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Only the duplex without overlap at the 5' end will allow extension by DNA polymerase in 3' to 5' direction. Following the extension of the OE-PCR reaction, the PCR mix or the eluted fragments of appropriate size are subject to normal PCR, using the outermost primers used in the initial, mutagenic PCR reactions.
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An extension of the 'colony-PCR' method (above), is the use of vector primers. Target DNA fragments (or cDNA) are first inserted into a cloning vector , and a single set of primers are designed for the areas of the vector flanking the insertion site.
Site saturation mutagenesis is a PCR based method used to target amino acids with significant roles in protein function. The two most common techniques for performing this are whole plasmid single PCR, and overlap extension PCR. Whole plasmid single PCR is also referred to as site directed mutagenesis (SDM).
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English: The use of PCR to splice two sequences together with primers that are complementary to both sequences. The dark blue & dark red lines represent the RNA Primers. The arrowheads represent the 3' -OH ends of the chai
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