised bands from a polyacrylamide gel without cloning. This method consists of electrophoresis with an agarose gel containing a base-specific DNA ligand to separate the equally sized fragments differing in base composition, and side-by-side comparison of the reamplified products from the experimental and control lane. Most of the cDNA fragments selected by this protocol provided readable sequences by direct sequencing and were confirmed to exhibit differential expression by Northern blot analysis or semiquantitative RT-PCR.
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PMID:Differential display with carboxy-X-rhodamine-labeled primers and the selection of differentially amplified cDNA fragments without cloning. 946 1

A multiplex real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) assay for the simultaneous detection of the H5 and H7 avian influenza hemagglutinin (HA) subtypes was developed with hydrolysis type probes labeled with the FAM (H5 probe) and ROX (H7 probe) reporter dyes. The sensitivity of the H5-H7 subtyping assay was determined, using in vitro transcribed RNA templates, to have a reproducible detection limit for H7 of approximately 10(4) HA gene copies and approximately 10(4)-10(5) HA gene copies of H5. A direct comparison of H5-H7 multiplex RRT-PCR with hemagglutination inhibition (HI) was performed with 83 AI RRT-PCR and virus isolation positive tracheal and cloacal swab samples obtained from various avian species and environmental swabs from live-bird markets in New York and New Jersey. Both multiplex RRT-PCR and HI agreed on the subtype determination of 79 (95.2%) of the 83 samples, of which 77 were positive for H7 and two were determined to be non-H5/non-H7 subtypes. No samples were determined to be the H5 subtype by either assay.
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PMID:Development of multiplex real-time RT-PCR as a diagnostic tool for avian influenza. 1457 17

To study promoters we usually use primer extension to map the transcription start site and a panel of PCR generated deletion mutants. This strategy is complex and time-consuming. Therefore, we decided to improve it by using Gateway and FLOE (Fluorescently Labeled Oligonucleotide Extension). In this report we developed the first luciferase reporter "destination vector" (GW luc basic) for the Gateway technology and tested its efficacy, accuracy and background level by transfecting two distant cell lines (THP1 monocytic and SH-SY5Y neural cells). This vector is a real advantage for the cloning of many PCR fragments and sustains reporter activity also in THP1 cells, which are known to be problematic for transfection/expression. FLOE is a straightforward method to map transcription start sites but a bias in the capillary electrophoretic migration pattern of ROX weight markers has been reported: ROX markers migrated as if they were some bp longer. We hypothesized that this could depend on the use of different enzymes for the two principal reactions (DNA polymerase for the dideoxy chain terminated reaction on DNA and reverse transcriptase for the primer extension on RNA). Therefore, we used the same reverse transcriptase enzyme on both reactions, demonstrating that the reported bias is not due to the use of different enzymes but is an intrinsic feature of the ROX markers. The proposed procedure is important not only because of the timeliness but also for the global impact on the study of the first layer of the gene regulation.
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PMID:Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. 1770 8