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)

A low-cost DNA sequencer was constructed based on a single helium-neon laser. The two-color peak-height encoded sequencing protocol, based on the use of T7 DNA polymerase in a manganese buffer, was used to generate samples. Two termination reactions were performed. In the first, a TAMRA (applied Biosystems)-labeled primer was extended in the presence of ddATP and ddCTP. The amounts of dideoxynucleotides were adjusted to produce a 3:1 peak height ratio. Similarly, a ROX (Applied Biosystems)-labeled primer was extended in the presence of ddGTP and ddTTP; the amounts of dideoxynucleotides was adjusted to produce a 3:1 peak height ratio. The pooled fragments were separated on a 4% T LongRanger gel operated at 39 degrees C. Over 500 bases of sequence were generated in 50 min.
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PMID:High-speed and high-accuracy DNA sequencing by capillary gel electrophoresis in a simple, low cost instrument. Two-color peak-height encoded sequencing at 40 degrees C. 798 30

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3 alpha,4 alpha-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to site-specifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.
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PMID:Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry. 1506 93

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