Sequence specific generation of a DNA panhandle permits PCR amplification of unknown flanking DNA.
We present a novel method for the PCR amplification of unknown DNA that flanks a known segment directly from human genomic DNA. PCR requires that primer annealing sites be present on each end of the DNA segment that is to be amplified. In this method, known DNA is placed on the uncharacterized side of the sequence of interest via DNA polymerase mediated generation of a PCR template that is shaped like a pan with a handle. Generation of this template permits specific amplification of the unknown sequence. Taq (DNA) polymerase was used to form the original template and to generate the PCR product. 2.2 kb of the beta-globin gene, and 657 bp of the 5' flanking region of the cystic fibrosis transmembrane conductance regulator gene, were amplified directly from human genomic DNA using primers that initially flank only one side of the region amplified. This method will provide a powerful tool for acquiring DNA sequence information.
Department of Pediatrics, University of Iowa College of Medicine, Iowa City 52242.
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Winistorfer
D001483:Base Sequence; D003001:Cloning, Molecular; D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005914:Globins / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics
C505032:CFTR protein, human; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005914:Globins; D004247:DNA