Amplification of 4-9-kb human genomic DNA flanking a known site using a panhandle PCR variant.
We present a method for the in vitro amplification of > 6.0 kb of DNA flanking a known site. This is accomplished by ligating an oligonucleotide to create an inverted repeat of a portion of the known sequence, followed by single-primer polymerase chain reaction (PCR) amplifications. This method generates a panhandle template following primer extension on the strand of interest. It does not involve template-directed extension from the ligated oligonucleotide, and it is carried out without DNA extractions. We have used this method to amplify 4.5-9.4 kb of DNA flanking the original primer annealing sites directly from human genomic DNA.
Jones
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DH
Department of Pediatrics, University of Iowa, Iowa City 52242, USA. douglas-h-jones@uiowa.edu
Winistorfer
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SC
D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D015894:Genome, Human; D005914:Globins / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D005914:Globins; D004247:DNA