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 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/intron 1 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.
Poppe
M
M
German Cancer Research Center, Heidelberg, Germany.
Hahm
B
B
Eickelbaum
W
W
Arand
M
M
Paweletz
N
N
Knehr
M
M
D000818:Animals; D001483:Base Sequence; D001709:Biotechnology; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005069:Evaluation Studies as Topic; D015698:Genomic Library; D006801:Humans; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic
D002868:Chromosomal Proteins, Non-Histone; D017931:DNA Primers; D018076:DNA, Complementary; C060905:centromere protein C; D004247:DNA