Use of PCR to screen for promoter elements in genomic DNA library clones

• Verfasst von: Poppe, Monika [VerfasserIn]
• Verfasst von: Paweletz, Neidhard [VerfasserIn]
• Titel: Use of PCR to screen for promoter elements in genomic DNA library clones
• Verf.angabe: M. Poppe, B. Hahm, W. Eickelbaum, M. Arand, N. Paweletz and M. Knehr
• E-Jahr: 2018
• Jahr: 22 Aug 2018
• Jahr des Originals: 1999
• Umfang: 9 S.
• Teil: volume:26
• Teil: year:1999
• Teil: number:4
• Teil: pages:718-726
• Teil: extent:9
• Fussnoten: Elektronische Reproduktion der Druckausgabe ; Gesehen am 15.04.2021
• Titel Quelle: Enthalten in: BioTechniques
• Ort Quelle: London, UK : Future Science Ltd, 1983
• Jahr Quelle: 1999
• Band/Heft Quelle: 26(1999), 4, Seite 718-726
• ISSN Quelle: 1940-9818
• DOI: doi:10.2144/99264rr01
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1754999476

Abstract

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.