Open AccessSequence specific generation of a DNA panhardle permits PCR amplication of unknown flanking DNA
Author(s) -
D. H. Jones,
Stanley C. Winistorfer
Publication year - 1992
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/20.3.595
Subject(s) - biology , primer (cosmetics) , genomic dna , primer dimer , polymerase chain reaction , inverse polymerase chain reaction , genetics , dna , microbiology and biotechnology , gene , dna sequencing , polymerase , in silico pcr , nested polymerase chain reaction , multiplex polymerase chain reaction , chemistry , organic chemistry
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.