Molecular Biotechnology

, Volume 2, Issue 1, pp 15-22

First online:

Inverse polymerase chain reaction

An efficient approach to cloning cDNA ends
  • Sheng-He HuangAffiliated withDivision of Infectious Diseases, Departments of Pediatrics and Microbiology, University of Southern California, Children’s Hospital Los Angeles

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The conventional polymerase chain reaction (PCR) requires that DNA sequences at both ends of the region to be amplified be known. Inverse PCR (IPCR) and anchored PCR overcome this limitation and amplify flanking unknown DNA sequences by utilizing inverse amplification and a universal primer, rcspectively. The major advantage of IPCR is that two gene-specific primers arc reserved for specific and efficient amplification of the unknown cDNA ends on the basis of a small stretch of known sequence. The protocol consists of five steps: reverse transcription, synthesis of second strand cDNA, circularization of double strand cDNA. reopen the circle DNA, and amplification of the inverse DNA fragment.

Index Entries

Inverse PCR flanking cDNA sequences reverse transcription inverse DNA fragment cDNA cloning