Use of PCR to screen for promoter elements in genomic DNA library clones
We report a modified PCR strategy to screen for promoter elements of genes of interest that is based upon consecutive rounds of PCR and appropriate subcloning. Following preliminary identification and sequencing of intron 1 by standardized PCR, the application of a suited cDNA/intron primer combinat...
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| Hauptverfasser: | , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
22 Aug 2018
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| In: |
BioTechniques
Year: 1999, Jahrgang: 26, Heft: 4, Pages: 718-726 |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/99264rr01 |
| Online-Zugang: | Verlag, lizenzpflichtig, Volltext: https://doi.org/10.2144/99264rr01 Verlag, lizenzpflichtig, Volltext: https://www.future-science.com/doi/10.2144/99264rr01 
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| Verfasserangaben: | M. Poppe, B. Hahm, W. Eickelbaum, M. Arand, N. Paweletz and M. Knehr |
| Zusammenfassung: | We report a modified PCR strategy to screen for promoter elements of genes of interest that is based upon consecutive rounds of PCR and appropriate subcloning. Following preliminary identification and sequencing of intron 1 by standardized PCR, the application of a suited cDNA/intron primer combination renders a succeeding PCR-mediated screening of cosmid or P1-derived artificial chromosome (PAC) libraries possible, thus identifying genomic clones comprising the searched promoter elements.We tested our approach in comparison with a commercially available promoter finder kit by searching the promoter elements of the CENP-C gene from the human and mouse genomes. Applying the kit system, we amplified the anticipated promoter from mouse, but failed in isolating human promoter elements. Our approach made use of a 5′-UTR/intron1 primer combination in the second round of PCR, enabling the identification of positive clones from genomic DNA within a human PAC library possible. Subcloning and final PCR amplification revealed the successful isolation of the human promoter.Therefore, we conclude that our approach might represent a helpful alternative to identify promoter elements, especially when prior art genome walking, STS-based strategies or anchored PCR failed. |
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| Beschreibung: | Elektronische Reproduktion der Druckausgabe Gesehen am 15.04.2021 |
| Beschreibung: | Online Resource |
| ISSN: | 1940-9818 |
| DOI: | 10.2144/99264rr01 |