Basic DNA and RNA Protocols

Volume 58 of the series Methods in Molecular Biology™ pp 293-301

Inverse Polymerase Chain Reaction

  • Daniel L. HartlAffiliated withDepartment of Organismic and Evolutionary Biology, Harvard University
  • , Howard OchmanAffiliated withDepartment of Genetics, Washington University School of Medicine

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The inverse polymerase chain reaction (IPCR) was the first extension of the conventional polymerase chain reaction to allow the amplification of unknown nucleotide sequences without recourse to conventional cloning. In the conventional polymerase chain reaction (PCR), synthetic oligonucleotides complementary to the ends of a known sequence are used to amplify the sequence (1,2). The primers are oriented with their 3′ ends facing each other, and the elongation of one primer creates a template for annealing the other primer. Repeated rounds of primer annealing, polymerization, and denaturation result in a geometric increase in the number of copies of the target sequence. However, regions outside the boundaries of the known sequence are inaccessible to direct amplification by PCR. Since DNA synthesis oriented toward a flanking region is not complemented by synthesis from the other direction, there is at most a linear increase in the number of copies of the flanking sequence.