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The overlap extension polymerase chain reaction (or OE-PCR) is a variant of PCR. It is also referred to as Splicing by overlap extension / Splicing by overhang extension (SOE) PCR. It is used to assemble multiple smaller double stranded DNA fragments into a larger DNA sequence.
A modification of this process, named Linear-After-The-Exponential-PCR (or LATE-PCR), uses a limiting primer with a higher melting temperature (T m) than the excess primer in order to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction. [3] See also overlap-extension PCR.
Overlap-extension PCR or Splicing by overlap extension (SOEing) : a genetic engineering technique that is used to splice together two or more DNA fragments that contain complementary sequences. It is used to join DNA pieces containing genes, regulatory sequences, or mutations; the technique enables creation of specific and long DNA constructs.
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).
The mammalian DNA does not come with these restriction sites, so they are built in by overlap extension PCR. The primers are designed to put the restriction sites carefully, so that the coding of the protein is in-frame, and a minimum of extra amino acids is implanted on either side of the protein.
They usually employ oligonucleotides of 40-50 nucleotides length that overlap each other. These oligonucleotides are designed to cover most of the sequence of both strands, and the full-length molecule is generated progressively by overlap extension (OE) PCR, [9] thermodynamically balanced inside-out (TBIO) PCR [10] or combined approaches. [11]
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This typically involves removing the stop codon from a cDNA sequence coding for the first protein, then appending the cDNA sequence of the second protein in frame through ligation or overlap extension PCR. That DNA sequence will then be expressed by a cell as a single protein. The protein can be engineered to include the full sequence of both ...