Amplification of 4–9-kb Human Genomic DNA Flanking a Known Site Using a Panhandle PCR Variant

Abstract

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.