Inverse polymerase chain reaction. An efficient approach to cloning cDNA ends.
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, respectively. The major advantage of IPCR is that two gene-specific primers are 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.
Huang
S H
SH
Department of Pediatrics, University of Southern California, Children's Hospital Los Angeles 90027.
D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary