Nonspecific, Nested Suppression PCR Method for Isolation of Unknown Flanking DNA (Cold-Start Method)

The ability of the polymerase chain reaction (PCR) to amplify DNA depends upon the existence of defined primer binding sites. Thus, to amplify a region of flanking unknown DNA sequence, a defined primer binding site must be created. Numerous strategies have been found to do this, such as addition of nucleotides or ligation of oligonucleotides to DNA ends, restriction of the flanking DNA followed by ligation of known DNA sequences and, (for cDNA amplification), ligation of RNA oligonucleotides to the RNA molecule followed by reverse transcription PCR (reviewed in ref. 1 ). All of these methods require considerable molecular biological processing of the source nucleic acid.