mutation further into a sequence than a 110 nt primer will allow, it is necessary to employ overlap extension PCR. In OE-PCR the sequence being modified is used to make two modified strands with the mutation at opposite ends, using the technique described above. After mixing and denaturation, the strands are allowed to anneal to produce three different combinations as detailed in the diagram. Only the duplex without overlap at the 5' end will allow extension by DNA polymerase in 3' to 5' direction. Following separation, the eluted fragments of appropriate size are subject to normal PCR, using the outermost primers used in the initial, mutagenic PCR reactions. References Molecular biology Laboratory techniques Polymerase chain reaction Genetic engineering Molecular biology techniques