Systematic analysis of intrinsic factors affecting differential display
Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annea...
| Veröffentlicht in: | BioTechniques. - 1993. - 32(2002), 4 vom: 05. Apr., Seite 762-4, 766 |
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| Format: | Aufsatz |
| Sprache: | English |
| Veröffentlicht: |
2002
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| Zugriff auf das übergeordnete Werk: | BioTechniques |
| Schlagworte: | Research Support, U.S. Gov't, P.H.S. Technical Report Journal Article |
| Zusammenfassung: | Differential display (DD) is a widely used method for identifying differentially expressed genes. To improve further the efficiency and reproducibility of the method, this report systematically examines four critical parameters of standard DD-PCR. Specifically, the study determined the optimal annealing temperature, elongation time, dNTP concentration, and arbitrary primer concentration. By using a thermal cycler that was capable of displaying a temperature gradient across a PCR plate, it was possible to determine (in a single experiment) the effect of different annealing temperatures. The optimal annealing temperaturefor a 13-mer arbitrary primer fell within a broad range of 40 degrees C-50 degrees C. Elongation times over a range of 30-120 s worked best. The optimal concentration for dNTPs was within a very broad range of 2-50 microM, with higher amounts allowing for greater pipetting accuracy. The most favorable concentration for the arbitrary primer was also within a broad range of 0.1-2.0 microM. A primer concentration below this range greatly reduced the efficiency of the amplification process. In conclusion, the experimental findings delineated the best possible DD conditions for a more reliable assessment of differential gene expression |
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| Beschreibung: | Date Completed 13.11.2002 Date Revised 28.09.2018 published: Print Citation Status MEDLINE |
| ISSN: | 0736-6205 |